CHARACTERIZATION OF THE MURINE ANGELMAN SYNDROME IMPRINTING CENTER

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1 CHARACTERIZATION OF THE MURINE ANGELMAN SYNDROME IMPRINTING CENTER By EMILY YVONNE SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

2 2010 Emily Y. Smith 2

3 To my mom and dad 3

4 ACKNOWLEDGMENTS I would like to thank all past and present members of the Resnick lab with whom I have had the pleasure to work with: Chris Futtner, Danielle Maatouk, Lori Kellam, Jessica Walrath, Karen Johnstone, Edwin Peery, Mike Lewis, and Amanda DuBose. Special thanks goes to our undergraduate assistants, Ryan Hallett and Chelsea Batten, who have spent countless hours helping with my research and completing many less than desirable lab duties. In addition, I would like to thank my mentor, Dr. Jim Resnick. He has provided me with incredible guidance in the lab, in my career, and has also given me invaluable life advice. I am eternally grateful for his extraordinary friendship and for always going out of his way to help, no matter the circumstances. The support he has given me and his other graduate students has been phenomenal and I could not have asked for a better mentor. Special thanks goes to my sister, brother-in-law, boyfriend, and friends for always providing great distractions from my research and reminding me of the most important things in life. Most importantly, I would like to thank my parents for their unconditional love and immeasurable support throughout my life. Words could never express my gratitude for everything they have done for me and for the great role models they have been in every aspect of life. 4

5 TABLE OF CONTENTS ACKNOWLEDGMENTS... 4 LIST OF TABLES... 8 LIST OF FIGURES... 9 ABSTRACT CHAPTER 1 INTRODUCTION page Genomic Imprinting The Discovery of Imprinted Genes The Epigenetics of Imprinting Prader-Willi and Angelman Syndromes Molecular Classes of PWS and AS The PWS/AS Locus MATERIALS AND METHODS Mouse Husbandry Transgenic Lines Genotyping RNA Isolation Northern Blot SnoRNA Northern Blot RT-PCR Southern Blot Primordial Germ Cell Purification High Molecular Weight Genomic DNA Preparation Bisulfite Sequence Analysis Bisulfite Conversion Bisulfite-Treated DNA Purification Bisulfite PCR Cloning PCR Products Plasmid Sequencing THE IMPRINTED BAC TRANSGENE Introduction Attempts to Identify the Murine AS-IC Founding the BAC Transgenic Model System Results

6 The 425Δ5-7 BAC Possesses a Functional AS-IC Imprinted expression of the 425Δ5-7 transgene Epigenetic imprinting marks on the 425Δ5-7 transgene Snrpn Upstream Exon Expression Patterns are Conserved on the 425Δ5-7A Transgene Upstream exon expression in the brain Upstream exon expression in the ovary Snrpn upstream exon expression is not restricted to the brain and ovary Snrpn Upstream Exons are Silent in the Germline Before the Establishment of Imprints Discussion IDENTIFICATION OF THE AS-IC Introduction Results Three Snrpn Upstream Exons Constitute the AS-IC on the 425D18 BAC Expression of the 425ΔU1-U3 transgene is not imprinted Epigenetic imprinting marks are absent on the 425ΔU1-U3 transgene Discussion A SINGLE UPSTREAM EXON IS SUFFICIENT TO IMPRINT THE BAC TRANSGENE Introduction Results The 425ΔU2/U3 BAC Displays AS-IC Activity The 425ΔU2/U3 BAC exhibits imprinted expression patterns The 425ΔU2/U3 BAC is epigenetically imprinted U1 Usage on the Imprinted 425ΔU2/U3 BAC Transgene U1 is not transcribed from the 425ΔU2/U3E transgene in newborn brain The 425ΔU2/U3E transgene does not express U1 in the ovary Discussion SILENCING OF THE ENDOGENOUS LOCUS BY A PATERNALLY TRANSMITTED TRANSGENE Introduction Results Imprint Analysis of the 425ΔU2/U3A Transgene: Uncoupling of the Epigenetic and Expression Imprints The 425ΔU2/U3A transgene does not display imprinted expression patterns The DNA methylation imprint is present on the 425ΔU2/U3A transgene U1 Usage From the 425ΔU2/U3A BAC Transgene Endogenous Snrpn is Silenced Upon Paternal Transmission of the 425ΔU2/U3A Transgene

7 The 425ΔU2/U3A transgene cannot silence Snrpn from another BAC transgene Silencing by the 425ΔU2/U3A transgene is independent of imprinting Regulation of Other Genes at the Endogenous PWS/AS Locus by the 425ΔU2/U3A Transgene The upstream cluster genes are not repressed by the 425ΔU2/U3A transgene The 425ΔU2/U3A transgene exerts varying effects on the downstream cluster genes Discussion CONCLUSIONS AND FUTURE DIRECTIONS APPENDIX: PCR PRIMER SET SEQUENCES LIST OF REFERENCEs BIOGRAPHICAL SKETCH

8 LIST OF TABLES Table page 6-1 Gene regulation at the PWS/AS locus by the 425ΔU2/U3A transgene A-1 List of primers used in PCR and RT-PCR experiments

9 LIST OF FIGURES Figure page 1-1 The imprint lifecycle Molecular classes of PWS and AS The PWS/AS domain Model for IC function at the PWS/AS locus Identifying the boundaries of the PWS-IC and AS-IC Organization of the SNPRN upstream exons Schematic representation of the Snrpn containing BAC transgenes Snrpn RT-PCR analysis of the 425Δ5-7 transgenic lines Schematic diagram of the Snrpn DMR Bisulfite sequence analysis of the 425Δ5-7H transgene Snrpn upstream exon expression from the 425Δ5-7A transgene Snrpn upstream exon expression in various tissues Snrpn upstream exon expression in the developing germline The modified 425D18 BAC transgenes Snrpn expression analysis of the 425ΔU1-U3D transgene Bisulfite sequence analysis of the 425ΔU1-U3D transgene Comparisons of the three modified 425D18 BAC transgenes Snrpn expression analysis of the 425ΔU2/U3E transgene Bisulfite sequence analysis of the 425ΔU2/U3E transgene Snrpn upstream exon usage from the 425ΔU2/U3E transgene Snrpn expression from the 425ΔU2/U3A transgene Bisulfite sequence analysis of the 425ΔU2/U3A transgene Snrpn upstream exon usage from the 425ΔU2/U3A transgene

10 6-4 Snrpn expression analysis with the 425U2/U3A and 380A paternally transmitted transgenes Endogenous Snprn repression in fetal gonads upon paternal transmission of the 425ΔU2/U3A transgene Upstream cluster gene expression in the 425ΔU2/U3A transgenic line Effects on Ube3a-ats and Ube3a expression in the 425ΔU2/U3A transgenic line SnoRNA expression in the 425ΔU2/U3A transgenic line SnoRNA expression analysis with the 425ΔU2/U3A and 380A maternally transmitted transgenes A working model for imprint establishment at the PWS-IC Structure of the GDF9 flox 380 BAC transgene Schematic diagram of the transcriptional terminator allele

11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF THE MURINE ANGELMAN SYNDROME IMPRINTING CENTER Chair: James Resnick Major: Medical Sciences Genetics By Emily Yvonne Smith May 2010 Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurological disorders resulting from improper gene expression from the imprinted domain on chromosome 15q11-q13, the PWS/AS locus. This locus is controlled by a bipartite imprinting center consisting of the PWS-IC and the AS-IC. Evidence suggests that the PWS-IC acts as a positive element to promote gene expression from the paternal allele. The AS-IC acts in the oocyte to inactivate the PWS-IC on the future maternal allele thus silencing the paternally expressed genes. The PWS-IC is located just 5 to and including exon one of SNRPN whereas the AS-IC is 35 kb upstream of SNRPN. Importantly, the AS-IC includes two of several SNRPN alternative upstream exons. The PWS/AS locus is well conserved in the mouse but a murine AS-IC remains uncharacterized. As in humans, the mouse Snrpn locus includes several upstream exons postulated to function in silencing the maternal allele. We have taken a transgenic approach to study the potential regulatory role of these alternative exons. To do so, we utilized the bacterial artificial chromosome (BAC) 425D18, which contains Snrpn and approximately 120 kb of 5 sequence in which three alternative upstream exons reside. We first confirmed that this BAC transgene displayed proper imprinted expression in multiple transgenic lines thus demonstrating 11

12 the presence of a functional AS-IC. Imprinting was further examined by analysis of the epigenetic status of the Snrpn differentially methylated region (DMR), which lies within the PWS-IC. To determine whether the upstream exons on the 425D18 BAC confer silencing upon maternal transmission, we used recombineering techniques to create targeted deletions of these exons. Deletion of the three upstream exons resulted in robust Snrpn expression upon both maternal and paternal transmission of the transgene as well as a loss of the epigenetic imprint at the Snrpn DMR. These results indicate that the three upstream exons comprise the AS-IC on the 425D18 BAC. Our data support a model in which transcription arising from the AS-IC and continuing through the PWS-IC results in epigenetic modification of the PWS-IC. Further experiments utilized this BAC transgenic system to investigate mechanisms of AS-IC action. 12

13 CHAPTER 1 INTRODUCTION Genomic Imprinting Sexual reproduction is a fundamental feature of life that generates genetic diversity through the production of offspring possessing two unique sets of chromosomes, one inherited maternally and one paternally inherited. Typically, homologous genes on these chromosomes are expressed at similar levels; however, there exists a small number of genes that do not display biallelic expression patterns, genes which are subject to a phenomenon termed genomic imprinting. Genomic imprinting is an epigenetic regulatory mechanism that acts at a subset of chromosomal regions and results in parent-of-origin specific monoallelic gene expression. The epigenetic imprint is a heritable mark that is established in gametes and maintained in somatic cells. Imprinted expression patterns are frequently both tissue and developmental-stage specific and appropriate control of these expression patterns is vital as imprinted genes regulate many aspects of growth and development. To date, 112 imprinted genes have been identified in mammals, 96 in the mouse and 53 in humans, including 37 which overlap (Morison et al., 2005). These imprinted genes are frequently found clustered together in specific regions of the genome, coordinately regulated by imprinting control centers (ICs) that direct allele specific differences in transcription, DNA methylation and histone modifications (Kitsberg et al., 1993; Lewis and Reik, 2006; Margueron et al., 2005; Razin and Cedar, 1994). The IC is a cis-acting DNA element that is capable of long-range regulation, however, the mechanisms of this regulation are still not fully understood (Ferguson-Smith and Surani, 2001). Furthermore, studies have shown that different imprinted regions are not necessarily subject to the same regulatory mechanisms. A major focus of current research in the field of genomic imprinting is on IC mechanisms as they act upon imprinted loci. 13

14 The Discovery of Imprinted Genes Evidence for genomic imprinting was first discovered in the mid-1980s through murine embryological and classical genetic experiments (Barton et al., 1984; McGrath and Solter, 1984). In these studies, diploid mouse embryos possessing either two female pronuclei (gynogenotes) or two male pronuclei (androgenotes) were created from one-cell-stage embryos via nuclear transplantation. The authors found that both androgenetic and gynogenetic embryos failed to complete normal embryogenesis, demonstrating that the maternal and paternal genetic contribution is not functionally equivalent. Both types of embryos were capable of developing to the blastocyst stage but died shortly after implantation. Embryonic growth, albeit delayed, was evident in the gynogenotes; however, these embryos lacked significant extraembryonic tissue and thus failed to survive to term. Conversely, androgenotes developed substantial extraembryonic tissue but exhibited little, if any, development of the embryo proper. Murine androgenetic development mimics that of the naturally occurring hydatidiform mole in humans, a paternally diploid conceptus that contains ample extraembryonic tissues but lacks a fetus (Bagshawe and Lawler, 1982). Additional genetic studies analyzing mice bearing uniparental disomies provided further evidence for the existence of genomic imprinting in mammals. The combination of results from a number of studies proved that for proper embryonic development, contributions from both parents are only necessary for certain chromosomal regions of the genome. Mice possessing uniparental disomies displayed normal survival rates and were phenotypically wild type for a majority of the chromosomes (Lyon et al., 1975). In contrast, embryos inheriting maternal or paternal disomies of certain chromosomes, those that are now known to contain imprinted loci, either failed to survive to term or survived but displayed developmental abnormalities (Cattanach and Kirk, 1985; Searle and Beechey, 1978). In addition, the developmental aberrations observed 14

15 in these mice varied markedly depending on the parental origin of the disomy. The failure of a duplication of one parental chromosome to complement the deficiency of the other suggested a differential functioning of alleles located within these chromosomes. These findings further demonstrated the existence of an imprinting mark at certain genomic loci, a heritable mark affecting gene activity in a parent-of-origin specific manner. The Epigenetics of Imprinting Since the existence of genomic imprinting was first revealed, intensive efforts have gone into discovering a mechanism for this phenomenon. Given that both parental alleles may be identical in DNA sequence, it follows that the differential expression of imprinted genes is controlled by an epigenetic regulatory mechanism. Epigenetic marks, including DNA methylation and histone modifications, provide a means for cells to generate diverse patterns of gene expression from identical DNA sequences. The majority of imprinted domains are coordinately regulated by cis-acting ICs that direct epigenetic modifications and expression patterns throughout the locus, oftentimes acting over regions of several megabases (Lewis and Reik, 2006). Although the exact mechanisms of IC regulation are not entirely clear, DNA methylation is recognized as playing a vital role in the process. Methylation of DNA is carried out by DNA methyltransferases (Dnmts) at the 5 position of cytosine residues in CpG dinucleotides. One function of this repressive epigenetic modification is to inhibit transcription at promoter regions (Bird, 2002). At imprinted loci, allele-specific differential DNA methylation is frequently observed at distinct sequences termed differentially methylated regions (DMRs). ICs typically contain DMRs as well as other allele-specific differences in chromatin structure such as DNase I hypersensitivity sites and covalent modifications of histone tails (Lewis and Reik, 2006). A recent study demonstrated a mechanistic link between DNA methylation and histone methylation 15

16 at several murine ICs, providing support for a model in which DNA methylation controls histone modifications at these loci (Henckel et al., 2009). Typically, repressive histone modifications including methylated lysines 9 and 27 on histone H3 (H3K9 and H3K27), reside on the methylated allele. The unmethylated allele generally displays activating histone modifications such as methylation of lysine 4 on histone H3 (H3K4) and acetylation of histones H3 and H4. The epigenetic marks that define the imprint are established at ICs in the germline, allowing for differential gene expression between parental alleles over the course of development. In order to be propagated throughout the generations, imprinting marks must go through a life cycle consisting of three stages: erasure, establishment and maintenance (Figure 1-1). It is essential for imprints to be erased and re-established in a parent-specific manner in the germline so that upon fertilization, each zygote contains one maternally imprinted set of chromosomes and one paternally imprinted set of chromosomes. Erasure of imprinting marks occurs in the primordial germ cells (PGCs) as they colonize the developing gonad between days post coitus (dpc) (Hajkova et al., 2002; Lee et al., 2002). Imprint erasure is associated with the demethylation of IC DMRs and well as with the biallelic expression or biallelic silencing of imprinted genes in PGCs by 12.5 dpc (Szabo and Mann, 1995). DNA demethylation during this time in development is part of a germ cell-specific global epigenetic reprogramming; DMRs are maintained in the soma at this stage of embryogenesis. The timing of imprint erasure is critical as it ensures both the maternally inherited and the paternally inherited alleles are at an equivalent epigenetic state before sexual differentiation of PGCs occurs at 13.5 dpc. It is not until after sexual differentiation that imprints are reestablished in a sex-specific manner in the germline. In the male embryo, paternally methylated DMRs are established in the mitotically arrested fetal prospermatogonia between dpc 16

17 (Davis et al., 2000). In the oocyte, maternally methylated DMRs are established after birth during the oocyte growth phase, corresponding with meiotic prophase I (Lucifero et al., 2004; Lucifero et al., 2002). Dnmt3a and Dnmt3l, two members of the de novo family of methyltransferases, are required for the acquisition of methylation imprints (Bourc'his and Bestor, 2004; Bourc'his et al., 2001; Hata et al., 2002; Kaneda et al., 2004). The one known exception is the Rasgrf1 DMR, which also requires Dnmt3b for establishment of DNA methylation (Kato et al., 2007). After genomic imprints are established in the germline, it is imperative that they are maintained following fertilization and stably inherited in the somatic cells throughout the course of multiple cell divisions. During pre-implantation development, the maternal and paternal genomes undergo extensive epigenetic reprogramming and the majority of DNA methylation is lost; however, imprinted genes are protected from this global demethylation (Tremblay et al., 1995). The maintenance methyltransferase, Dnmt1, acts on hemimethylated DNA after replication to methylate CpG dinucleotides on the newly synthesized strand. Dnmt1 is required to maintain methylation imprints in both pre- as well as post-implantation development (Hirasawa et al., 2008; Li et al., 1993). How Dnmt1 activity is specified to imprinted regions in pre-implantation embryos is still under investigation. Prader-Willi and Angelman Syndromes Due to their monoallelic expression patterns, imprinted genes are functionally hemizygous, resulting in a genetic vulnerability that contributes to developmental disorders and disease, carcinogenesis, and embryonic lethality. My research is focused on two distinct neurogenetic imprinting disorders, PWS and AS. The prevalence of each disorder is approximately one in every 15,000 live births. Patients with either syndrome exhibit significant developmental and behavioral problems. PWS is characterized by an initial failure to thrive in infancy which 17

18 evolves into severe hyperphagia and obesity within the first few years of life (Holm et al., 1993). Other traits include hypogonadism, small hands and feet, short stature, mild to moderate mental retardation and obsessive-compulsive disorder. AS clinical manifestations include severe mental retardation, profound speech impairment, sleep disorders, microcephaly, gait ataxia, and seizures (Williams et al., 2001). AS patients also display behavioral abnormalities including a happy disposition with frequent laughter and smiling, uplifted hand-flapping, and hyperactivity (Williams et al., 2006). Both PWS and AS are the result of improper gene expression from the imprinted locus on chromosome 15q11-q13, a region known as the PWS/AS locus. This locus contains the paternally expressed genes MKRN3, MAGEL2, NDN, SNURF/SNRPN (referred to henceforth as SNRPN), a UBE3A-antisense transcript (UBE3A-ATS), and multiple small nucleolar RNA (snorna) encoding genes. UBE3A, an E6-associated-protein ubiquitin-protein ligase, is also located within the PWS/AS domain. This gene is biallelically expressed in non-neuronal tissues but preferentially expressed from the maternal allele in portions of the brain (Albrecht et al., 1997; Rougeulle et al., 1997; Vu and Hoffman, 1997). Mutations leading to a loss of UBE3A function from the maternal chromosome result in AS (Kishino et al., 1997; Matsuura et al., 1997). PWS has classically been thought to be the result of a loss of multiple paternal gene products from the PWS/AS locus; however, two recently described individuals, one diagnosed with PWS and one with several PWS-associated characteristics, both bear microdeletions limited to the HBII-85 snorna C/D cluster thereby challenging this notion (de Smith et al., 2009; Sahoo et al., 2008). Molecular Classes of PWS and AS There are several genetic mechanisms that lead to AS and PWS, each resulting in a loss of gene expression from the normally active allele (Figure 1-2). In PWS, it is a loss of paternal 18

19 gene expression from the PWS/AS locus and in AS it is a loss of maternal gene expression from this region. Approximately 70% of PWS cases are a result of a large interstitial deletion on paternal chromosome 15 while roughly 29% of PWS patients exhibit maternal uniparental disomy (UPD) of chromosome 15. A third molecular class of PWS, representing approximately 1% of patients, is defined as an imprinting defect (ID) in which the paternal chromosome possesses a maternal imprint at the PWS/AS locus. Rare patients display balanced chromosomal translocations where HBII-85 snorna expression is absent (Wirth et al., 2001). These translocation patients, in addition to the HBII-85 snorna-containing microdeletion patients mentioned previously, provide evidence that this snorna cluster plays a key role in the pathology of PWS. There are five distinct molecular classes of AS with varying phenotypic severities depending on the origin of the defect. The first class, representing about 70% of patients, is a large de novo deletion of maternal chromosome 15. Five percent of patients fall into the second molecular class of AS, bearing a UPD of paternal chromosome 15. The third class of AS represents the ID patients, those possessing a paternal imprint at the PWS/AS locus on the maternal chromosome (Reis et al., 1994). This class constitutes less than 5% of all AS cases. Class four is caused by a mutation of UBE3A, comprising 10% of cases, and the final 10% of cases (class five) are of an unknown origin. Patients in classes one and five display the most severe phenotypes while those in classes two and three exhibit less severe clinical manifestations. The PWS/AS Locus The PWS/AS imprinted domain is located within a 2 Mb region on the long arm of chromosome 15 (Figure 1-3A). This locus contains two imprinted gene clusters commonly referred to as the downstream and the upstream cluster based on their position relative to the IC. 19

20 The upstream cluster contains the paternally expressed MKRN3, MAGEL2, and NDN genes. The downstream cluster is comprised of the paternally expressed SNRPN, several snornas including HBII-85, HBII-52, and HBII-13, as well as UBE3A-ATS. The paternally expressed downstream gene products are thought to be processed from one single large transcript of over 460 kb that originates at SNRPN exon one (Runte et al., 2001). Imprinting of the paternally expressed genes in both the upstream as well as the downstream cluster appears to be ubiquitous throughout the different tissues of the body. Contrasting to these paternal expression patterns are the expression patterns of two other genes, UBE3A and ATP10A, which are also located within the downstream cluster. ATP10A displays variable imprinted expression in the brain with some individuals demonstrating biallelic expression and others exhibiting monoallelic expression in this tissue (Hogart et al., 2008). As mentioned previously, UBE3A shows preferential expression from the maternal allele exclusively in brain tissues. Located between the upstream and downstream imprinted gene clusters is a cis-acting bipartite IC, comprised of the PWS-IC and the AS-IC, that regulates both epigenetic reprogramming as well as gene expression at this locus (Figure 1-4) (Buiting et al., 1995b; Dittrich et al., 1996). Prevailing models of IC function suggest that the PWS-IC serves as a positive element to activate gene expression from the paternal allele. The AS-IC is posited to act as a negative element that directs inhibitory epigenetic modifications at the PWS-IC during oogenesis, thereby silencing the paternally expressed genes on the future maternal allele (Brannan and Bartolomei, 1999; Dittrich et al., 1996; Shemer et al., 2000). Rare PWS and AS patients display IDs at the PWS/AS domain due to mutations or microdeletions of the IC (Buiting et al., 1995a). By analyzing the shortest region of overlap of these microdeletions, the boundaries of the PWS-IC and AS-IC have been defined. The PWS-IC 20

21 is located within a region of less than 4.3 kb 5 to and including exon one of SNRPN (Figure 1-5A) (Ohta et al., 1999b; Sutcliffe et al., 1994). The SNPRN DMR is a CpG island contained within the PWS-IC that is methylated specifically on the maternal allele, representing an essential epigenetic imprint at this locus. The paternal SNRPN DMR is unmethylated and displays additional epigenetic marks indicative of open chromatin structure including high levels of histone H3 and H4 acetylation as well as histone H3 lysine 4 methylation (Fulmer-Smentek and Francke, 2001; Saitoh and Wada, 2000). In PWS ID cases without a PWS-IC deletion, the SNPRN DMR is hypermethylated on both alleles, indicating a maternal imprint on the paternal chromosome, and expression of the paternal genes is absent (Ohta et al., 1999b). Mapping of microdeletions in AS ID patients has localized the AS-IC to a sequence of 0.88 kb approximately 35 kb upstream of the PWS-IC (Figure 1-5B) (Buiting et al., 1999; Ohta et al., 1999a; Saitoh et al., 1996). These patients display hypomethylation of the maternal PWS- IC along with biallelic expression of the paternal genes. Notably, within the 0.88 kb of AS-IC sequence are two of several alternative upstream exons of SNRPN, u5 and u6 (Figure 1-6A) (Farber et al., 1999; Wawrzik et al., 2009). These SNRPN alternative exons are the result of multiple duplication events of the IC region. Several alternatively spliced transcripts, termed IC/SNRPN transcripts, originate from these upstream exons and are postulated to have a role in silencing the PWS-IC on the maternal allele (Dittrich et al., 1996). The PWS/AS locus is highly conserved in both gene order and expression patterns at the orthologous region on central mouse chromosome 7, thereby providing an excellent model for studying imprinting mechanisms within this domain (Figure 1-3B). The PWS-IC location and function are also conserved, allowing for in-depth investigation into the role of this element in imprinting regulation. Hindering these studies is the absence of an identifiable murine AS-IC 21

22 element. No conserved sequence from the human AS-IC exists in the mouse however, the murine Snprn does possess a number of alternative upstream exons similar to the human locus. Nine have been identified to date, termed U1-U9, spanning over 450 kb upstream of Snrpn exon one (Figure 1-6B) (Bressler et al., 2001; Landers et al., 2004). These exons generate multiple transcripts, most frequently splicing into exon two of Snrpn but also splicing downstream of the Snrpn gene as well. Notably, while Snrpn is widely expressed in adult tissues, published reports have shown that the upstream exons are transcribed specifically from the paternal allele in the brain as well as in the oocyte and granulosa cells (Bressler et al., 2001; Le Meur et al., 2005; Mapendano et al., 2006). Significantly, transcription in the oocyte corresponds with the establishment of the maternal imprint at the PWS-IC. The focus of my research is to identify and characterize the murine AS-IC. I have investigated the role of the Snrpn alternative upstream exons in establishing the maternal imprint in the oocyte. Mainly, I explored the function of transcription from the upstream exons in the epigenetic modification of the PWS-IC on the maternal allele. A recent study from Chotalia et al. demonstrated that transcription through the Gnas DMR in growing oocytes is required to establish maternal germline methylation imprints at this domain (Chotalia et al., 2009). This study supports a role for transcription in establishing imprints in the female germline. My experimental approach utilizes bacterial artificial chromosome (BAC) transgenes containing Snrpn along with a significant amount of upstream sequence to study a potential regulatory role of the upstream exons in genomic imprinting mechanisms. 22

23 Figure 1-1. The imprint lifecycle. Genomic imprints go through three stages during mammalian development: establishment, maintenance and erasure. Imprints are first established in a sex-specific manner in the germline during gametogenesis. Upon fertilization, imprints must be maintained in the soma of the developing embryo. Contrastingly, in the primordial germ cells imprints are erased between dpc during a germline-specific epigenetic reprogramming event. After erasure, female embryos re-establish maternal imprinting marks in the oocytes and male embryos re-establish paternal imprinting marks in the sperm. 23

24 Figure 1-2. Molecular classes of PWS and AS. PWS and AS result from improper gene expression from chromosome 15q11-q13, the PWS/AS locus (grey bar). PWS is caused by a loss of paternal gene products while AS is caused by a lack of UBE3A (green bar) expression from the maternal chromosome. A large interstitial deletion including the PWS/AS locus on the paternal chromosome (blue bar) or a maternal uniparental disomy of chromsome 15 cause PWS. Additionally, a microdeletion or mutation of the PWS-IC on the paternal chromosome so that it behaves as if inherited maternally also leads to PWS. Large deletions on maternal chromsome 15 (red bar) including the q11-q13 region, a paternal uniparental disomy, or maternal UBE3A defects result in AS. Also, a microdeletion or mutation of the maternal AS-IC leads to paternal imprints on the maternal chromosome, causing AS. 24

25 Figure 1-3. The PWS/AS domain. This locus is highly conserved in gene order and expression patterns from human chromosome 15q11-13 to central mouse chromosome 7. A) The human PWS/AS locus. B) The murine PWS/AS locus. This domain consists of two gene clusters, an upstream and a downstream cluster (in relation to the IC). The downstream cluster of paternally expressed genes is thought to be transcribed as a single long transcript from which multiple gene products are processed. Arrows represent the direction of transcription. 25

26 Figure 1-4. Model for IC function at the PWS/AS locus. The bipartite IC consists of the PWS-IC and the AS-IC. The PWS-IC functions as a positive element to activate expression of both the upstream cluster genes and the paternally expressed downstream cluster genes. On the maternal allele, the AS-IC acts as a negative element to inhibit the PWS-IC and thereby silence the paternally expressed genes. 26

27 Figure 1-5. Identifying the boundaries of the PWS-IC and AS-IC. A) Microdeletions were mapped from PWS patients displaying imprinting defects. The boundaries of the PWS-IC were defined by the shortest region of overlap (SRO) of these deletions. The PWS-IC is located within 4.3 kb of sequence just 5 to and including SNRPN exon one. B) Microdeletions from AS patients exhibiting imprinting defects were mapped to identify the AS-IC. The SRO was mapped to a region of 0.88 kb approximately 35 kb upstream of the PWS-IC. 27

28 Figure 1-6. Organization of the SNPRN upstream exons. Schematic diagrams of the A) human SNRPN locus and the B) murine Snrpn locus. Verified transcription start sites are indicated by arrows. 28

29 CHAPTER 2 MATERIALS AND METHODS Mouse Husbandry Transgenic Lines The 425D18 BAC was obtained from the Roswell Park Cancer Institute RPCI-23 murine BAC library, which was derived from the C57BL/6J strain. BAC recombineering as well as preliminary BAC injections were performed by Chris Futtner at the University of Florida (Futtner, 2007). Additional injections were performed by the UF Mouse Models Core Facility. All BACs were injected as supercoils into the male pronucleus of fertilized oocytes, which were obtained from superovulated FVB/N female mice. The resulting pups were screened for the transgene at three weeks of age by PCR. At eight weeks of age, transgenic founders were mated with wild-type partners to establish the lines and ensure germline transmission of the transgene. Lines were initially maintained on the FVB background however, for bisulfite sequence analysis, the lines were crossed for two generations to the B6.cast.c7 strain. This is a congenic strain that has a Mus musculus domesticus C57BL/6J background but contains a region of chromosome 7 from the species Mus musculus castaneous (cast) (Wakeland et al., 1997). By performing this cross, we were able to obtain transgenic mice on a homozygous cast c7 background, allowing us to distinguish the endogenous alleles from the transgene in our bisulfite sequence analysis. Genotyping At three weeks of age, pups were weaned. During this process they were sex separated, ear punched for identification and tail clipped for genotyping. Genomic DNA was isolated from tail biopsies by overnight incubation at 55 C in 0.4 ml tail lysis buffer (100 mm Tris ph 8.5, 5 mm EDTA, 0.2% SDS, 200 mm NaCl) supplemented with 100 ug/ml proteinase K. Phenol:chloroform:isoamyl alcohol (25:24:1) DNA extraction was performed followed by 29

30 ethanol precipitation. Purified genomic DNA was subject to PCR with the appropriate primer set for the desired genotyping. For the 425 BAC transgene, the x5-7del-f1/r2 primer set (Appendix A) was developed. These primers were designed to allow for differentiation between the modified BAC Snrpn, which possesses a deletion between exons five to seven, and the endogenous locus. The primer set flanks the deletion so that the transgene product is approximately 0.8 kb while the endogenous product is about 2.3 kb. For detection of the cast allele, a PCR amplification of the Ndn gene was performed followed by a restriction endonuclease digest. The Ndnpoly-F/R primer set was used (Appendix A) to amplify a region of the Ndn gene containing a polymorphic AvaII site between the cast allele and the domesticus allele. The AvaII site exists on the domesticus allele, at position 117 of the Ndn transcript, but not on the cast allele. Thus, after AvaII digestion of the PCR reactions at 37 C for three hours, the digests were electrophoresed on an agarose gel and the two alleles differentiated based on band size. RNA Isolation RNA was extracted from murine neonatal whole brains as well as various adult tissues using RNA-Bee reagent (Tel-Test, Inc.) per manufacturer s instructions. For each tissue sample, approximately 4 ml of RNA-Bee was added followed by homogenization with a Polytron homogenizer. Next 0.4 ml chloroform was added, samples were shaken vigorously, placed on ice for five minutes and centrifuged at 12,000g for 15 minutes. After centrifugation, the RNA was contained in the upper aqueous phase, separated from the DNA and proteins in the inter and organic phases. The aqueous phase was thus transferred to a new tube, combined with an equal volume of isopropanol, placed on ice for 15 minutes, and centrifuged at 12,000g for 15 minutes 30

31 to pellet the RNA precipitate. Each sample was then washed in 75% ethanol, dried briefly under vacuum, and resuspended in 100 ul diethylpyrocarbonate (DEPC)-treated water. Northern Blot Purified RNA from whole neonatal mouse brains was used for Northern blot analyses to investigate gene expression patterns. Four volumes of formaldehyde sample buffer were added to 10 ug of total RNA. Samples were heat denatured for five minutes at 65 C, placed on ice, and run on 1% agarose formaldehyde gels for several hours at 70 volts. The gels were soaked in 20X SSC for 30 minutes and RNA transferred onto Hybond N+ nylon membranes in 10X SSC overnight. The following day, the membranes were washed in 2X SSC and baked at 80 C for two hours in a vacuum oven. Prehybridization was carried out in 15 ml Church and Gilbert hybridization buffer (1% BSA, 1 mm EDTA ph 8.0, 0.25 M sodium phosphate buffer ph 7.2, 7% SDS) at 65 C for two hours (Church and Gilbert, 1984). DNA probes were prepared with the Prime-It II random primer labeling kit (Stratagene) and α 32 P-dCTP (Perkin-Elmer), purified with the QIAquick nucleotide removal kit (Qiagen), and subsequently denatured by boiling for five minutes. After prehybridization, the membranes were hybridized overnight at 65 C in 10 ml Church and Gilbert hybridization buffer combined with denatured probe. The following day, membranes were washed two times for 20 minutes each at 65 C in 0.2X SSCP/0.1% SDS and exposed to film. SnoRNA Northern Blot Northern blot analyses were also performed to investigate snorna expression. For detection of these small RNA molecules, total RNA was separated on 8% denaturing polyacrylamide gels (7M urea, 1X TBE buffer). Gels were pre-run in 1X TBE buffer for 30 minutes at 250 volts. For each sample, 10 ug of RNA was combined with formaldehyde sample buffer, heated to 95 C for five minutes, placed on ice and then run on the gel at 250 volts for 31

32 approximately two hours. The gels were equilibrated on ice in 0.5X TBE for 20 minutes and subsequently transferred to Hybond N+ nylon membranes using a semi-dry blotting apparatus (Trans-blot SD, BioRad). Electroblotting was carried out at 20 volts for one hour and membranes were then baked at 80 C overnight in a vacuum oven. Membranes were prehybridized in Church and Gilbert hybridization buffer for two hours at 58 C. Oligonucleotide probes were made by end-labeling reactions using T4 polynucleotide kinase (Invitrogen) and γ 32 P-ATP (Perkin-Elmer). Oligonucleotides, complementary to snorna sequences, used in probe synthesis were as follows: MBII TTCCGATGAGAGTGGCGGTACAGA-3, MBII CCTCAGCGTAATCCTATTGAGCATGAA-3, and 5.8S rrna 5 - TCCTGCAATTCACATTAATTCTCGCAGCTAGC-3. Probes were purified using the QIAquick nucleotide removal kit (Qiagen). Membranes were hybridized with purified probe overnight at 58 C in 10 ml Church and Gilbert hybridization buffer. The following day, membranes were washed three times for 15 minutes each at room temperature in 0.2X SSCP/0.1%SDS and exposed to film. RT-PCR RNA isolated from several tissue types was subject to gene expression analysis by RT- PCR. RNA samples were DNase treated prior to reverse transcription to eliminate genomic DNA contamination. For each sample, 10 ug of RNA was treated with DNase I (Invitrogen) for 15 minutes at room temperature. The reaction was stopped by the addition of EDTA and ten minutes incubation at 65 C. Next, the samples were divided into two aliquots of 5 ug each. One aliquot was subject to reverse transcription while the other, generated for use as a control, was treated in parallel but in the absence of reverse transcriptase to ensure any PCR product observed was amplified from the cdna. First strand cdna was synthesized by adding 1 ul of 500 ug/ml random primers (Invitrogen) to 5 ug RNA and bringing up to 26.4 ul total volume with sterile 32

33 ddh 2 O. Samples were heated to 68 C for three minutes and placed on ice. Then, 1.6 ul of a mixture of 2.5mM dntps, 8 ul reverse transcriptase buffer, 2 ul of 100 mm DTT, 1ul RNase OUT, and 1 ul Superscript II reverse transcriptase (Invitrogen) were added to each sample. Reactions were carried out at 37 C for 60 minutes. One microliter of cdna was used as template in PCR reactions under the following conditions: 10 mm Tris-HCl, 50 mm KCl, 1.5 mm MgCl 2, four dntps at mm each, 10% betaine, 1.5 units Taq DNA polymerase (NEB), and the appropriate primers (Appendix A) at 10 um each. Reactions were subject to an initial denaturation at 95 C for five minutes followed by 34 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 60 s. An extension step of five minutes at 72 C completed the reaction. Southern Blot RT-PCR products were subject to Southern blotting procedures as described (Sambrook et al., 1989). Samples were run on 1.2% agarose/0.5x TBE gels at 95 volts for approximately one hour. The DNA was denatured by placing the gel in alkali solution (1.5 M NaCl and 0.5 N NaOH) for 45 minutes and then in neutralizing solution (1.5 M NaCl and 1 M Tris ph 7.4) for 90 minutes. The DNA was subsequently transferred onto Hybond N+ nylon membranes in 10X SSC overnight. The following day, membranes were washed in 2X SSC and baked at 80 C for two hours in a vacuum oven. Prehybridization was carried out in 15 ml Church and Gilbert hybridization buffer (1% BSA, 1 mm EDTA ph 8.0, 0.25 M sodium phosphate buffer ph 7.2, 7% SDS) at 65 C for two hours (Church and Gilbert, 1984). DNA probes were prepared with the Prime-It II random primer labeling kit (Stratagene) and α 32 P-dCTP (Perkin-Elmer), purified with the QIAquick nucleotide removal kit (Qiagen), and subsequently denatured by boiling for five minutes. After prehybridization, the membranes were hybridized overnight at 65 C in 10 ml Church and Gilbert hybridization buffer combined with denatured probe. The following day, 33

34 membranes were washed two times for 20 minutes each at 65 C in 0.2X SSCP/0.1% SDS and exposed to film. Primordial Germ Cell Purification PGCs were immunomagnetically purified from embryos at 13.5 dpc for gene expression analysis. Initially, timed matings were set up and females examined for copulatory plugs. Noon on the day a plug was first visible was reasoned to be 0.5 dpc in embryonic development. At 13.5 dpc embryos were removed from the mothers uteri and placed into 1X PBS. The fetal gonads were then dissected out, sex segregated, and each sex pooled together for purification. Around eight sets of gonads were desired per sex per purification. Once collected, 500 ul of trypsin-edta was added to each sample and the tissue incubated for five minutes at 37 C. Following incubation, the samples were triturated to break up the tissue and then centrifuged at 2,000 rpm for two minutes. After centrifugation the trypisn-edta was removed and 1 ml of PBS-DNase (1X PBS, 5 mm EDTA, 0.5% BSA, and 20 ug/ml DNase) was added. Samples were mixed, centrifuged, and then the PBS-DNase solution was removed. Subsequently, 160 ul of fresh PBS-DNase was added and the tissue was again triturated in order to obtain a single cell suspension. Samples were placed on ice and 40 ul of the TG-1 antibody, a primary mouse IgM antibody that binds to the cell surface of PGCs, was added followed by a 30 minute incubation. After this incubation the cells were washed three times with PBS-DNase and incubated for 30 minutes on ice in a rat anti-mouse IgM secondary antibody (Miltenyi Biotech # ). This secondary antibody has an iron bead conjugated to it for purification of PGCs through MACs columns on the magnetic MACs Separator (Miltenyi Biotech). For PGC purification, the MACs columns were placed on a Miltenyi Mini-MACs magnet and prewashed with 1X PBS/3% DNase equilibration buffer. Each cell suspension was passed 34

35 through the column three times to ensure that the majority of the PGCs had adhered to the column. The immunodepleted fraction, which contained the somatic cells of the fetal gonad, was collected for use as a control. Columns were then washed four times with PBS-DNase. The PGCs were subsequently eluted from the columns in PBS-DNase after removal from the magnet. PGC purity was assayed by alkaline phosphatase staining. High Molecular Weight Genomic DNA Preparation Genomic DNA was extracted from mouse neonatal whole brains for Southern blot analysis and bisulfite sequence analysis. Frozen brains were homogenized with a dounce homogenizer in 2.5 ml of 1X SSC, 1% SDS, 0.25 mg/ml Pronase E (Sigma). The homogenates were poured into 15 ml round bottom Falcon tubes and the homogenizer washed with an additional 2.5 ml homogenizing solution, which was then combined with the homogenate. Samples were vortexed and placed at 37 C for one hour. After incubation, 5 ml of phenol:chloroform:isoamyl alcohol (25:24:1) was added and samples were vortexed and spun for five minutes at 2500 rpm. The DNA-containing aqueous layer was collected and the extraction repeated two more times, once as above and once with chloroform alone. After the chloroform extraction, the DNA was ethanol precipitated and resuspended in 400 ul ddh 2 O. Next, 25 ul of 2 mg/ml RNase A was added and the samples were incubated for 30 minutes at 37 C. Then 25 ul of 5 mg/ml Pronase E was added and samples again incubated at 37 C for 30 minutes. The phenol:chloroform:isoamyl alcohol extractions and ethanol precipitation was repeated as described above. The purified genomic DNA was subsequently resuspended in 200 ul ddh 2 O, incubated at 55 C for an hour and then allowed to sit at room temperature overnight to ensure resuspension. 35

36 Bisulfite Sequence Analysis Bisulfite Conversion Murine genomic DNA was subject to bisulfite sequence analysis in order to determine the methylation status of a region within the Snrpn DMR (Clark et al., 1994). For each sample, 5 ng of genomic DNA was denatured in a solution of 0.3 M NaOH for 30 minutes at 37 C. The denatured DNA was then combined with 2.0 M sodium metabisulfite/10 mm hydroquinone solution, for a final concentration of 1.5 M bisulfite/0.5 mm hydroquinone, and incubated at 55 C for hours. Bisulfite-Treated DNA Purification After bisulfite conversion, free bisulfite was removed with the Promega Wizard DNA clean-up system per manufacturer s instructions. DNA was eluted in 45 ul ddh 2 O and 5 ul 3 M NaOH was added for a final concentration of 0.3 M. After a 15 minute incubation at 37 C, the DNA was ethanol precipitated overnight at -80 C. The next day, the DNA was pelleted by centrifugation, washed in 70% ethanol and resuspended in 100 ul ddh 2 O. Bisulfite PCR Following purification, the bisulfite-converted genomic DNA was subject to PCR amplification. Primers were designed to anneal to the bisulfite-converted DNA sequence. A 364 bp region within the Snrpn DMR, which spans 14 CpG dinucleotides, was amplified with primers W18 and W19 (Appendix A). PCR conditions were as follows: 10 mm Tris-HCl, 50 mm KCl, 1.5 mm MgCl 2, four dntps at mm each, 1.5 units Jumpstart Taq DNA polymerase (Sigma), and the appropriate primers at 10 um each. To each 25 ul reaction, 1ul of template DNA was added. The PCR reactions were subject to an initial denaturation step at 95 C for 15 minutes followed by 38 cycles of: 94 C for 45 s, 54 C for 60 s and 72 C for 90 s. A 36

37 10 minute final extension at 72 C completed the reactions. PCR products were run on 1.2% low melt agarose gels containing ethidium bromide. The appropriate sized bands were excised and purified with the Wizard DNA clean-up system. Three microliters of the 40 ul purifications were run on 1.2% gels to verify DNA quality before cloning. Cloning PCR Products The purified bisulfite PCR products were ligated into the pgem-t Easy Vector System (Promega) per manufacturer s protocol. Briefly, 5 ul of 2X rapid ligation buffer, 1 ul pgem-t Easy vector, 1 ul purified DNA, 2 ul ddh 2 O, and 1 ul of T4 ligase were combined and incubated at 4 C overnight. The following day, the ligations were transformed into XL1-Blue subcloning-grade competent cells (Stratagene). For each sample, 6 ul of the ligation reaction was added to a 50 ul aliquot of bacteria and placed on ice for 20 minutes. The bacteria were then heat shocked at 42 C for 45 seconds, placed on ice for two minutes and subsequently shaken for 30 minutes at 37 C in 0.9 ml of SOC medium. The transfomations were plated onto Luria-Bertani (LB) plates (1% tryptone, 0.5% yeast extract, 1% sodium chloride and 1.5% agar) containing 50 ug/ml ampicillin. In addition, the plates were supplemented with 100 ul of 100 mm IPTG and 25 ul of 40 mg/ml X-Gal for blue-white colony selection (Sambrook et al., 1989). Plates were incubated overnight at 37 C. The next day, white colonies were picked and cultured overnight at 37 C in 3 ml LB broth, consisting of 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 50 ug/ml ampicillin. Plasmid DNA was purified from the cultures using the QIAprep Spin Miniprep Kit (Qiagen) as directed. 37

38 Plasmid Sequencing Purified plasmids were sequenced using ABI Prism BigDye Terminator reagent (Applied Biosystems) and run on an ABI Prism 377XL Automated DNA Sequencer at the Center for Epigenetics DNA Sequence Core (University of Florida). Sequencing reactions were set up as follows: 1 ul of 3.2 pmol/ul SP6 primer, 2 ul of 5X sequencing buffer, 2 ul DNA, 2 ul BigDye Terminator, and 3 ul ddh 2 O. Reactions were subject to PCR for 24 cycles of: 96 C for 30 s, 50 C for 15 s and 60 C for 4 minutes. After PCR, the reactions were purified by passage through Performa DTR Gel Filtration Columns (Edge Biosystems) and taken to the Center for Epigenetics for sequencing. Sequence files were analyzed using Sequencher 4.2 (Gene Codes Corporation). 38

39 Attempts to Identify the Murine AS-IC CHAPTER 3 THE IMPRINTED BAC TRANSGENE Introduction Imprinted genes characteristically reside in clusters that are coordinately regulated in cis by IC elements. These imprinted domains typically span over one megabase of DNA and thus the ICs must be capable of exerting their control across extensive amounts of sequence. A bipartite IC consisting of the PWS-IC and the AS- IC exerts control over the entire PWS/AS locus, a domain encompassing approximately two megabases of sequence. Although the human AS-IC has been localized to a region about 35 kb upstream of the PWS-IC, the location of the murine AS-IC remains unknown. Several targeted deletion approaches to knock out AS-IC function have proven unsuccessful, including two deletions made by our lab that removed sequences at the same location as the human AS-IC (relative to the PWS-IC). These knockouts consisted of an 8.2 kb deletion, from -29 to -37 relative to Snrpn exon one, and a 12.8 kb deletion, from -37 to -24 relative to Snrpn exon one (Peery et al., 2007). Both knockout models were viable and fertile with no discernable phenotype. Notably, these deletions left all of the currently identified Snrpn alternative upstream exons intact. The Beaudet lab also made mutations in the region upstream of Snrpn in an attempt to knockout AS-IC function (Wu et al., 2006). One of these mutations consisted of a large deletion encompassing 80 kb of sequence from -13 to -93 relative to Snrpn exon one, a region containing two of the most proximal Snrpn upstream exons but not U2 or U4-U9. This deletion generated mice displaying an imprinting defect with incomplete penetrance. Upon maternal inheritance of this deletion, the DNA methylation imprint at the maternal PWS-IC was lost or partially lost in over half of the pups analyzed. There was no detectable imprinting defect when the paternal 39

40 chromosome carried this deletion. The other mutation generated in this study was described as an insertion/duplication mutation 13 kb upstream of Snrpn exon one, designated the IC an allele. This insertion included Hprt exons three to nine, a loxp site, a puromycin resistance (Pur R ) cassette transcribed in the opposite orientation of Snrpn, and a six kb duplication of target site sequence. Paternal transmission of the IC an allele had no consequence. In contrast, maternal inheritance of the IC an allele resulted in biallelic expression of Snrpn, a loss of DNA methylation at the maternal PWS-IC, and a dramatic reduction in Ube3a expression in the brain. Thus, the maternally inherited IC an mutant displays several phenotypes associated with loss of AS-IC function. The mechanism of interference with AS-IC activity created by the IC an insertion/duplication remains unclear. One possibility is that transcription from the Pur R cassette interferes with production of the Snrpn upstream exon transcripts. In additional efforts to identify the AS-IC and characterize the mechanism utilized in imprinting regulation at the PWS/AS locus, several transgenic mouse models have been generated. In these models, an imprinted transgene is defined as displaying 1) Snrpn expression upon paternal but not maternal transmission and 2) DNA methylation at the transgene PWS-IC exclusively after maternal transmission. Previously, our lab produced transgenic lines from a P1 phage clone containing Snrpn as well as 33 kb of 5 flanking sequence, sequence lacking any of the identified alternative upstream exons. Imprinting of this transgene varied based on copy number with a single copy line expressing Snrpn upon both maternal and paternal inheritance but with a two-copy line displaying maternally imprinted Snrpn expression (Blaydes et al., 1999). This model indicated that all the elements required for AS-IC function are not present on the transgene but that in multi-copy this deficiency is overcome and imprinting is observed. One 40

41 explanation for this result is that the extra copy of the transgene adds back genomic sequence necessary for imprinting to occur. Another transgenic study, published by Shemer et al., also utilized a Snrpn P1 phage clone. In contrast to our transgene, this transgene contained approximately 50 kb of sequence 5 to Snrpn, including the most proximal upstream exon, U1. Two high copy number lines were examined: one line containing ten copies and the other containing 20 copies of the transgene. DNA methylation analysis using methylation sensitive restriction enzyme digests showed that this transgene was methylated at two sites within the PWS-IC upon maternal but not paternal inheritance (Shemer et al., 2000). Analysis of transgene expression in this study was complicated by the presence of endogenous Snrpn background. The transgene was reported as being imprinted, however, close inspection of figure 2C in this paper suggests biparental Snrpn expression albeit at a much higher level after paternal transmission relative to the expression level after maternal transmission. Overall, these mouse models for identifying the AS-IC and investigating mechanisms of regulation have proven unsuccessful and have yielded perplexing results. Several deletion models have failed to completely knockout IC function while traditional transgenic models have provided inconclusive results. Therefore, we have taken a different transgenic approach, one utilizing BAC transgenes, to identify the murine AS-IC. Founding the BAC Transgenic Model System BAC transgenes have many advantages over conventional plasmid transgenes when studying imprinted loci, mainly due to their large size. Up to 300 kb of genomic sequence can be contained in one BAC thus providing a great vector for examining interactions between ICs and the genes they regulate, which can be located hundreds of kilobases away. The large amount of genomic sequence that can be incorporated into one BAC increases the probability that any other 41

42 existing elements necessary for proper imprinting will be included in the transgene. Another advantage of BAC transgenes is that their large size typically results in integration at low copy number, which is important as previous studies have shown that transgene copy number affects imprinting at the PWS/AS locus. Furthermore, smaller plasmid based transgenes are frequently influenced by the chromatin state at the integration site as well as by the flanking endogenous sequence. These position effects cause differences in expression patterns among multiple transgenic lines, producing conflicting results and making for cumbersome analyses. The substantial amount of sequence contained in the BAC vectors can act as a buffer against these effects, allowing for accurate and consistent expression patterns. One additional advantage of BAC constructs is that they are easily modified via BAC recombination techniques. This simple procedure allows for manipulation of the transgene, which can be useful for distinguishing it from the endogenous locus or for examining the effects of modifications to the transgene such as a deletion of regulatory sequences. To study imprinting mechanisms at the PWS/AS locus and identify the murine AS-IC, we created a BAC transgenic model system. Our system is based on the discovery of an imprinted BAC transgene, a discovery indicating that all the genetic information necessary for imprinting at this locus is contained within the transgene. Once an imprinted transgene was discovered, implying that a functional AS-IC is located within this transgene sequence, modifications could be made to knockout IC function. Loss of imprinting on a modified transgene would thereby identify the AS-IC and provide a model to investigate IC mechanisms. To create our model system, we screened the Roswell Park Cancer Institute (RPCI) RPCI- 23 murine BAC library, which was derived from the C57BL/6J mouse strain, for clones containing the entire Snrpn gene (Chamberlain, 2003). In addition to Snrpn, we looked for 42

43 clones with varying amounts of upstream sequence as we hypothesized that the AS-IC is contained within the Snrpn upstream exons. Twenty BACs were identified and of those, three chosen for further study: 380J10, 425D18, and 215A9. The 380J10 BAC contains Snrpn as well as 16 kb of sequence upstream and 140 kb of sequence downstream. The 425D18 BAC spans from 120 kb 5 to 65 kb 3 relative to Snrpn while the 215A9 BAC extends 150 kb 5 and 20 kb 3 to Snrpn. Importantly, the 380J10 BAC possesses no upstream exons whereas the 425D18 BAC includes U1, U2, and U3 and the 215A9 BAC includes U1, U2, U3 and U4 (Figure 3-1). Multiple lines of transgenics were generated from these three BACs and each line was examined for Snrpn expression patterns. In order to analyze imprinting in these transgenics, we had to be able to distinguish expression of the transgene from endogenous Snrpn expression. Therefore, we made use of a Snrpn mutant previously constructed by our lab, the ΔSmN mouse line. This mutant carries a deletion between exons five to seven of the Snrpn gene, resulting in the absence of standard Snrpn transcript and instead producing low levels of a larger fusion transcript that includes Snrpn exons one to four and the neomycin resistance gene, which was inserted into the deletion during targeting (Yang et al., 1998). Paternal transmission of the ΔSmN allele abolishes endogenous Snrpn expression allowing for analysis of transgene expression without endogenous background. Results from these experiments showed that the 380J10 BAC, the BAC lacking Snrpn upstream exons, expresses Snrpn upon both maternal and paternal transmission, demonstrating that the transgene does not contain sufficient sequence to confer imprinting. Conversely, both the 215A9 BAC and the 425D18 BAC displayed imprinted expression patterns, indicating that there is a functional AS-IC located within these transgenes (Futtner, 2007). 43

44 As these transgenic studies showed that the 120 kb of sequence upstream of Snrpn on the 425D18 BAC is sufficient to establish imprinting, we chose to make this BAC the basis of our model system to identify the AS-IC. To simplify expression analysis of the transgene and eliminate the need for complex breeding schemes with lines bearing deletions in Snrpn or the PWS-IC, we used BAC recombineering techniques to modify the transgene. We made a deletion of approximately 242 bps of coding sequence between Snrpn exons five to seven. We chose this deletion because it is the same deletion that was made previously in the lab at the endogenous locus (the ΔSmN mutation) and it proved to have no effect on imprinting at the locus. We termed this modified transgene 425Δ5-7. After thorough analysis of this transgene, further modifications were made to identify the AS-IC as will be discussed in the upcoming chapters. Results The 425Δ5-7 BAC Possesses a Functional AS-IC Imprinted expression of the 425Δ5-7 transgene To begin our study, it was imperative to first examine the 425Δ5-7 BAC to ensure that it was properly imprinted, displaying appropriate epigenetic imprinting marks as well as imprinted expression patterns. The BAC was injected the into fertilized FVB/N oocytes and the oocytes subsequently implanted into pseudopregnant females in an attempt to obtain multiple founders (Futtner, 2007). Injections were performed by Chris Futtner, a previous graduate student in the lab. From these injections, we were able to establish three transgenic lines suitable for analysis: line A, H, and I. Because copy number influenced the imprinted status of previously analyzed transgenic lines, we deemed it necessary to obtain at least one single copy line for our studies. Copy number was analyzed via Southern blot on high molecular weight genomic DNA isolated from postnatal day 1 (P1) whole brains. Line A was determined to be a single copy line while lines H and I were found to bear multiple copies of the transgene (data not shown). 44

45 After determining the copy number of each line, we investigated expression patterns to verify the imprinted status of the 425Δ5-7 BAC. We expected to find imprinted expression such that offspring inheriting the transgene maternally would silence Snrpn but offspring inheriting the transgene paternally would express Snrpn. As this transgene bears a deletion in the body of Snrpn, a simple RT-PCR was performed to determine expression of the transgene while at the same time distinguishing it from the background endogenous expression. To distinguish between the two transcripts, we designed primers in regions flanking the deletion (N2.1-F/N6.2- R) (Appendix A). Our forward primer sequence is located within Snrpn exon four and our reverse primer sequence is located within exon eight, generating amplicons of 513 bps from the endogenous transcript and 350 bps from the transgenic transcript (Figure 3-2A). We set up matings to obtain offspring with either a maternally or paternally inherited transgene. On the day of birth, litters were collected and brains harvested from the pups. Total RNA from P1 whole brains was extracted and subject to DNase treatment followed by reverse transcription to produce cdna. The cdnas were then used as templates in RT-PCR reactions to analyze Snprn expression. Our results showed that the transgene was expressed upon paternal transmission but silenced upon maternal transmission in each of the three lines (Figure 3-2B-D). Given that multiple lines demonstrated the same expression patterns, we concluded that the 425Δ5-7 BAC was imprinted, as was the unmodified 425D18 BAC transgene. For the majority of our further studies with this transgene, we used the 425Δ5-7A line since a single copy line was desirable for analysis. Epigenetic imprinting marks on the 425Δ5-7 transgene A common and functionally important feature of imprinted loci is the presence of differential epigenetic marks at imprinting control regions. These epigenetic marks are essential for the establishment and maintenance of imprinting. Notably, within the PWS-IC resides the 45

46 Snrpn DMR, which is hypermethylated on the maternal allele and unmethylated on the paternal allele in somatic cells (Gabriel et al., 1998; Shemer et al., 1997). This is a germline DMR, meaning the methylation imprint is erased in fetal germ cells and reapplied in a sex-specific manner during gametogenesis. The Snrpn DMR obtains its maternal-specific methylation imprint postnatally during the oocyte growth phase (Lucifero, Mann et al. 2004). To investigate whether the 425Δ5-7 BAC undergoes appropriate epigenetic reprogramming in the germline, we examined methylation at the Snrpn DMR in newborn brain DNA using genomic bisulfite sequence analysis (Clark et al., 1994). Bisulfite treatment of DNA results in the deamination of unmethylated cytosine residues, converting them to uracil nucleotides while methylated cytosines remain unaffected by the treatment. After bisulfite treatment, DNA is amplified by PCR and the unmethylated cytosines are represented as thymines in the amplicon. By cloning the PCR product and sequencing individual clones, the methylation status of a region of interest can be determined. For our analysis, we amplified a fragment of 364 bps that included the 5 flanking region of Snrpn as well as exon one (Figure 3-3). This sequence is located within the Snrpn DMR and spans 14 CpG dinucleotides. For this experiment to be informative, we had to have the ability to distinguish the transgene sequences from the endogenous sequences; therefore, we transferred the 425Δ5-7 transgene onto a C57BL/6J line congenic for the PWS/AS domain of Mus musculus castaneus chromosome 7 (B6.cast.c7) (Wakeland et al., 1997). We then utilized sequence polymorphisms between the C57BL/6J derived transgene and the endogenous cast alleles, one of which was located within our 364 bp amplicon. Our initial analysis was performed on the 425Δ5-7H line, which we crossed to the B6.cast.c7 line. Subsequently, we mated the F1 offspring to generate pups for collection of newborn brains. Whole brain genomic DNA was isolated and bisulfite sequence analysis 46

47 performed on samples that were positive for the transgene and that also exhibited cast c7 homozygosity as indicated by PCR genotyping experiments. Sequences were examined for the presence of a single nucleotide polymorphism (SNP) at base number 69 of the Snrpn DMR amplicon. The C57BL/6J transgene was identified by a guanine nucleotide while the endogenous cast alleles were identified by a thymine at this position. We compared data between paternally and maternally inherited transgenic brains after performing experiments on at least two brains per mode of inheritance. Our results from the sequence analysis were as expected, the paternally transmitted 425Δ5-7H transgene displayed hypomethylation of the Snrpn DMR while the maternally transmitted transgene possessed a hypermethylated DMR (Figure 3-4A). The endogenous alleles displayed a mixture of methylated and unmethylated clones as both the maternal and paternal alleles are represented in these sequences (Figure 3-4B). Taken together, this methylation data along with the expression analysis confirm that BAC 425Δ5-7 harbors AS-IC activity. We are in the process of repeating the genomic bisulfite sequencing experiment with the 425Δ5-7A line as we want to verify our methylation data with this single copy transgenic line. We expect the results to be the same as those obtained with the 425Δ5-7H line; the transgene will exhibit hypomethylation of the Snrpn DMR upon paternal transmission but display hypermethylation of the DMR upon maternal transmission. These results, combined with the 425Δ5-7H methylation data, will provide compelling evidence indicating that the appropriate epigenetic imprinting marks are established on the 425Δ5-7 transgene at the Snrpn DMR, a DMR that is located within the PWS-IC. Snrpn Upstream Exon Expression Patterns are Conserved on the 425Δ5-7A Transgene In our proposed model for AS-IC function, transcription of the Snrpn alternative upstream exons is vital for epigenetically modifying the PWS-IC on the maternal allele and thereby 47

48 silencing the paternally expressed genes. As the 425Δ5-7 transgene displays appropriate imprinted expression and epigenetic imprinting marks, we hypothesized that upstream exon expression from this transgene would mimic the endogenous upstream exon expression patterns. Specifically, we expected to find upstream exon containing transcripts from the transgene exclusively in oocytes and in postnatal brain upon paternal transmission as published reports have shown (Bressler et al., 2001; Le Meur et al., 2005; Mapendano et al., 2006). As stated earlier, we chose the 425Δ5-7A line for our analysis as this was a single copy line. To examine upstream exon expression patterns, we needed a way to distinguish the transgene-generated transcript from the endogenous alleles. This was made possible by a LoxP site that remained in the coding region of Snrpn after recombineering the deletion in exons five through seven. We designed an RT-PCR experiment with a primer set (SnrpnU1-F2/Lox Tg-R2) that included a forward primer in U1 and a reverse primer in the LoxP sequence (Appendix A), generating amplicons exclusively from the transgene (Figure 3-5A). RT-PCRs were Southern blotted with a probe for Snrpn U1 to exon three sequence to intensify signal strength. Upstream exon expression in the brain Initially, we performed RT-PCR on newborn whole brain cdnas from pups inheriting the transgene either maternally or paternally. As expected, transcripts derived from the transgene that included U1 were detected in brain tissue after paternal transmission. Lower levels of expression were detected after maternal transmission (Figure 3-5B). This leaky expression is expected, as imprinting is never 100% efficient. The observed expression patterns mimic upstream exon expression from the endogenous locus in the brain. Upstream exon expression in the ovary Next, we analyzed upstream exon expression from the transgene in the ovary. Detecting expression in this tissue is important as the epigenetic imprint is established at the PWS-IC in 48

49 growing oocytes. Thus, transcription of the Snrpn upstream exons, originating from the transgene, in growing oocytes would provide supporting evidence for our hypothesis on the AS- IC identity and function. In a study from the Trasler lab investigating the timing of DNA methylation imprint establishment in the maternal germline, the Snrpn DMR was found to acquire methylation in growing oocytes between 5-25 days postpartum (dpp) (Lucifero et al., 2004). Therefore, we examined ovaries from three-week-old female mice, the age at which imprints are being established in the oocytes, for upstream exon expression. We harvested ovaries from four transgenic females (maternal transmission of the transgene) and two nontransgenic littermates for negative control tissue. As all alleles in the oocytes are reset as maternal alleles, the mode of parental inheritance of the transgene was irrelevant. We performed RT-PCR using the U1-LoxP primer set on cdnas generated from ovary RNA. We then Southern blotted the gel with a probe consisting of Snrpn U1-exon three sequence to intensify the signal. Significantly, we detected upstream exon containing transcripts derived from the transgene in the ovary (Figure 3-5C). Snrpn upstream exon expression is not restricted to the brain and ovary To complete our Snprn upstream exon expression comparisons between the transgene and the endogenous locus, we performed RT-PCR on a panel of tissues from an adult 425Δ5-7A paternally transmitted transgenic male. We made cdnas from brain, testis, kidney, heart, and liver tissue and used 425Δ5-7A transgenic ovary cdna as a positive control. We first performed RT-PCR looking specifically at upstream exon expression from the transgene by using the U1-LoxP primer set and subsequently Southern blotting the gel with the Snrpn U1- exon three probe. Surprisingly, we discovered that upstream exon-containing transcripts were present in every tissue analyzed with low levels of expression in testis, kidney, and liver and high levels of expression in ovary, brain, and heart (Figure 3-6). We next analyzed upstream exon 49

50 expression from the endogenous allele by RT-PCR with the same forward U1primer (U1-F2) but a reverse primer in Snrpn exon five (5 RACE Ex5-R), which consisted of sequence lying within the exon five to seven deletion region (Appendix A). Again, we Southern blotted this gel with the Snrpn U1-exon three probe. In contrast to published reports, we found expression of the endogenous upstream exons in all tissues analyzed (Figure 3-6). These expression patterns essentially mimicked those of the transgene, with high levels of expression in the ovary, brain, and heart but lower levels in the kidney and liver. However, unlike the transgene-specific RT- PCR, the testis did not show expression of the standard sized product from the endogenous locus. A larger product was observed, most likely representing an alternatively spliced transcript. A smaller band was also seen in the kidney with the transgene-specific primer set, again most likely representing an alternatively spliced transcript. The ovary expression data together with the expression data from transgenic brain samples and the transgenic tissue panel demonstrate that upstream exon expression from the imprinted 425Δ5-7A transgene recapitulates the endogenous expression pattern. We also discovered that, contrary to previous reports, upstream exon expression is activated in many tissues, albeit at varying levels. Snrpn Upstream Exons are Silent in the Germline Before the Establishment of Imprints Further support for our theory on the role of Snrpn upstream exon transcription in imprinting the PWS/AS locus was provided upon analysis of upstream exon expression in the developing germline. Snrpn is expressed biallelically in 13.5 dpc PGCs, as imprints are erased in the germline in post-migratory germ cells between 10.5 to 12.5 dpc (Szabo et al., 2002; Szabo and Mann, 1995). Therefore, we examined 13.5 dpc PGCs for the presence of Snrpn upstream exon containing transcripts. We expected not to find these upstream exon-containing transcripts during this stage of germ cell development since imprinting is not established until after birth in 50

51 the maternal germline. We performed RT-PCR with a primer set spanning from U1 to Snrpn exon three (Appendix A) on wild type, sex-segregated, purified 13.5 dpc PGC cdnas as well as on adult ovary and testis cdnas (Figure 3-7A). The gel was Southern blotted with a probe of the same sequence to intensify the signal strength. From this experiment, we discovered that neither male nor female 13.5 dpc PGCs express Snrpn upstream exon U1 (Figure 3-7B). The ovary, used as a positive control, did show expression of U1 while the negative control testis tissue showed no U1 expression. We then performed RT-PCR on the same cdnas using the primer set spanning Snrpn exon four to eight (Appendix A). This experiment verified that Snrpn is expressed in each of these tissue types and an Hprt RT-PCR reaction was performed to control for cdna quality. These data demonstrate that upstream exon expression is not activated in the germline before imprints are established. Discussion Years of research have gone into investigating maternal imprinting mechanisms at the PWS/AS locus with little success. Attempts to identify the murine AS-IC via conventional transgene and knockout models have resulted in only small insights into imprinting mechanisms at this locus. Thus, we have taken an alternative approach to investigate these mechanisms using a BAC transgene system. Our model for maternal imprint establishment at this locus is based upon transcription from the Snrpn alternative upstream exons. Therefore, we utilized a transgene containing Snrpn and 120 kb of upstream sequence, including three of the alternative upstream exons. We showed that this transgene is properly imprinted in multiple transgenic lines, indicating that there is a functional AS-IC contained within its sequence. Proper imprinting was demonstrated by the expression of Snrpn from the transgene after paternal transmission but not after maternal transmission. Additionally, appropriate epigenetic imprinting of the transgene 51

52 was shown by the presence of DNA methylation at the Snrpn DMR exclusively after maternal transmission. Our model for the establishment of maternal imprints at the PWS-IC involves transcription from the Snrpn upstream exons in oocytes; therefore, we investigated transcription of these exons from the transgene in ovary. As maternal imprints are established at the PWS-IC between 5-25 dpp, it was vital for the imprinted transgene to display expression of the upstream exons during this time in development for our model to stand. Our discovery of transcripts derived from the transgene that included the upstream exons in the ovary at 21 dpp provided significant support for our model. Additionally, absence of U1 containing transcripts in wild type PGCs after imprints have been erased but before their reestablishment also lends support to our model. Future experiments will investigate the timing of transcriptional activation of the upstream exoncontaining transcripts in growing oocytes at the endogenous locus. We can then compare the timing of transcriptional activation with the establishment of maternal imprints at the PWS-IC. In addition, we will examine the activation of Snrpn upstream exon transcription from the 425Δ5-7 transgene in growing oocytes to determine if it mimics activation of the endogenous locus. We can then examine the methylation imprint at the transgene PWS-IC in these oocytes to see if it corresponds with the appearance of transcripts from the upstream exons, or shortly thereafter, and also if it mimics the establishment of methylation imprints at the endogenous PWS-IC. Consistent with our hypothesis for the role of transcription from the Snrpn upstream exons in maternal imprinting mechanisms are the attempted AS-IC knockout mouse models and the IC an insertion/duplication mouse (Peery et al., 2007; Wu et al., 2006). Two of the three knockout mice, the 8.2 kb and the 12.8 kb deletion mice had all Snrpn upstream exons intact and showed 52

53 no disruption of imprinting. The 80 kb deletion spanned only the proximal two upstream exons and these mice showed incomplete penetrance in eliminating AS-IC function. We hypothesize that the more distal upstream exons are less efficient in acting as an AS-IC as compared to the proximal upstream exons. Furthermore, the IC an insertion/duplication allele, when inherited maternally, bears no imprint the PWS-IC. The insertion includes a Pur R gene that is transcribed in the opposite orientation of Snrpn. Several reasons may underlie the disruption of AS-IC function on the IC an allele. Our model, which posits that transcription from the Snrpn upstream exons through the PWS-IC is required for maternal imprinting, might predict that transcription from the Pur R cassette disrupts transcription from the upstream exons through the PWS-IC. Further experiments with this model to determine if Snrpn upstream exon-containing transcripts from the IC an allele are present in growing oocytes would be very informative. The absence of these transcripts coupled with the lack of maternal imprinting at the PWS/AS locus would support our proposed model of maternal imprinting mechanisms. When comparing Snrpn upstream exon expression patterns from the transgene and the endogenous alleles, we unexpectedly discovered that upstream exon expression is not restricted to the brain and ovary as was previously reported. Varying results could be due to strain differences between our mice and mice used in the labs from which these reports came from. Additionally, the sensitivity of experimental techniques utilized could cause discrepancies. Our analysis used FVB/N mice and RT-PCR consisting of 35 cycles followed by Southern blotting. Bressler et al. reported that Snrpn upstream exon expression was repressed in kidney, heart, liver, lung, spleen and muscle but expressed from the paternal allele in the brain (Bressler et al., 2001). In contrast to our experiments, this study used 129/SvEv mice and RT-PCR without Southern blotting. Le Meur et al. also reported brain-specific upstream exon expression upon 53

54 RT-PCR analysis of liver, testis, ovary and brain tissues (Le Meur et al., 2005). However, this study used C57BL/6 mice and the authors also did not perform Southern blots of the RT-PCR products. In addition, this report included a Northern blot on various adult tissues and in situ hybridizations on testis and ovary sections; these experiments further demonstrated upstream exon expression exclusively in the brain. These conflicting results relative to our studies were not a total surprise as their assays were designed to only identify transcripts containing upstream exon two while our studies examined U1 expression. Lastly, the study by Mapendano et al. showed Snrpn upstream exon usage specifically from the paternal allele in the brain as well as in oocytes and granulosa cells (Mapendano et al., 2006). The authors of this study did not indicate the strain of mouse used in their experiments, which is very critical information when evaluating conflicting data. It would be very useful to construct tissue panels from several strains of mice and compare upstream exon expression patterns between the strains. In the upcoming chapters, we will further investigate the role of the upstream exons in the AS-IC activity displayed on the 425Δ5-7 transgene as our system provides a simple way to modify the transgene via BAC recombineering techniques. 54

55 Figure 3-1. Schematic representation of the Snrpn containing BAC transgenes. The three BACs from which transgenic lines were made, 380J10, 425D18, and 215A9, are displayed below the endogenous locus. The amount of genomic sequence upstream and downstream of the Snrpn gene on each BAC is indicated. 380J10 contains no U exons, 425D18 possesses U1, U3, and U2, while 215A9 includes U1-U4. 55

56 Figure 3-2. Snrpn RT-PCR analysis of the 425Δ5-7 transgenic lines. A) Schematic diagram of our Snrpn RT-PCR experimental design. The top bar represents the endogenous Snrpn locus (exons one to ten) and the bottom bar represents the modified BAC 425Δ5-7 transgene. Black arrows designate RT-PCR primers. RT-PCR was performed on newborn brains from three separate transgenic lines: B) 425Δ5-7A, C) 425Δ5-7H, and D) 425Δ5-7I. The endogenous Snrpn amplicon is 513 bps while the transgene transcript generates a 350 bp product. Two transgenic brains from both maternal and paternal transmission were analyzed for each line. Non-transgenic littermate brain cdnas were used as controls. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. 56

57 Figure 3-3. Schematic diagram of the Snrpn DMR. The DMR lies within the PWS-IC. It is methylated on the maternal allele and unmethylated on the paternal allele. The region examined with bisulfite sequence analysis is displayed beneath the locus. The bisulfite amplicon spans from base -175 to +189 relative to the Snrpn transcription start site. This sequence contains 14 CpG dinucleotides, which are displayed as lollipops (black lollipops represent methylated CpG dinucleotides, white lollipops represent unmethylated CpG dinucleotides). 57

58 Figure 3-4. Bisulfite sequence analysis of the 425Δ5-7H transgene. A region of the Snrpn DMR that contains 14 CpG dinucleotides was examined. Each CpG is represented by a circle. Methylated CpGs are displayed as black circles and unmethylated CpGs are shown as white circles. Each row represents an individually sequenced clone. A) Transgenic alleles: the majority of the maternally transmitted alleles are hypermethylated while the paternally transmitted alleles are mainly unmethylated. B) The endogenous alleles represent both the maternal and paternal locus and thus are a mixture of methylated and unmethylated sequences. 58

59 Figure 3-5. Snrpn upstream exon expression from the 425Δ5-7A transgene. A) Schematic diagram of the RT-PCR strategy used to amplify upstream exon containing transcripts exclusively from the transgene. The Snrpn locus is depicted including upstream exons U1, U3, and U2 as well as exons one to ten. The deletion spanning a portion of exon five to exon seven is shown by a green box, within it a LoxP site. The forward primer for RT-PCR is located in U1 (red arrow) and the reverse primer is located within the LoxP sequence (green arrow). RT-PCRs were Southern blotted with a probe for Snrpn U1 to exon three sequence to intensify signal strength. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. B) U1 expression from the transgene in newborn brain RNA. Two transgenic brains from both maternal and paternal transmission were analyzed. Non-transgenic littermate brain cdnas were used as controls. C) U1 expression from the transgene in ovaries from three-week-old female mice. Four sets of transgenic ovaries were assayed as well as two sets of nontransgenic control ovaries. 59

60 Figure 3-6. Snrpn upstream exon expression in various tissues. Snrpn upstream exon expression was analyzed by RT-PCR and subsequent Southern blot with a probe for U1-exon three. The top panel represents U1 expression from the transgene (primers shown by arrows in schematic diagram on the left) on cdnas from several tissues harvested from a paternally transmitted 425Δ5-7A transgenic male mouse. Ovary cdna from an age-matched female transgenic was used as a positive control. The middle panel represents U1 expression exclusively from the endogenous locus (primers shown in schematic diagram on left by arrows). RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. 60

61 Figure 3-7. Snrpn upstream exon expression in the developing germline. A) Schematic diagram of the RT-PCR strategy used to amplify Snrpn upstream exon-containing transcripts. The Snrpn locus is depicted including upstream exons U1, U3, and U2 in addition to exons one to ten. The forward primer for RT-PCR is located in U1 (red arrow) and the reverse primer is located within Snrpn exon three (black arrow). B) U1 expression in the germline. The top row represents transcripts from U1 to Snrpn exon three. This RT-PCR was Southern blotted with a probe for the same sequence to intensify the signal. The middle row represents expression of Snrpn using primers spanning exons four to eight and the bottom row is an Hprt control reaction for cdna quality. Adult testis and ovary as well as 13.5 dpc purified female and male PGC cdnas were used as templates (depicted by +). -RT samples (depicted by -) were used as controls for genomic DNA contamination. 61

62 CHAPTER 4 IDENTIFICATION OF THE AS-IC Introduction Once we determined that AS-IC activity was present on the 425Δ5-7 BAC transgene, we had a base upon which to build our model system. After showing that the 425Δ5-7 transgene was properly imprinted, our experimental design was to make deletions within this transgene to determine the elements required to confer imprinting to the locus. Our hypothesis proposes that it is the active transcription, originating at the Snrpn upstream exons and continuing through the PWS-IC, in growing oocytes that establishes the maternal imprint at the PWS/AS locus. The imprinted 425Δ5-7 BAC contains three of the nine identified Snprn upstream exons including U1, U3, and U2. In relation to Snrpn exon one, the upstream exons are located at -43 kb (U1), - 75 kb (U3), and -96 kb (U2). In the previous chapter, we showed that transcripts originating from the 425Δ5-7 transgene that contained upstream exon U1 were present in ovaries during the time at which maternal imprints were being established in oocytes. Thus, our next series of experiments were aimed at determining whether the Snrpn upstream exons are required for the BAC transgene to be imprinted. To determine whether these transcripts are necessary for maternal imprinting, we utilized BAC recombineering techniques to make targeted deletions of all three of the upstream exons contained within the 425Δ5-7 BAC (Figure 4-1). For each exon, a 2.5 kb region of homology was removed in attempts to eliminate any potential promoter function. Recombineering was performed by Chris Futtner as described and the modified transgene was termed 425ΔU1-U3 (Futtner, 2007). 62

63 Results Three Snrpn Upstream Exons Constitute the AS-IC on the 425D18 BAC Expression of the 425ΔU1-U3 transgene is not imprinted Injections of the 425ΔU1-U3 BAC DNA into the pronuclei of fertilized FVB oocytes were initially performed by Chris Futtner. Four founders were obtained and lines A, B, C, and D were established. We investigated Snrpn expression patterns in these lines to determine the imprinted status of the 425ΔU1-U3 BAC. We expected to find an absence of imprinting due to the removal of the three upstream exons. If this were the case, offspring inheriting the transgene either maternally or paternally would express Snrpn. As with the 425Δ5-7 lines, expression of the transgene was examined in newborn whole brains by RT-PCR. We utilized the primer set located in Snrpn exons four and eight to distinguish transgenic Snrpn expression from the endogenous Snrpn (Appendix A). We obtained brains from newborn pups with either a maternally or paternally inherited transgene and generated cdnas for use as templates in RT- PCR reactions. Of the four 425ΔU1-U3 lines, only one was suitable for further analysis. Line A did not express Snrpn from the transgene upon maternal or paternal transmission. This was most likely due to position effects at the insertion site or breakage of the transgene upon integration at a point incompatible for Snrpn expression, which is possible since the BACs were injected as supercoils. Unfortunately, lines B and C were lost before expression analysis could be completed. Preliminary data did show that line B expressed Snrpn from the transgene upon maternal transmission but no conclusions could be made since other samples were not available from this line and thus paternal transmission could not be tested (Futtner, 2007). That left only one line available for complete analysis, line D. Our results from analysis of this line showed 63

64 that the 425ΔU1-U3 transgene is expressed upon both paternal and maternal transmission, demonstrating an absence of imprinting on this modified BAC (Figure 4-2). When performing studies with transgenics, it is crucial to demonstrate consistent results with independent lines. Therefore, because we had only one suitable line for analysis from the initial injections, we performed another set of injections through the newly established UF Mouse Models Core Facility. From these injections we were able to establish two new lines: 425ΔU1-U3 E and F. Unfortunately, attempts to analyze imprinting in these lines proved cumbersome. The E line displayed an incomplete penetrance such that Snrpn was imprinted in a majority of maternally inherited transgenic brains but expressed in a small number (data not shown). We hypothesized that this was due to a copy number effect similar to what was seen in our Snrpn phage clone transgenic line (Blaydes et al., 1999). We were also unable to analyze imprinting in the 425ΔU1-U3F line as we discovered a female-specific embryonic lethal phenotype that prevented us from examining maternal transmission of the transgene. In order to establish another 425ΔU1-U3 transgenic line to complete our analysis, a third round of BAC injections was performed by the UF Mouse Models Core Facility. From this, three more transgenic lines were created: 425ΔU1-U3 G, H, and I. We are currently in the process of analyzing these three lines in order to verify our results from the 425ΔU1-U3D BAC transgenic line. Epigenetic imprinting marks are absent on the 425ΔU1-U3 transgene Next, we investigated whether the 425ΔU1-U3 BAC possessed the appropriate epigenetic imprint at the PWS-IC. We did so by examining the methylation status of the Snrpn DMR in newborn brain DNA via genomic bisulfite sequence analysis. As expression of the 425ΔU1-U3 transgene was not imprinted, we expected to find a lack of epigenetic imprinting marks at the transgene PWS-IC. The 425ΔU1-U3D line was used as the representative line for this modified 64

65 BAC transgene in bisulfite sequencing experiments. For our methylation analysis, we examined the same region of the Snrpn DMR that was analyzed on the 425Δ5-7 transgene (Figure 3-3). We transferred the 425ΔU1-U3D transgene onto the B6.cast.c7 background and mated the F1 mice to generate pups for newborn brain collection. Whole brain genomic DNA was isolated and bisulfite sequence analysis performed on samples that were positive for the transgene and that also exhibited cast c7 homozygosity. We compared data between paternally and maternally inherited transgenic brains after performing the bisulfite sequencing experiments on at least two brains per mode of inheritance. Our results from the sequence analysis were as expected, the paternally and maternally transmitted 425ΔU1-U3D transgenes both displayed hypomethylation of the Snrpn DMR (Figure 4-3A). The endogenous alleles displayed a mixture of methylated and unmethylated clones, representing both the maternal and paternal alleles (Figure 4-3B). These results prove that the maternal DNA methylation imprint is not present in newborn brain tissue on the 425 BAC transgene after deletion of the Snrpn upstream exons. Discussion We have shown that unlike the 425Δ5-7 transgene, the 425ΔU1-U3 transgene is expressed upon both paternal and maternal inheritance. Furthermore, the Snrpn DMR is hypomethylated on the transgene after both paternal and maternal transmission, demonstrating a loss of the maternal epigenetic imprint coinciding with the deletion of the upstream exons. These results confirm that imprinting of the 425Δ5-7 transgene is lost upon removal of the three Snprn upstream exons contained within it, providing compelling evidence that the upstream exons play a major role in AS-IC function. Although we showed that the 425ΔU1-U3 transgene is not imprinted in newborn brain samples, we have not determined whether the deletion of the upstream exons causes a defect in establishing the imprint at the PWS-IC or if it merely causes a failure to maintain the imprint 65

66 after it is established. There are reports that provide evidence suggesting that methylation imprints are unstable post-zygotically, thus signifying the importance of maintenance mechanisms and a potential role for the AS-IC in methylation maintenance at the PWS-IC. One such study was published from our lab; we generated and analyzed a mutant mouse, termed the PWS-IC HS mutant, that had the murine PWS-IC replaced with the human PWS-IC (Johnstone et al., 2006). Following maternal inheritance of the PWS-IC HS allele, the DNA methylation imprint was properly established at the human PWS-IC during oogenesis. However, this imprint was absent in newborn brain samples, demonstrating a failure in imprint maintenance at the human PWS-IC sequence and thereby indicating post-zygotic instability of the methylation imprint. A discovery of Snrpn upstream exon function in imprint maintenance at the PWS-IC would be a very significant finding. Dnmt1 is known to maintain methylation imprints in preimplantation development, a time during which global epigenetic reprogramming is occurring and the majority of DNA methylation is erased (Hirasawa et al., 2008; Li et al., 1993). How this DNA methyltransferase s activity is specified to imprinted regions in pre-implantation embryos is unclear. One possibility is that transcription originating from the upstream exons and continuing through the PWS-IC during pre-implantation development promotes the recruitment of Dnmt1-containing complexes. A plausible factor in such a mechanism is Zfp57, a member of the KREB zinc finger family of transcriptional repressors. KAP1/TIF1β, a required co-repressor, interacts with factors in this family via the KREB box domain and recruits complexes associated with DNA methylation and repressive histone modifications (Abrink et al., 2001; Ayyanathan et al., 2003; Friedman et al., 1996; Schultz et al., 2002; Wiznerowicz et al., 2007). A role for Zfp57 in imprinting at the PWS/AS locus was revealed by analyzing Zfp57 knockout mice. Li et al. produced this knockout and discovered that Zfp57 homozygous mutant 66

67 females failed to establish the maternal DNA methylation imprint in the germline at the PWS-IC (Li et al., 2008). In addition, homozygous mutant (-/-) and maternally transmitted heterozygous mutant (-/+) pre-implantation embryos also lacked the DNA methylation imprint. In contrast, paternally transmitted heterozygous mutant (+/-) pre-implantation embryos did possess methylation at the maternal PWS-IC. Surprisingly, they also found that approximately half of the (-/+) embryos acquired a DNA methylation imprint at the PWS-IC by midgestation, suggesting that the imprint could be re-established in post-implantation development in these heterozygotes. Li et al. also demonstrated that Zfp57 binds to the PWS-IC in murine ES cells. Thus, the authors speculate that zygotic Zfp57 directs methylation to the PWS-IC around the time of implantation, the time in embryonic development during which de novo DNA methylation acquisition occurs. We are currently preparing experiments to determine the expression patterns of the Snrpn upstream exons over the course of early embryogenesis and will thereby determine if this transcription possibly plays a role in imprint maintenance. Finding that the upstream exon transcript endures post-zygotically may suggest that the active transcription of the upstream exons or the transcript itself cooperates in directing the methylation maintaining apparatus to the PWS-IC. This potential mechanism could include the recruitment of Zfp57 and its associated repressive factors. Our BAC transgenic system will allow us to directly compare the epigenetic status of the PWS-IC in the presence and absence of the Snrpn upstream exons. Analysis of the 425ΔU1-U3 transgene suggests that the AS-IC activity on the 425D18 BAC originates from the upstream exons. Future experiments will examine the epigenetic imprint on the 425ΔU1-U3 transgene at the PWS-IC in the maternal germline. This will be accomplished through bisulfite sequence 67

68 analysis of mature oocytes from transgenic females. We will determine whether the imprint is initially established in oocytes but not maintained during the course of development or whether the imprint is never established in the maternal germline. These experiments will reveal a function for the upstream exons either in imprint establishment or maintenance. 68

69 Figure 4-1. The modified 425D18 BAC transgenes. The 425Δ5-7 transgene is depicted above with the three upstream exons shown in red and Snrpn exons one to ten in blue. The 425ΔU1-U3 transgene was modified by BAC recombineering and bears deletions of each of the three upstream exons. Each deletion is 2.5 kb. 69

70 Figure 4-2. Snrpn expression analysis of the 425ΔU1-U3D transgene. RT-PCR was performed on newborn brains with a primer set spanning from Snrpn exons four to eight, flanking the exon five to seven deletion. The endogenous Snrpn amplicon is 513 bps while the transgene product is 350 bps. Two transgenic brains from both maternal and paternal transmission were analyzed. Non-transgenic littermate brain cdnas were used as controls. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. 70

71 Figure 4-3. Bisulfite sequence analysis of the 425ΔU1-U3D transgene. A portion of the Snrpn DMR containing 14 CpG dinucleotides was analyzed. Each CpG is represented by a circle. Methylated CpGs are displayed as black circles and unmethylated CpGs are shown as white circles. Each row represents an individually sequenced clone. A) Transgenic alleles: the majority of both the maternally transmitted and paternally transmitted alleles are unmethylated. B) The endogenous alleles represent both the maternal and paternal locus and thus are a mixture of methylated and unmethylated clones. 71

72 CHAPTER 5 A SINGLE UPSTREAM EXON IS SUFFICIENT TO IMPRINT THE BAC TRANSGENE Introduction Our experimental approach to identify the murine AS-IC was based upon finding an imprinted transgene and then modifying this transgene to knockout imprinting center function. The 425D18 BAC showed proper imprinted expression as a transgene and hence was used as the foundation for our model system. We made a deletion within the body of Snrpn on the 425D18 BAC in order to differentiate endogenous expression from transgene expression, designating this modified BAC as the 425Δ5-7 BAC. Subsequently, we made targeted deletions of each of the three Snrpn upstream exons contained within this imprinted transgene in order to determine whether these exons were required to generate the maternal imprint. Upon deletion of the three upstream exons from the 425Δ5-7 BAC transgene, we discovered that AS-IC activity was lost, prompting us to ask whether one upstream exon was sufficient to imprint this transgene? Consequently, our next step was to delete two of the three upstream exons contained within the 425Δ5-7 BAC to determine if one Snrpn upstream exon would be adequate to maintain AS-IC activity on the transgene. We chose to first investigate whether Snrpn upstream exon U1 was itself sufficient to imprint the BAC transgene as our experiments analyzing upstream exon expression were all based upon U1. Another reason we chose to analyze U1 function in the absence of the other upstream exons was due to the data generated from previous studies performed on the imprinted 215A9 BAC transgenic lines. The 215A9 BAC extends an additional 30 kb upstream of the 425D18 BAC and includes Snrpn upstream exons U1, U2, U3, and U4. We discovered that the 215B line, which showed proper imprinting, had undergone a truncation roughly 40 kb upstream of Snrpn exon one (Chamberlain, 2003). For this transgenic line, it was critical to determine 72

73 whether the remaining 5 BAC sequence contained U1, which is located 43 kb upstream of Snrpn, as this was the only identified upstream exon that would be present on the truncated transgene. RT-PCR analysis on newborn brains with a paternally transmitted transgene revealed that U1 was present on the transgene and also expressed (data not shown). These results suggested that Snrpn upstream exon U1 is sufficient to imprint the BAC transgene. To study Snrpn upstream exon U1 function in the absence of other upstream exons, we used BAC recombineering to delete the other two upstream exons contained within the 425D18 BAC sequence. We made targeted deletions of 2.5 kb each for both U3 and U2, leaving U1 intact (Figure 5-1). The recombineering was performed by Chris Futtner as described and the modified transgene was termed 425ΔU2/U3 (Futtner, 2007). Results The 425ΔU2/U3 BAC Displays AS-IC Activity Injections of the 425ΔU2/U3 BAC DNA into the pronuclei of fertilized FVB oocytes were also initially performed by Chris Futtner. From these injections, one founder was obtained and line A was established. This line had a peculiar phenotype however and will be discussed at length in the next chapter. In order to generate additional lines for analysis, we had the UF Mouse Models Core Facility perform another set of injections with the 425ΔU2/U3 BAC DNA. We were able to establish only one new line from this round of injections: 425ΔU2/U3E. The 425ΔU2/U3 BAC exhibits imprinted expression patterns We started our investigation of the 425ΔU2/U3E line by analyzing Snrpn expression patterns to determine the imprinted status of this modified BAC. Critical to the imprinted status of this transgene is copy number since tandem copies of the transgene could potentially create artificial imprinting. Thus, we first investigated the copy number of the 425ΔU2/U3E line by Southern blot analysis and determined that it was indeed single copy (data not shown). After 73

74 copy number analysis, expression of the transgene was examined in P1 whole brains by RT-PCR with the Snrpn exon four to eight primer set (Appendix A) as was done with the previously studied transgenic lines. We harvested brains from newborn pups with either a maternally or paternally inherited transgene and generated cdnas for use as templates in RT-PCR reactions. The RT-PCR analysis showed that the 425ΔU2/U3E transgene was expressed upon paternal transmission but silenced upon maternal transmission. Thus, this transgene bearing only Snrpn upstream exon U1 demonstrated proper imprinted expression patterns (Figure 5-2). The 425ΔU2/U3 BAC is epigenetically imprinted We next examined the epigenetic imprint at the PWS-IC on the 425ΔU2/U3E transgene by analyzing the methylation status of the Snrpn DMR. As expression of the 425ΔU2/U3E transgene is imprinted, we expected to find the appropriate epigenetic imprinting marks at the transgene PWS-IC. This would mean that we would see an unmethylated Snrpn DMR on the paternally transmitted transgene and a methylated DMR on the maternally transmitted transgene. We investigated the methylation status of the PWS-IC via genomic bisulfite sequence analysis on newborn brain DNA, analyzing the same region of the Snrpn DMR that was analyzed on the 425Δ5-7 and 425ΔU1-U3 transgenes (Figure 3-3). We transferred the 425ΔU2/U3E transgene onto the B6.cast.c7 background and mated the F1 mice to generate pups from which to collect newborn brains. Whole brain genomic DNA was isolated and bisulfite sequence analysis performed on samples that were positive for the transgene and that also exhibited cast c7 homozygosity. We compared data between paternally and maternally inherited transgenic brains after performing experiments on at least two brains per mode of inheritance. Our results from the sequence analysis were as expected, the paternally transmitted 425ΔU2/U3E transgene displayed very low methylation at the Snrpn DMR and the maternally transmitted transgene demonstrated hypermethylation at this DMR (Figure 5-3A). The endogenous alleles displayed a 74

75 mixture of methylated and unmethylated clones, presumably representing both the maternal and paternal alleles (Figure 5-3B). These results prove that the maternal DNA methylation imprint is present on the BAC transgene when only Snrpn upstream exon U1 is intact. From our Snrpn expression analysis and DNA methylation analysis, we can conclude that the 425ΔU2/U3E transgene is properly imprinted. These preliminary results suggest that U1 is sufficient to imprint the 425D18 BAC without contributions from the other upstream exons. Thus, these data strongly imply that a functional AS-IC is contained within this one alternative upstream exon. U1 Usage on the Imprinted 425ΔU2/U3 BAC Transgene Our hypothesis for AS-IC function proposes that it is the active transcription of the Snrpn alternative upstream exons on the maternal allele that promotes the epigenetic modification of the PWS-IC during oogenesis. Mapendano et al. demonstrated that transcription of the upstream exons in the oocyte corresponds with the establishment of the maternal imprint at the PWS-IC (Mapendano et al., 2006). We have demonstrated that the properly imprinted 425Δ5-7A transgene also expresses upstream exon containing transcripts in the ovary at the time of imprint establishment (Figure 3-5C). Furthermore, this transgene displays imprinted expression of the upstream exons in the brain such that they are highly expressed from the paternal allele but only show leaky expression from the maternal allele as is the case for the endogenous locus. To determine whether the 425ΔU2/U3E transgene retains these upstream exon expression patterns, we examined Snrpn upstream exon U1 expression dynamics from this transgene in the ovary as well as in the brain. To detect U1 expression from the transgene, we performed RT- PCR utilizing the U1-LoxP primer set that distinguishes transgene expression from endogenous expression (Appendix A). We then Southern blotted the PCR products with probe consisting of Snrpn upstream exon U1 through exon three to amplify the signal. 75

76 U1 is not transcribed from the 425ΔU2/U3E transgene in newborn brain Initially, we analyzed newborn whole brain cdnas from pups inheriting the transgene either maternally or paternally to determine U1 expression patterns. As the 425ΔU2/U3E transgene is properly imprinted, we expected to find U1 expression in the paternally inherited transgenic brains but only low levels of expression in the maternally inherited transgenic samples. Unexpectedly, transcripts derived from the transgene that included U1 were not detected in brain tissue after paternal or maternal transmission (Figure 5-4A). The 425ΔU2/U3E transgene does not express U1 in the ovary Although we were surprised to find no Snrpn upstream exon U1 transcripts originating from the transgene in the paternally inherited P1 brain samples, the more critical question was whether they were present in the ovaries of female transgenics during the time at which imprints were being established in the oocytes. Therefore, we analyzed ovaries from three transgenic females and one non-transgenic littermate at three weeks of age for U1 expression. We found that Snrpn upstream exon one was not transcribed from the transgene in the three-week-old ovaries (Figure 5-4B). Thus, the imprinted 425ΔU2/U3E transgene does not express Snrpn upstream exon U1 in P1 brain or in oocytes during maternal imprint establishment. Discussion The data presented in this chapter for the 425ΔU2/U3E transgenic line confirm the imprinted status of this modified BAC as a single copy transgene. We have shown that this transgene displays maternally imprinted expression as well as the appropriate DNA methylation imprinting marks at the maternal PWS-IC. These results, together with the data demonstrating the truncated 215B BAC transgene is imprinted and that the 425ΔU1-U3 BAC transgene is not imprinted, provide convincing evidence that Snrpn upstream exon U1 is sufficient to imprint the PWS-IC. Unfortunately, we cannot make any definitive conclusions based on the data from the 76

77 425ΔU2/U3E line since we do not have a second transgenic line to verify our results. Our future plans include another round of injections of the 425ΔU2/U3 BAC in conjunction with the UF Mouse Models Core. While the 425ΔU2/U3E transgene displayed appropriate imprinting, we were surprised to find that Snrpn upstream exon U1 was not expressed from this transgene in the brain or in the ovary during the oocyte growth stage. This could suggest that it is the U1 sequence that is sufficient for imprinting the BAC transgene rather than transcription from the upstream exons; however, this conclusion is premature. Because BACs break at random sites upon integration into the genome, it is possible that the 425ΔU2/U3E BAC transgene broke upon integration at a site 3 to U1 thereby resulting in a transgene lacking any Snrpn upstream exons. How then could transcription proceed through the PWS-IC? We suggest that the transgene integrated just downstream of a promoter that is active in growing oocytes so that transcription through the PWS-IC of the 425ΔU2/U3E BAC transgene occurs during oogenesis, contributing to the maternal imprint. Critical to this interpretation is that the 425ΔU2/U3E transgene is single copy. To provide evidence in support of our hypothesis, we need to examine the structure of the 425ΔU2/U3E transgene to determine whether it is intact and if it does in fact contain U1. Finding that U1 is in place on the transgene would strongly suggest that it is the U1 sequence that leads to maternal imprinting of the transgene. We also need to perform a 5 RACE experiment with transgenic ovaries from three-week-old females in order to determine if there are Snrpn-containing transcripts originating from upstream of the transgene PWS-IC. The 5 RACE experiment is important for two reasons: i) to identify any potential promoter activity at the integration site that would lead to transcription through the transgene PWS-IC and ii) to verify that there are no additional unidentified upstream exons contained within the 425D18 77

78 BAC that are actively transcribed in growing oocytes, as this could lead to imprinting of the transgene as well. The presence of previously unidentified exons on the 425D18 BAC that are transcribed during oogenesis is highly unlikely however, as we expect this would lead to imprinting of the 425ΔU1-U3D BAC transgene. Further experiments with additional single copy 425ΔU2/U3 transgenic lines are imperative to determine whether Snrpn upstream exon U1 is sufficient to imprint the 425D18 BAC transgene in the absence of other upstream exons. If U1 is sufficient, these experiments will also provide important insights into mechanisms of action of this alternative upstream exon in maternal imprinting at the PWS-IC. 78

79 Figure 5-1. Comparisons of the three modified 425D18 BAC transgenes. The 425Δ5-7 transgene is depicted at the top. The three upstream exons are shown in red and Snrpn exons one to ten in blue. The 425ΔU1-U3 transgene, displayed in the middle, was modified by BAC recombineering and bears deletions of each of the three upstream exons. The 425ΔU2/U3 transgene is depicted on the bottom. This transgene possesses deletions of U2 and U3 while U1 remains intact. Each upstream exon deletion is 2.5 kb. 79

80 Figure 5-2. Snrpn expression analysis of the 425ΔU2/U3E transgene. RT-PCR was performed on newborn brains with a primer set spanning Snrpn exons four to eight, flanking the exon five to seven deletion. The endogenous Snrpn amplicon is 513 bps while the transgene product is 350 bps. Two transgenic brains from both maternal and paternal transmission were analyzed. Non-transgenic littermate brain cdnas were used as controls. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. 80

81 Figure 5-3. Bisulfite sequence analysis of the 425ΔU2/U3E transgene. A portion of the Snrpn DMR containing 14 CpG dinucleotides was analyzed. Each CpG is represented by a circle. Methylated CpGs are displayed as black circles and unmethylated CpGs are shown as white circles. Each row represents an individually sequenced clone. A) Transgenic alleles: the maternally transmitted alleles are hypermethylated while the paternally transmitted alleles are hypomethylated. B) The endogenous alleles represent both the maternal and paternal locus and thus are a mixture of methylated and unmethylated clones. 81

82 Figure 5-4. Snrpn upstream exon usage from the 425ΔU2/U3E transgene. RT-PCR was performed to analyze Snrpn upstream exon U1 expression from the 425ΔU2/U3E transgene. The products were Southern blotted with a probe for Snrpn upstream exon U1 to exon three to intensify the signal. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. A) U1 expression from the transgene in newborn brain RNA. Two transgenic brains from both maternal and paternal transmission were analyzed and non-transgenic littermate brain cdnas were used as controls. cdna from a paternally inherited 425Δ5-7A transgenic brain was used as a positive control (+). B) U1 expression from the transgene in ovaries from three-week-old female mice. Three sets of transgenic ovaries were assayed as well as one set of non-transgenic control ovaries. 82

83 CHAPTER 6 SILENCING OF THE ENDOGENOUS LOCUS BY A PATERNALLY TRANSMITTED TRANSGENE Introduction In our attempts to knockout AS-IC function on the 425Δ5-7 transgene and identify the minimal elements necessary for imprinting, we created the 425ΔU2/U3 BAC. This modified BAC had upstream exons U2 and U3 removed so that only one upstream exon remained, U1. We initially established one transgenic line, denoted line A, and upon analysis of this line we discovered aberrant gene regulation at the endogenous Snrpn locus. We then established a second transgenic line from this BAC, 425ΔU2/U3E, and found that this line did not display the same unusual characteristics; this line was properly imprinted and our complete analysis is detailed in chapter five. Thus, we declared this abnormal phenotype as unique to the 425ΔU2/U3A line and not a result of the structure of the 425ΔU2/U3 transgene itself. This chapter describes the observations we have made in relation to the aberrant gene regulation in the 425ΔU2/U3A line at the endogenous PWS/AS locus. Results Imprint Analysis of the 425ΔU2/U3A Transgene: Uncoupling of the Epigenetic and Expression Imprints The 425ΔU2/U3A transgene does not display imprinted expression patterns We began our investigation of the 425ΔU2/U3A line by analyzing the expression patterns of this transgene. We did so via RT-PCR on P1 brains possessing either a maternally or a paternally inherited transgene. By using the Snrpn primer set spanning exons four to eight (Appendix A), we found that the transgene was expressed upon both maternal and paternal transmission, albeit at lower levels after maternal transmission (Figure 6-1A). Thus, unlike the 83

84 425ΔU2/U3E transgenic line, the 425ΔU2/U3A line did not display imprinted expression of the transgene. The DNA methylation imprint is present on the 425ΔU2/U3A transgene Next, we analyzed the Snrpn DMR on the 425ΔU2/U3A transgene for the presence of the DNA methylation imprint. Because the transgene did not show imprinted expression, we expected that the epigenetic imprint would be absent and thus the Snrpn DMR would be unmethylated upon both paternal and maternal transmission of the transgene. We examined the methylation status of the DMR by genomic bisulfite sequence analysis on newborn brain DNA exactly as described for the previously analyzed transgenic lines. Our results from the sequence analysis were as expected for the paternally transmitted 425ΔU2/U3A transgene as an unmethylated Snrpn DMR was displayed (Figure 6-2A). However, we were surprised to discover that the maternally transmitted 425ΔU2/U3A transgene was hypermethylated at the Snrpn DMR, demonstrating an uncoupling of the methylation imprint from the expression imprint. The endogenous maternal and paternal alleles displayed a mixture of methylated and unmethylated clones (Figure 6-2B). These results showed that the maternal DNA methylation imprint is present on the BAC transgene when only Snrpn upstream exon U1 is intact, in agreement with the data generated from the 425ΔU2/U3E line. However, in contrast to the 425ΔU2/U3E transgene, this methylation imprint is not correlated with the expression patterns of the 425ΔU2/U3A transgene, which lacks imprinted expression. U1 Usage From the 425ΔU2/U3A BAC Transgene After the imprinting analysis of the 425ΔU2/U3A transgene generated such unexpected results, we decided to investigate the Snrpn upstream exon expression patterns from this transgene. We began this investigation by performing RT-PCR on P1 brain cdnas bearing either a paternally or maternally inherited transgene. We used the U1-LoxP primer set that 84

85 distinguishes transgene expression from the endogenous alleles (Appendix A) and subsequently Southern blotted the PCR products with a probe consisting of Snrpn upstream exon one through exon three to intensify the signal. As the 425ΔU2/U3E transgene does not express U1 in newborn brain, we expected to see the same result from the 425ΔU2/U3A transgene. However, we did not obtain these results. Instead, we found that U1 is highly expressed after paternal transmission of the 425ΔU2/U3A transgene and also at a very low level after maternal transmission (Figure 6-3). This expression mimics the U1 expression patterns from the imprinted 425Δ5-7A transgene (Figure 3-5B). Experiments are underway to determine U1 expression patterns from the 425ΔU2/U3A transgene in the ovary. Endogenous Snrpn is Silenced Upon Paternal Transmission of the 425ΔU2/U3A Transgene Our analysis of Snrpn expression from the 425ΔU2/U3A transgene generated even more surprising results. We observed repression of the endogenous Snrpn upon paternal transmission of the transgene; however, this silencing was not detected after maternal transmission of the transgene (Figure 6-1A). To confirm that this silencing was real and not just a PCR anomaly, we performed a Northern blot on transgenic newborn whole brain RNAs, comparing the 425ΔU2/U3A transgenic samples to the 425Δ5-7A transgenics. We analyzed two brains with paternally inherited transgenes from the 425Δ5-7A line and one brain with a maternally inherited transgene. For the 425ΔU2/U3A line, we analyzed two transgenic brains for each parental transmission along with non-transgenic littermate controls. We used a probe containing Snrpn exons one through five, which detects both endogenous and transgenic Snrpn. The transgene Snrpn transcript is approximately 150 bp smaller than the endogenous Snrpn due to the deletion in exons five to seven and thus a doublet of bands is displayed when Snrpn is expressed from both the transgene and the endogenous locus. Results from our Northern blot experiment showed that the endogenous Snrpn is indeed silenced upon paternal transmission of the 85

86 425ΔU2/U3A transgene, demonstrated by the absence of the upper band of the doublet in the two paternally inherited transgenic samples (Figure 6-1B). The same is not true for the maternally inherited transgenic brains as the endogenous Snrpn band is present as well as the transgenic Snrpn band. This Northern blot also confirms our expression data for the 425Δ5-7A line, showing that this transgene is paternally expressed and maternally imprinted. The 425ΔU2/U3A transgene cannot silence Snrpn from another BAC transgene To further investigate the silencing potential of the 425ΔU2/U3A transgene when paternally transmitted, we crossed this line to another Snrpn BAC transgenic line, a 380J10 transgenic line. We sought to determine whether the 425ΔU2/U3A transgene could silence Snrpn from any locus or only the endogenous locus. As discussed in earlier chapters, the 380J10 BAC expresses Snrpn upon both maternal and paternal transmission, demonstrating the presence of the PWS-IC but a lack of an AS-IC. The 380J10 BAC transgene does not have the deletion in Snrpn exons five through seven and therefore RT-PCR analysis will differentiate between the two transgenes but not between the 380J10 transgene and the endogenous allele. To examine the effects of the 425ΔU2/U3A transgene on the 380A transgene, we performed a preliminary mating that generated a male bearing both transgenes. We then performed RT-PCR with the Snrpn exon four to eight primer set (Appendix A) on P1 brain cdnas produced by mating this dual transgenic male to a wild type female. We assayed two brains each bearing both transgenes, one brain with only the 380A transgene, one brain with only the 425ΔU2/U3A transgene, and two non-transgenic littermate samples. The brains possessing both transgenes showed Snrpn expression from both the 425ΔU2/U3A transgene and the 380A transgene, assuming the larger amplicon was from the 380A transgene and not the endogenous allele (Figure 6-4). We obtained the same result when the 380A transgene was transmitted maternally, demonstrating that the 425ΔU2/U3A transgene was not capable of silencing Snrpn from the 380A BAC transgene (data 86

87 not shown). From these experiments, we determined that the paternally transmitted 425ΔU2/U3A transgene cannot repress the 380A transgene and therefore is not acting by identifying and repressing any Snrpn locus. Silencing by the 425ΔU2/U3A transgene is independent of imprinting We next investigated whether the 425ΔU2/U3A transgene was creating an imprint at the PWS-IC and thereby silencing Snrpn upon paternal transmission of the transgene via an imprinting mechanism. To see if this were the case, we looked at Snrpn expression in 14.5 dpc gonads with a paternally inherited 425ΔU2/U3A transgene. The germline undergoes imprint erasure between dpc and thus the germ cell-containing fetal gonads would not possess imprints at 14.5 dpc. Because imprints are absent in the germline at this time of development, Snrpn is expressed from both the maternal and paternal allele. Therefore, we made use of a PWS-IC deletion mouse (ΔIC) created previously by our lab for this experiment. The ΔIC mutant bears a 35 kb deletion consisting of Snrpn exons one through six and 16 kb of sequence 5 to Snrpn. Upon paternal transmission of the ΔIC allele, there is a complete absence of paternal gene expression from the PWS/AS locus (Yang et al., 1998). Thus, we mated a 425ΔU2/U3A transgenic male to a ΔIC female to ablate Snrpn expression from the maternal allele in the resulting 14.5 dpc germ cells, allowing for analysis of repression of Snrpn only from the paternal endogenous locus. We dissected out fetal gonads from the 14.5 dpc embryos generated by this mating, genotyped a portion of each embryo, and made cdna from the gonadal tissue for RT-PCR analysis. We examined four sets of gonads from one litter of embryos: one wild type (+/+;-/-), one with a paternally transmitted 425ΔU2/U3A transgene (+/+;-/Tg), one with a maternal ΔIC allele but no transgene (ΔIC/+;-/-), and one with both the maternal ΔIC and the transgene (ΔIC/+;-/Tg). We performed RT-PCR for Snrpn exons four to eight on these cdnas, thereby allowing for differentiation of the transgene and endogenous 87

88 transcripts. We discovered that upon paternal transmission of the 425ΔU2/U3A transgene and maternal transmission of the ΔIC allele, we had no endogenous Snrpn expression. We did however observe Snprn expression from the transgene. These results demonstrated that the paternally transmitted 425ΔU2/U3A transgene does not silence the endogenous Snrpn by the same mechanism responsible for imprinting the locus. Furthermore, we found that the fetal gonads with the 425ΔU2/U3A transgene and both Snrpn endogenous alleles intact (+/+;-/Tg) displayed no endogenous Snrpn expression. Therefore, the paternally transmitted 425ΔU2/U3A transgene has the ability to silence both endogenous Snrpn alleles. Regulation of Other Genes at the Endogenous PWS/AS Locus by the 425ΔU2/U3A Transgene The PWS-IC is widely believed to be a positive acting element that activates genes on the paternal allele. After finding that the 425ΔU2/U3A transgene repressed endogenous Snrpn expression upon paternal transmission, we sought to determine whether it was interfering with regulation of the other paternally expressed genes at the PWS/AS locus. This result would suggest that the transgene was disrupting function of the PWS-IC. The upstream cluster genes are not repressed by the 425ΔU2/U3A transgene We started our investigation by examining the upstream cluster genes: Ndn, Frat3, Magel2, and Mkrn3. To analyze Ndn expression, we examined the same Northern blot that was used to determine Snrpn expression patterns in the 425ΔU2/U3A line. After stripping the blot and hybridizing it with a probe for Ndn, we found that there was no silencing of this gene in the 425ΔU2/U3A transgenic brains (Figure 6-1B). Next, we utilized RT-PCR techniques to determine whether mice bearing the 425ΔU2/U3A transgene were experiencing silencing of Frat3, Magel2, or Mkrn3. We used the same P1 brain cdna samples that were used in the initial Snrpn expression analysis of this 88

89 transgenic line as templates in RT-PCR reactions with primer sets specific for each gene: Frat3 F/R, Ngl2 F/R, and Zfp Poly F/R for Frat3, Magel2, and Mkrn3 respectively (Appendix A). The 425ΔU2/U3A transgenic brains did not display repression of any of the upstream cluster genes thus demonstrating that the transgene is not acting to silence the PWS-IC (Figure 6-6). The 425ΔU2/U3A transgene exerts varying effects on the downstream cluster genes Next, we analyzed several of the paternally expressed downstream cluster genes, including the Ube3a-ats transcript and the MBII-85 and MBII-52 snornas, to determine the effects of the 425ΔU2/U3A transgene on their expression. To examine Ube3a-ats expression, we performed RT-PCR with the Ube3a-ats M-F-bis and UEAS-1 primer set (Appendix A) on P1 whole brain RNAs bearing either a maternally transmitted or paternally transmitted 425ΔU2/U3A transgene. We discovered that the paternally transmitted 425ΔU2/U3A transgene repressed the Ube3a-ats transcript but the maternally transmitted transgene did not (Figure 6-7A). Previous studies have provided evidence suggesting that the paternally imprinted Ube3a is regulated in cis by the paternally expressed Ube3a-ats transcript (Chamberlain and Brannan, 2001; Johnstone et al., 2006; Kishino, 2006; Rougeulle et al., 1998). Therefore, we examined the allelic expression of Ube3a in paternally transmitted 425ΔU2/U3A transgenic P1 brains. We expected to see an increase in Ube3a expression from the paternal allele as a result of the downregulation of the Ube3a-ats transcript. To look at allelic expression of Ube3a, we made use of a SNP between the Mus musculus domesticus and Mus musculus castaneous strains that is contained within the Ube3a coding region (Chamberlain and Brannan, 2001). This SNP is located within a Tsp509I restriction site that is retained in the domesticus sequence but eliminated in the cast sequence. We generated the samples for our experiment by crossing a B6.cast.c7 female to a 425ΔU2/U3A transgenic male and collecting P1 brains from the resulting pups. We then performed RT-PCR on these brains, analyzing two transgenic samples and two 89

90 non-transgenic littermate samples as controls. We used the primer set Ube3a 5F/6R (Appendix A), which amplifies a product of 246 bps spanning the polymorphism. Another Tsp509I site is contained within this amplicon, regardless of the strain, so that the cast product is cut into two fragments upon digestion with this enzyme: 225 bps and 21 bps. The domesticus product is cut into three fragments by Tsp509I digest: 138 bps, 87 bps, and 21 bps (Figure 6-7B). After digestion of our PCR products, we ran them on a gel and analyzed the ratio of paternal:maternal fragments to determine whether the transgenic samples had an increase in Ube3a expression from the paternal allele. Surprisingly, this was not the case. There was no increase in the ratio of paternal:maternal Ube3a expression in the 425ΔU2/U3A transgenic brains as compared to the wild type control samples (Figure 6-7B). This experiment showed that even with repression of the paternally expressed Ube3a-ats transcript by the paternally transmitted 425ΔU2/U3A transgene, there is no resulting increase in Ube3a expression from the paternal allele. We next analyzed snorna expression in the 425ΔU2/U3A transgenic line. MBII-85 and MBII-52 are two of the snornas located within the PWS/AS domain, both residing 3 to Snrpn. Both snornas exist as arrays of tandem repeats, each covering approximately 100 to 200 kb of sequence (Cavaillé et al., 2000). The 3 end of the 425D18 BAC extends through a portion of the MBII-85 cluster but does not include MBII-52. To examine the effect of the 425ΔU2/U3A transgene on snorna expression, we performed a Northern blot comparing expression in P1 brains among wild type pups, 425Δ5-7A transgenics, and 425ΔU2/U3A transgenics. We hybridized the same blot with oligo probes for MBII-85, MBII-52, and 5.8s rrna, which served as a loading control, stripping the membrane between each hybridization. Our initial analysis showed a decrease in MBII-85 expression upon maternal transmission of the 425ΔU2/U3A 90

91 transgene (Figure 6-8A). No repression of MBII-52 was observed and no silencing of either snorna was displayed in the brains with a paternally inherited 425ΔU2/U3A transgene. To verify our results, we repeated the experiment, this time with a panel of brains bearing a maternally transmitted 425ΔU2/U3A transgene. We obtained nine P1 brains with a maternally inherited transgene as well as two wild type control samples. Our transgenic samples originated from four distinct mating cages and included brains from six different litters. In addition, we harvested brains from newborn pups bearing a paternal ΔIC allele and a maternal 425ΔU2/U3A transgene. The PWS-IC deletion on the paternal allele served to ablate the endogenous snorna expression so that we could determine the level of expression from the transgene without the endogenous background. From this experiment, we concluded that there was virtually no expression of the snornas from the maternally transmitted 425ΔU2/U3A transgene (Figure 6-8B). The level of expression of MBII-85 is very low and approximately equal to that of MBII- 52. Thus, since the MBII-52 expression cannot be from the transgene, it must be leaky expression from the maternal allele. The same is most likely true for the low level of MBII-85 expression observed in the presence of the paternal ΔIC allele, as this leaky maternal expression was demonstrated previously in our lab by analysis of MBII-85 expression in brains bearing a paternally transmitted ΔIC mutation (Chamberlain et al., 2004). Additionally, we unexpectedly discovered that only two of the nine 425ΔU2/U3A transgenic brains demonstrated repression of MBII-85 (Figure 6-8B). In contrast to our first experiment, these two brains also displayed down-regulation of MBII-52. These samples were from the same mating cage but different litters. Furthermore, two transgenic littermates of one of these samples were included in this Northern blot and they did not show any repression of the snornas. 91

92 We next decided to analyze the effect of the maternally transmitted 425ΔU2/U3A transgene on snorna expression from another BAC transgene, the 380A transgene. The 380A transgene spans the MBII-85 snorna cluster but does not contain any of the MBII-52 cluster. Again, we were curious as to whether the silencing effect on the snornas only occurred at the endogenous locus or if it had a more extensive repression capacity. For this experiment, we generated P1 brain samples with a number of different genotypes. To determine the expression level of snorna from the 380A transgene without endogenous background, it was necessary to have a paternally transmitted ΔIC allele. Therefore, we crossed the 425ΔU2/U3A transgene to the 380A line to generate a female carrying both transgenes. We then mated this female to a male carrying the ΔIC mutation and screened the resulting pups for the presence of both transgenes as well as the ΔIC allele. After harvesting the newborn brains, we performed a Northern blot on the whole brain RNA, assaying samples of the following genotypes: wild type, 380/+, 380/ΔIC, 425/+, 425/ ΔIC, and 380, 425/ ΔIC where 380 represents the 380A transgene and 425 represents the 425ΔU2/U3A transgene. The maternally transmitted 425ΔU2/U3A transgenic sample (425/+) did not display repression of the endogenous snornas in this experiment. The 380/ΔIC sample verified that MBII-85 is expressed from the maternally transmitted 380A transgene but at a lower level than the endogenous expression. The two samples containing both the 380A and the 425ΔU2/U3A transgenes on a paternal ΔIC background (380, 425/ ΔIC) both showed levels of snorna expression equivalent to that of the 380/ΔIC sample thereby demonstrating a lack of repression of the 380A transgene by the 425ΔU2/U3A transgene (Figure 6-9). Discussion This chapter outlines the unusual characteristics we have observed in the 425ΔU2/U3A BAC transgenic line. Unlike the 425ΔU2/U3E line, this line does not display imprinted 92

93 expression of the transgene; it is expressed in P1 brains when inherited both maternally and paternally although the level of expression upon maternal transmission is fairly low relative to expression upon paternal transmission. The 425ΔU2/U3A transgene does however demonstrate the proper DNA methylation imprint at the PWS-IC, as it is unmethylated upon paternal transmission but hypermethylated upon maternal transmission. This is not the first example of an uncoupling of the methylation imprint from the expression imprint. In a study by Xin et al., Dnmt1 -/- ES cells were analyzed for Snrpn expression via RNA fluorescence in situ hybridization and for PWS-IC methylation by genomic bisulfite sequencing. This study demonstrated that even in the complete absence of DNA methylation at the PWS-IC, Snrpn still displayed monoallelic expression (Xin et al., 2003). Xin et al. also determined that the Dnmt1 -/- ES cells had maintained histone methylation at H3K9, a repressive epigenetic mark present at the PWS- IC specifically on the maternal allele. We would eventually like to analyze the histone modifications at the PWS-IC on the maternally transmitted 425ΔU2/U3A BAC to determine whether repressive histone modifications are present. We expect that because Snrpn is expressed from the maternally transmitted 425ΔU2/U3A transgene, the histone modifications at this locus would be transcriptionally permissive; however, DNA methylation at the maternal PWS-IC is dependent on G9a histone methyltransferase, a chromatin remodeling enzyme that methylates histone H3K9 and H3K27, both repressive epigenetic marks (Xin et al., 2003). In addition, Henckel et al. demonstrated a dependence of repressive histone modifications on DNA methylation at the PWS-IC by showing that Dnmt3l m-/- embryos (embryos from homozygous mutant Dnmt3l mothers) lacking DNA methylation at the maternal Snrpn DMR also displayed reduced histone methylation in this region (Henckel et al., 2009). Due to the high copy number of the 425ΔU2/U3A transgene, it is possible that the factors necessary for maintaining Snrpn 93

94 silencing become exhausted and allow for a low level of Snrpn expression from the maternally inherited transgene. Another difference observed between the 425ΔU2/U3E line and the 425ΔU2/U3A line was the expression of Snrpn upstream exon U1 in the brain. The imprinted 425ΔU2/U3E transgene lacked upstream exon expression in the brain but the 425ΔU2/U3A transgene displayed high levels of upstream exon expression in P1 brain upon paternal transmission and low levels upon maternal transmission. The U1 expression patterns displayed by the 425ΔU2/U3A BAC in the brain mimic those of the imprinted 425Δ5-7A transgene. If the 425ΔU2/U3E transgene is intact, then the variation in upstream exon expression between the two 425ΔU2/U3 lines is likely due to differences in copy number as the A line is high copy and the E line is single copy. With the removal of 5 kb of sequence for the deletion of U2 and U3, the 425ΔU2/U3 transgene itself may not have all the necessary elements for Snrpn upstream exon U1 expression in the brain; however in multi-copy, it may be able to overcome this deficiency thereby allowing for U1 expression. Finally, the most peculiar phenotype demonstrated by the 425ΔU2/U3A transgene was the aberrant effect on gene regulation at the endogenous locus. A summary of these effects on the PWS/AS locus is outlined in Table 1. We discovered that the paternally transmitted transgene silenced endogenous Snrpn but the maternally transmitted transgene had no observable effect on Snrpn expression. This paternally transmitted transgene also had a repressive effect on Ube3aats expression but not on any other gene that we examined within the PWS/AS domain. One possibility as to why only Snrpn and Ube3a-ats are down-regulated by this paternally transmitted transgene could be due to the presence of different splice variants of the Snrpn transcript. The 425ΔU2/U3A transgene may have a direct impact on one splice variant, one containing Snrpn 94

95 and Ube3a-ats, but not affect others. The most widely accepted model for expression of the downstream cluster genes is that they are transcribed as a single long transcript, originating at the Snrpn promoter, from which multiple paternal gene products are spliced (Landers et al., 2004). However, a recent study by Vitali et al. provides evidence suggesting that the MBII-52 and MBII-85 snornas are produced from at least two independent transcription units (Vitali et al.). Thus, it is possible that the paternally transmitted 425ΔU2/U3A transgene represses only transcriptional units producing the Snrpn and Ube3a-ats transcripts. In contrast to the phenotype of the paternally transmitted 425ΔU2/U3A transgene, the maternally transmitted transgene had a partial penetrance in silencing the snornas. Drawing any conclusions from these experiments proved difficult as consistent results were never obtained. Some transgenic brain samples displayed repression of only MBII-85 and not MBII- 52, others showed repression of both, and the majority of samples demonstrated no downregulation of the snornas. We were able to provide strong evidence suggesting that the 425ΔU2/U3A transgenemediated silencing was independent of imprinting. The upstream cluster genes were not repressed in any of the 425ΔU2/U3A transgenics and this transgene was also not able to silence the 380A transgene, which contains a PWS-IC. These results demonstrated that the transgene was not acting via repression of the PWS-IC, whether endogenous or exogenous. In addition, we showed that the paternally transmitted 425ΔU2/U3A transgene silenced the endogenous Snrpn in 14.5 dpc fetal gonads. Imprints are erased in germ cells by 13.5 dpc thus these data further demonstrate that the Snrpn repression is independent of imprinting. Moreover, we were able to show that silencing of the endogenous Snrpn was not correlated with hypermethylation of the 95

96 paternal Snrpn DMR, as the endogenous locus showed no increase in methylation in our bisulfite sequence analysis of paternally inherited 425ΔU2/U3A transgenic brains. We were also able to determine that the 425ΔU2/U3A transgene was not creating a paramutation-like effect. This was demonstrated in our analysis of non-transgenic littermate samples that came from a transgenic father, none of which showed silencing of the endogenous Snrpn. Additionally, non-transgenic brain samples from subsequent generations did not display endogenous Snrpn repression, ruling out paramutation. This is not the first example of a high-copy number transgene affecting the endogenous locus. Hatada et al. produced mice transgenic for the imprinted U2af1-rs1 gene, which is typically methylated and silenced on the maternal allele and unmethylated and expressed from the paternal allele. They analyzed two high-copy number lines and found that a portion of the offspring bearing a paternally transmitted transgene displayed aberrant methylation of the endogenous paternal U2af1-rs1 allele (Hatada et al., 1997). Unexpectedly, they discovered that a fraction of the non-transgenic littermates also demonstrated this methylation at the endogenous paternal allele. In addition to being methylated, the paternal U2af1-rs1 was also silenced. This phenotype was not a result of paramutation as the non-transgenic mice bearing aberrant methylation of the paternal U2af1-rs1 did not have offspring possessing a methylated paternal allele when crossed to wild type females. The authors hypothesized that a trans-acting factor interacts with U2af1-rs1 in the testis to prevent methylation of the paternal allele. In the presence of the high-copy transgene, this factor is titrated, allowing for methylation of a fraction of the paternal alleles. While the results of this study are not the same as our observations with the 425ΔU2/U3A transgenic line, they still demonstrate that a high-copy number transgene has the ability to interfere with regulation of the endogenous locus. 96

97 Figure 6-1. Snrpn expression from the 425ΔU2/U3A transgene. Experiments were performed on P1 whole brains. A) RT-PCR for Snrpn exons four to eight. The endogenous Snrpn amplicon is 513 bps while the transgene product is 350 bps. Two transgenic brains from both maternal and paternal transmission were analyzed. Non-transgenic littermate brains were used as controls. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. B) Northern blot analysis was performed for Snrpn expression as well as the upstream cluster gene Ndn. The 425ΔU2/U3A line was compared to the 425Δ5-7A line. A Β-Actin probe was used as a loading control. 97

98 Figure 6-2. Bisulfite sequence analysis of the 425ΔU2/U3A transgene. A portion of the Snrpn DMR containing 14 CpG dinucleotides was analyzed. Each CpG is represented by a circle. Methylated CpGs are displayed as black circles and unmethylated CpGs are shown as white circles. Each row represents an individually sequenced clone. A) Transgenic alleles: the majority of the maternally transmitted alleles are methylated while all of the paternally transmitted alleles are unmethylated. B) The endogenous alleles represent both the maternal and paternal DMRs. 98

99 Figure 6-3. Snrpn upstream exon usage from the 425ΔU2/U3A transgene. RT-PCR was performed on newborn brain RNA to analyze Snrpn upstream exon U1 expression from the 425ΔU2/U3A transgene. The products were Southern blotted with a probe for Snrpn upstream exon U1 to exon three to intensify the signal. Two transgenic brains from both maternal and paternal transmission were analyzed and nontransgenic littermate brain cdnas were used as controls. RT samples were included to control for the presence of genomic DNA contamination. Hprt RT-PCR was performed on these cdnas to confirm sample quality and is shown in Figure 6-1A. 99

100 Figure 6-4. Snrpn expression analysis with the 425U2/U3A and 380A paternally transmitted transgenes. Snrpn RT-PCR was performed on newborn whole brain cdnas bearing both the 425U2/U3A and 380A paternally transmitted transgenes. Controls include littermate brains bearing either the 425ΔU2/U3A or 380A transgene alone as well as non-transgenic littermate samples. The endogenous Snrpn and 380A Snrpn amplicons are 513 bps while the 425ΔU2/U3A transgene product is 350 bps. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT- PCR was run to test cdna quality. 100

101 Figure 6-5. Endogenous Snprn repression in fetal gonads upon paternal transmission of the 425ΔU2/U3A transgene. We perfored RT-PCR for Snrpn expression using primers spanning exons four to eight on 14.5 dpc fetal gonads. Gonads were of the following genotypes: WT: wild type, -/Tg: paternally inherited 425ΔU2/U3A transgene, ΔIC/+: maternally inherited PWS-IC deletion, ΔIC/+;-/Tg: maternally inherited PWS-IC deletion and paternally inherited 425ΔU2/U3A transgene. The endogenous Snrpn amplicon is 513 bps while the transgene product is 350 bps. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT- PCR was run to test cdna quality. 101

102 Figure 6-6. Upstream cluster gene expression in the 425ΔU2/U3A transgenic line. RT-PCR analysis was performed on transgenic P1 brain samples to examine Frat3, Magel2, and Mkrn3 expression. Two transgenic brains from both maternal and paternal transmission were analyzed and non-transgenic littermate brains were used as controls. Hprt RT-PCR was run to test cdna quality. 102

103 Figure 6-7. Effects on Ube3a-ats and Ube3a expression in the 425ΔU2/U3A transgenic line. RT-PCR analysis was performed on transgenic P1 brain samples to examine Ube3aats and Ube3a expression. A) Ube3a-ats expression: two transgenic brains from both maternal and paternal transmission were analyzed and non-transgenic littermate brains were used as controls. RT samples were included to control for the presence of genomic DNA contamination and Hprt RT-PCR was run to test cdna quality. B) Allele-specific Ube3a expression analysis. Two P1 brains with a paternally inherited 425ΔU2/U3A transgene were analyzed for increased paternal:maternal Ube3a expression as compared to wild type samples. RT-PCR followed by a Tsp509I restriction digest was performed to distinguish between expression from the maternal and paternal alleles. The SNP between the paternal domesticus allele and the maternal castaneous allele is contained within a Tsp509I site that is eliminated in the castaneous strain (shown in the schematic diagram to the right of the gel). The maternal Ube3a is represented by a 225 bp amplicon and the paternal Ube3a is represented by 138 and 87 bp amplicons. 103

104 Figure 6-8. SnoRNA expression in the 425ΔU2/U3A transgenic line. Northern blots were performed on P1 whole brain RNA and probed with oligos for MBII-85 and MBII s rrna probe was utilized as a loading control. A) Comparison of snorna expression between the 425Δ5-7A and 425ΔU2/U3A transgenic lines. Two wild type brains (-/-) were run as controls. B) SnoRNA expression was analyzed in a panel of maternally transmitted 425ΔU2/U3A transgenic brain samples. Two brains bearing a paternally transmitted PWS-IC deletion and the maternally transmitted 425ΔU2/U3A transgene (Tg/-; +/ΔIC) were assayed to determine expression levels from the transgene and two wild type brains (WT) were run as controls. 104

105 Figure 6-9. SnoRNA expression analysis with the 425ΔU2/U3A and 380A maternally transmitted transgenes. Northern blots were performed on P1 whole brain RNA and probed with oligos for MBII-85 and MBII s rrna probe was utilized as a loading control. Samples included: a wild type brain (WT), brains bearing either the maternally transmitted 380A or 425ΔU2/U3A transgene ((380/+) or (425/+)), each of these transgenes maternally transmitted with a paternally transmitted PWS-IC deletion ((380/ΔIC) or (425/ΔIC)), and both of the transgenes maternally transmitted with a paternal PWS-IC deletion allele (380,425/ΔIC). 105

106 Table 6-1. Gene regulation at the PWS/AS locus by the 425ΔU2/U3A transgene Gene Maternal transmission Paternal transmission Ndn No observable effect No observable effect Frat3 No observable effect No observable effect Magel2 No observable effect No observable effect Mkrn3 No observable effect No observable effect Snrpn No observable effect Repressed MBII-85 Variable No observable effect MBII-52 Variable No observable effect Ube3a-ats No observable effect Repressed Ube3a TBD No observable effect 106

107 CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS Maternal imprinting mechanisms have eluded investigators for decades, despite intensive efforts and abundant resources. Mouse models have proven an invaluable asset for studying genomic imprinting mechanisms, especially for imprinted domains that are highly conserved between human and mouse as is the PWS/AS locus. However, the use of mouse models to study mechanisms of gene regulation at the PWS/AS locus has been frustrated due to the lack of an identifiable murine AS-IC. This study has contributed to the understanding of maternal imprinting at the PWS/AS locus by identifying a murine AS-IC and examining its function. Based on the results of our studies, we have proposed a working model for imprint establishment at the PWS-IC that is focused on transcription of the Snrpn upstream exons. Central to our model is the timing of two events in germline development: i) imprint erasure in PGCs between dpc and ii) re-establishment of imprints in the postnatal maternal germline during the oocyte growth phase (Sasaki and Matsui, 2008). Our model is as follows: the Snrpn upstream exons are silent after imprint erasure in PGCs; however, just prior to the establishment of imprinting in the maternal germline, the upstream exons are activated and transcription from these exons through the PWS-IC commences. Transcription from the upstream exons through the PWS-IC in the growing oocytes promotes epigenetic modification of this element. The repressive epigenetic modifications are retained throughout mitosis and the PWS-IC remains inactivated on the maternal allele, thereby silencing the paternally expressed genes that reside within the PWS/AS locus (Figure 7-1). While we have shown that the Snrpn upstream exons act as a functional AS-IC on the 425D18 BAC, we still have much to learn about their role in imprinting. Our experiments suggest that the upstream exons are necessary for maternal imprinting at the PWS-IC but they 107

108 have not fully revealed the mechanism of action that these exons employ. Future experiments are critical to determine whether it is the upstream exon sequence or their promoter function that is necessary to imprint the PWS-IC. Experiments that will differentiate between the two mechanisms are already well underway in our lab. To determine whether transcription through the PWS-IC during oogenesis is sufficient to establish an imprint at the PWS-IC in the absence of any upstream exon sequence, we made a modified BAC transgene, replacing the Snrpn upstream exons with an oocyte-specific promoter (Figure 7-2). This construct is based on the 380J10 BAC, the BAC that contains a complete PWS-IC but no Snrpn upstream exons and is not imprinted in several independent transgenic lines (Chamberlain, 2003). To generate our desired construct, we first utilized BAC recombineering to make a deletion between Snrpn exons five to seven, the same deletion as was made for each of the 425 BAC transgenes, so that we could easily distinguish between expression from the transgene and the endogenous locus. We then placed 0.4 kb of the oocyte-specific Gdf9 promoter at the 5 end of this BAC. A study by Yan et al. demonstrated that this 0.4 kb fragment of the Gdf9 promoter was sufficient for robust oocytespecific transgene expression throughout the critical stage of oocyte growth thus representing a seemingly ideal promoter for our artificial system (Yan et al., 2006). We flanked the Gdf9 promoter fragment with LoxFAS sequences so that the promoter could be removed to create an isogenic control transgenic line. This construct has been recombineered and awaits injection. Once lines are established, we can determine whether transcription from the Gdf9 promoter through the PWS-IC during oogenesis is sufficient to imprint the transgene. This result would indicate that it is not the Snrpn upstream exon sequence that is critical for establishing the maternal imprint but rather their promoter function. 108

109 We have designed an additional construct to differentiate between imprinting mechanisms involving upstream exon-directed transcription and upstream exon sequence. This construct leaves the Snrpn upstream exons intact but disrupts transcription between these upstream exons and the PWS-IC. Instead of making use of BAC transgenes for this experiment, we altered our approach and designed a targeting construct for homologous recombination at the endogenous locus (Figure 7-3). For the targeting vector, we made use of a 1.2 kb region of the rabbit β- globin gene, a proven RNA polymerase II transcriptional terminator (Sleutels et al., 2002). We floxed this terminator sequence with LoxFAS sites and inserted it into a SalI restriction site that is approximately 13 kb upstream of Snrpn exon one, the same site that Wu et al. utilized for their IC an insertion/duplication mouse that displayed a loss of Snrpn imprinting (Wu et al., 2006). As our hypothesis proposes that it is the transcription of the Snrpn upstream exons through the PWS-IC during oogenesis that establishes the maternal imprint, we expect that the presence of the terminator will cause a loss of imprinting at the PWS-IC upon maternal transmission. This result would indicate it is the promoter function of the upstream exons, not their sequence, that directs maternal imprinting. There are several potential mechanisms by which transcription from the upstream exons could lead to epigenetic modification of the PWS-IC. These silencing mechanisms can be divided into two categories: i) those that require the RNA transcript or ii) those requiring active transcription through the PWS-IC. One potential mechanism, which would fall into the second category, is that transcription through the PWS-IC during oogenesis produces an open chromatin conformation, allowing access for the DNA methylation complexes as well as other silencing complexes. Alternatively, the transcript itself may act to recruit chromatin-modifying complexes to the PWS-IC. A compelling possibility is that the transcript may function in a similar manner 109

110 to Xist, the long noncoding RNA (lncrna) that is required for X chromosome inactivation (XCI). Xist is transcribed specifically from the future inactive X chromosome (X i ) at the onset of XCI. This transcript accumulates along the chromosome in cis to initiate gene silencing by targeting repressive chromatin modifications to the locus (Clemson et al., 1996; Kohlmaier et al., 2004). The exact mechanism by which the Xist transcript recruits the chromatin-modifying complexes to the future X i remains unclear. LncRNAs have become a major focus in the study of imprinting mechanisms and recent investigations are providing clues about potential Xist-like silencing mechanisms. Two lncrnas, Air and Kcnq1ot1, are required for the epigenetic repression of multiple genes in cis at their respective imprinted domains (Umlauf et al., 2008). Several reports provide evidence suggesting that these lncrnas function via a RNA-directed targeting mechanism in which they localize to chromatin and recruit histone methyltransferases and other chromatin modifying complexes to imprinted loci in cis. Both the Air and Kcnq1ot1 lncrnas are paternally expressed and function to silence paternally imprinted genes in the placenta. Upon disruption of transcription of Air or Kcnq1ot1, the imprinted gene clusters that these lncrnas regulate become biallelically expressed (Fitzpatrick et al., 2002; Mancini-Dinardo et al., 2006; Shin et al., 2008; Sleutels et al., 2002; Wutz et al., 2001; Zwart et al., 2001). Independent reports have shown that both the Air and Kcnq1ot1 transcripts accumulate at promoter regions of their target genes (Nagano et al., 2008; Pandey et al., 2008). The same studies demonstrated that these lncrnas co-immunoprecipitate with the histone methyltransferase G9a, which methylates histone H3K9 and H3K27. These repressive epigenetic marks are enriched on the paternal allele at promoter regions of Air and Kcnq1ot1 target genes. In addition, Kcnq1ot1 was found to coimmunoprecipitate with members of the polycomb repressive complex 2 (PRC2), a complex that 110

111 initiates epigenetic gene silencing. Taken together, the results of these studies suggest an imprinting mechanism involving RNA-directed targeting that requires the lncrna transcript. This mechanism could be applied to the lncrnas transcribed from the alternative Snrpn upstream exons at the PWS/AS locus in the growing oocytes. Alternatively, we cannot rule out a maternal imprinting mechanism requiring active transcription through the PWS-IC. In yeast, the process of transcriptional elongation was shown to generate repressive histone modifications within the transcript body so as to prevent spurious transcription initiation (Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh et al., 2005). This could be translated to a mechanism of regulation at the PWS/AS locus wherein the active transcription from the Snrpn upstream exons through the PWS-IC during oogenesis establishes the repressive chromatin structure at this regulatory element. Future experiments based on the information presented here will lead to an even greater understanding of maternal imprinting mechanisms at the PWS/AS locus. These mechanisms may be applicable to other imprinted domains as well, generating a better comprehension of the intricacies of genomic imprinting. With the knowledge gained through these studies, effective treatments can be designed for patients suffering from PWS, AS, and a wide range of other imprinting disorders. 111

112 Figure 7-1. A working model for imprint establishment at the PWS-IC. Top: schematic diagram of the paternal PWS/AS locus. Arrows indicate the direction of transcription. The PWS-IC is represented by an orange pentagon. Below the locus is a magnified view of the region surrounding Snrpn over the course of development. After imprints are erased in fetal germ cells, the Snrpn upstream exons are silent on both alleles. In growing oocytes, transcription is initiated from the upstream exons, corresponding with the timing of imprint establishment in the maternal germline. This transcription proceeds through the PWS-IC, promoting the epigenetic modification of this element (black lollipops represent methylated CpG dinucleotides). The PWS-IC on the future maternal allele is thereby inactivated, rendering the paternally expressed genes silent. 112

113 Figure 7-2. Structure of the GDF9 flox 380 BAC transgene. Top: the endogenous locus is depicted with the 380J10 BAC underneath to show the boundaries of the BAC sequence in relation to Snrpn. Bottom: the 380J10 BAC was modified via BAC recombination to possess the deletion between Snrpn exons five to seven. To generate the GDF9 flox 380 BAC, we inserted the Gdf9 promoter (purple box) at the 5 boundary of the 380J10 BAC. We flanked this promoter sequence with LoxFAS sites (green triangles). 113

114 Figure 7-3. Schematic diagram of the transcriptional terminator allele. The transcriptional terminator (TT) will be inserted approximately 13 kb upstream of Snrpn exon one, 3 to the alternative upstream exons, and flanked by LoxFAS sites (green triangles). 114