Chromosome-wide Analysis of Parental Allele-specific. Chromatin and DNA Methylation

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1 MCB Accepts, published online ahead of print on 1 February 0 Mol. Cell. Biol. doi:./mcb.001- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Chromosome-wide Analysis of Parental Allele-specific Chromatin and DNA Methylation Purnima Singh 1, Xiwei Wu, Dong-Hoon Lee 1, Arthur X. Li, Tibor A. Rauch,&, Gerd P. Pfeifer, Jeffrey R. Mann,* and Piroska E. Szabó 1$ 1 Department of Molecular and Cellular Biology, Information Sciences, Cancer Biology, and Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, USA $Author for correspondence: Tel: -01- Fax: pszabo@coh.org $ Present address: Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois * Present address: Murdoch Children s Research Institute, The Royal Children s Hospital, Parkville, Victoria, and Department of Zoology, The University of Melbourne, Victoria, Australia. 1 Running title: Chromosome wide allele-specific chromatin analysis Key words: Epigenetics/chromatin/imprinting/ChIP on chip Characters:, excluding Methods and References - 1 -

2 ABSTRACT To reveal the extent of domain-wide epigenetic features at imprinted gene clusters we performed a high-resolution allele-specific chromatin analysis of over 0 megabases of maternally or paternally duplicated distal chromosomes and 1 in MEFs. We found that reciprocal allelespecific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of HKme (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H1/Igf domain. A paternal allele-specific gap was found along Kcnq1ot1 interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allelespecific chromatin marks at the Peg1 and Slca DMRs, Cdkn1c upstream region and at the Inpp_v DMR and paternal allele-specific CTCF binding at the Peg1 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor HKac and [CTCF or HKme] in one allele and HKme in the reciprocal allele, using a sliding window approach, recognized with precision, the parental allelespecificity of known imprinted genes, H1, Igf, Igfas, Cdkn1c, Kcnq1ot1, Inppf_v on Chr and Peg1 and Slca on Chr1. Chromatin features, therefore, can unequivocally identify genes with imprinted expression. - -

3 INTRODUCTION Imprinted genes are monoallelically expressed according to parental inheritance. The maternally and paternally inherited alleles are distinguished epigenetically by DNA methylation and histone modifications. Parental allele-specific DNA methylation at germ line DMRs is established in the male and female germ lines. The hyper- and hypomethylated alleles of DMRs largely coincide with repressive and active histone covalent modifications, respectively. DNA methylation (,,,, ) and chromatin differences (,, 1,,, 0) are important for parental allelespecific gene expression. Chromatin is usually probed with allele-specific ChIP assays at discrete genomic positions at the transcription start sites of imprinted genes or at DMRs. The extent of the allele-specific chromatin at imprinted domains is not known except for the Igfr/Airn imprinted locus on Chr1, where the two alleles were investigated using ChIP on chip methodology in hemizygous mouse embryo fibroblasts (MEFs) carrying the 0 kb T hp deletion in one allele (). To assess the chromatin of the parental alleles separately we used mouse embryo fibroblasts (MEFs) that carried maternal and paternal duplication of distal Chr, MatDup.dist and PatDup.dist (,,, ) respectively (Figure 1B and C). These resulted from the intercrosses of mice heterozygous for a reciprocal T(;1)H translocation at the TH breakpoint (). MatDup.dist MEFs carry maternal duplication and paternal deficiency for the translocated distal Chr, as well as paternal duplication and maternal deficiency of distal Chr1 (1, 1). PatDup.dist MEFs carry paternal duplication and maternal deficiency for the distal Chr regions, as well as maternal duplication and paternal deficiency of distal Chr1. The MatDup.dist and PatDup.dist genotypes are associated with fetal or embryonic lethality, respectively (,, 1, ) due to misexpression of imprinted genes. To obtain MEFs at 1. dpc, the PatDup.dist embryos had been rescued by an Ascl transgene (, 1). MEFs provided sufficient number of cells for ChIP on chip experiments. - -

4 The duplicated chromosome segments in MatDup.dist and PatDup.dist MEFs harbor five germ line DMRs (Figure 1A-C): the paternally methylated H1/Igf ICR (,,, ); the maternally methylated KvDMR1 (1,, ); and maternally methylated Inppf_v DMR () in distal Chr; and the maternally methylated Slca; and Peg1 DMRs in distal Chr1 (, ). The distal portion of mouse chromosome contains two imprinted domains representing two major mechanisms known to regulate imprinted gene expression (Figure 1D). The H1-Ig region is controlled by allele-specific enhancer blocking due to CTCF insulator protein binding in the imprinting control region (ICR) (,,,,,, 0, 1, ). The unmethylated maternal DMR allele elicits insulation between the H1 and the Igf reciprocally expressed imprinted genes, with the H1 gene expressed exclusively from the maternal chromosome (, 1, ) and Igf from the paternal chromosome (1). The Igfas non-coding RNA is also likely regulated similarly (). Ins is paternally expressed, but only in the yolk sac (). The Cdkn1c/Kcnq1 domain (Figure 1D) is controlled by the maternally methylated ICR, the Kv differentially methylated region (KvDMR1) (1,, ). The unmethylated paternal allele produces a noncoding RNA, Kcnq1ot1 (). Transcription of this RNA is required for repressing the paternal allele of an array of maternally expressed imprinted genes in the placenta (,, ). The duplicated chromosomes in MatDup.dist and PatDup.dist MEFs additionally harbor the paternally expressed Ampd (), Inppf_v, and Inppf_v (1, ) on Chr and the maternally expressed Kcnk (), Trappc (1) and paternally expressed Peg1 (), and Slca (, ) imprinted genes on distal Chr1. The parental-allele-specific imprinted expression of the H1, Igf, Cdkn1c, Kcnq1ot1, Peg1 and Slca transcripts is ubiquitous. Phlda is maternally biased in most fetal organs (). Other imprinted genes exhibit parental-allele-specific expression in specific tissues: Ins in the yolk sac (), Inpp_v (1) and Trappc in the brain (1), Tspan, Cd1, Tssc, Nap1l, Tnfrsf, Osbpl and Dhcr in the placenta (, 1, 1, 1,,,, ), and are either not expressed or biallelically expressed in other organs ( (). Allele - -

5 specific gene expression can also be developmentally regulated: Kcnq1 is maternally expressed in the embryo at. dpc () but becomes biallelic at later fetal stages in a tissue-specific manner (,, 1) and is silent later in development (). Slca1 is maternally biased in the embryo and fetus but not in the adult (1). We performed ChIP-on-chip and MIRA-on-chip analyses in combination with hybridization to Nimblegen tiling arrays to map the chromatin features and DNA methylation status along the duplicated chromosomal segments and provide a panoramic map of the allelespecific epigenetic features, chromatin and DNA methylation, at the major imprinted domains and scattered imprinted genes on mouse chromosomes and

6 MATERIALS AND METHODS MatDup.dist and PatDup.dist MEFs MEFs were derived from 1. dpc embryos. PatDup.dist embryos that would otherwise die at. dpc were rescued for this purpose with an Ascl transgene (). The injected P1 clone spanned kilobases. Its location is shown in Figure Chromatin Immunoprecipitation Chromatin was prepared from MatDup.dist and PatDup.dist primary MEFs (1) as described earlier (0). The chromatin was crosslinked for min (N-ChIP) or min (X-ChIP) with formaldehyde and sonicated in lysis buffer (). An aliquot of the chromatin was reversecrosslinked, quantified by OD measurement and the efficiency of sonication was assessed on agarose gel. Sonicated chromatin was then diluted to 0. mg/ml concentration and snap-frozen in small aliquots. One aliquot was thawed on the day of ChIP. The chromatin immunoprecipitation was performed as described previously (0) with minor modifications. Pre-blocked A/G beads from Santa Cruz (Cat#sc-00) were used for capturing the precipitated chromatin. The antibodies used in the ChIP assays are listed in Table S MIRA The methylated fraction of sonicated genomic DNA was captured using recombinant MBDL1 and MBDb proteins as described earlier (, ). LM-PCR Amplification LM-PCR was done to amplify ChIP- and MIRA- enriched DNA as previously described (1) with minor modifications. - -

7 ChIP-on-chip Custom-designed NimbleGen tiling arrays covering the distal arm of mouse chromosome (00_Szabó_CoH_mm_chr_chip), central chromosome (C _MMTiling Set 1) and distal chromosome 1 (C-1-01_MM Tiling Set 1) were used for the histone modification profile analysis. The array for distal Chr covers the region,000,00-1,1,0, and for distal Chr1:,0,000-,0,000. Amplified ChIP DNA fractions were compared with amplified input DNA. The labeling of DNA, microarray hybridization, and scanning were performed by the NimbleGen Service Group (Reykjavik, Iceland). Data were extracted from scanned images by using NimbleScan. extraction software (NimbleGen Systems) RNA isolation and cdna preparation RNA was isolated from MatDup.dist and PatDup.dist MEFs using RNA-Bee according to manufacturer s instructions (Tel-Test). The pellet was dissolved in DEPC water containing RNasin (Promega) and mm DTT. Contaminating DNA was removed with the DNA-free Kit (Ambion). ds cdna was prepared from µg of MatDup.dist and PatDup.dist MEF total RNA with oligo dt primers using SuperScript Double-Stranded cdna Synthesis Kit (Invitrogen) according to NimbleGen Arrays User s Guide ( The ds cdna was purified using QIAquick PCR Purification Kit (Qiagen) and was hybridized to custom Nimblegen Tiling Arrays (00_Szabó_CoH_mm_chr_chip) at City of Hope Functional Genomics core facility. Allele-specific peak identification We first performed quantile normalization on the distal Chr NimbleGen log ratio data. Next, peaks were defined in each sample as four consecutive probes with log ratio above th - -

8 percentiles on each array, allowing one probe gap. For each peak present in MatDup.dist samples, the median log ratios of probes falling into the peak region in the corresponding PatDup.dist sample were calculated and compared to the median log ratios of these probes in MatDup.dist samples. The peaks with signal differences of more than -fold between MatDupd.dist and PatDup.dist samples were designated as allele-specific peaks. - -

9 RESULTS Measuring allele-specific histone chromatin at a chromosome-wide scale To reveal the domain-wide allele-specific epigenetic features of imprinted genes with highresolution along Chr and Cr1, we used Matdup.dist and PatDup.dist MEFs (Figure 1 B and C). These cells allowed us to separately asses the maternal and paternal alleles along the duplicated chromosomal segments. We performed ChIP-on-chip and MIRA-on-chip analyses in combination with LM-PCR amplification and hybridization to Nimblegen tiling arrays to map the chromatin features and DNA methylation. Antibodies for well-known active (HKac, HKme, HKme) and repressive (HKme and HKme) histone tail modifications and also for histone globular domain marks (HK1ac, HKme and HKme) were used in ChIP to map chromatin. We recently showed that HK1ac and HKme marks are typically found in the unmethylated allele of DMRs whereas HKme is predominantly found in the methylated alleles (). CTCF ChIP was used to map insulators/chromatin barriers. Chromatin and methylation data were cross-referenced with gene expression patterns. Affymetrix RNA microarrays confirmed the allele-specific expression of known imprinted genes along the duplicated chromosome segments (Figure S1). ChIP with the elongation-type RNA polymerase II (PolII) antibody and double stranded (ds) cdna hybridization on the Nimblegen arrays was also used to visualize biallelic and allele-specific gene expression along the duplicated chromosome regions and potentially uncover novel unannotated ncrnas. 1 Analysis of the H1/Igf imprinted domain The reciprocal maternal and paternal allele-specific expression of H1 and Igf transcripts was confirmed in RNA microarray (Figure S1) and was visualized by ds cdna hybridization to custom Nimblegen arrays containing Mb region of distal chr. MatDup.dist MEFs exhibited - -

10 strong signal for H1 but no signal for Igf whereas PatDup.Dist. MEFs exhibited strong signal for Igf and Igfas but not for H1 (Figure ). No transcript signal was present for Ins. The PolII signal was very clear along the Igf transcript but was hard to detect along H1 (not shown). As expected, DNA methylation was found at the ICR in PatDup.dist but not in MatDup.dist MEFs and DNA methylation was biased toward the paternal allele at the Igf locus (1). The robust CTCF peak at the ICR in MatDup.dist MEFs was in agreement with our in vivo footprinting data obtained using the same cells (1) and ChIP analysis in normal MEFs (0, ). The MIRA peak coincided with strong HKme and HKme peaks at the ICR in PatDup.dist MEFs as in ChIP-SNuPE results using normal cells (). The CTCF peak coincided with HKac, HKme and HKme peaks. The previously reported maternal allele-specific CTCF peak in neonatal liver chromatin at the Igf DMR1 () was not apparent in MatDup.dist MEFs (Figure ), most likely due to cell type-specific differences in CTCF binding. This peak was absent in normal MEFs (0). The expressed allele of imprinted genes, i.e. the maternal allele for H1 and paternal allele for Igf and Igfas, was rich in HKac, HK1ac, HKme and HKme and was slightly enriched in HKme in MatDup.dist. and PatDup.dist MEFs, respectively. The silent alleles of imprinted genes, i.e. the paternal allele for H1 and maternal allele for Igf and Igfas, lacked all these active chromatin peaks but exhibited HKme marks in PatDup.dist and MatDup.dist MEFs, respectively. HKme showed maternal allele-specificity at the Igf promoter/gene body. HKme was biallelic in normal MEFs at the ICR (0, ) and in MatDup.dist-PatDup.dist MEFs (Figure ), except for one maternal-allele-specific peak at the telomeric end of the ICR. The ChIP-chip data were verified by real-time PCR at the H1/Igf ICR, the H1 promoter and the Igf DMR regions (Figure SA and B). When we looked at the panoramic picture of the domain, we observed a novel feature, a stretch of over 0 kilobase (kb) long maternally biased HKme enrichment encompassing the entire imprinted domain (Figure SB). Extended regions of HKme enrichment were identified by an algorithm, broad local enrichment (BLOC) in MEFs at imprinted and non- - -

11 imprinted regions (0, ) but allele-specific features of BLOCS have not been reported. We confirmed the HKme BLOC at two positions in the H1/Igf intergenic region (Figure SC). This marking excluded the active H1 promoter, confirming the data obtained in normal MEFs (Figure ) (0). Panoramic view of the region centromeric to H1 (Figure SA) shows that the maternally biased BLOC extends beyond the neighboring non-imprinted gene, Mrpl, and stops before Tnnt, but Mrpl gene itself is excluded Analysis of he Kcnq1/Cdkn1c imprinted domain We confirmed the allele-specific expression of Cdkn1c by RNA microarray (Figure S1). Additionally, the maternally expressed Cdkn1c and paternally expressed Kcnq1ot1 transcripts were visualized by ds cdna hybridization (Figure ). We found that the ds cdna occupies a window of about kb at the Kcnq1ot1 transcript. This is very close to the previously reported kb (), but was shorter than the recently reported 1 kb (). The longer transcript was mapped in 1. dpc placenta. It is possible that the different size Kcnq1ot1 transcripts have different regulatory potentials. Contrary to previous findings of monoallelic Kcnq1 expression in late mouse embryos (), we did not detect Kcnq1 expression in MEFs (Figure S1 and SE). The Ascl gene was not interpreted because an Ascl transgene was used to rescue the lethality of PatDup.dist MEFs and the hybridization signals may come from the P1 clone integrated elsewhere (). The other imprinted genes of this domain were not expressed in MatDup.dist and PatDup.dist MEFs (Th, Tspan, Kcnq1, Slca1), or were expressed equally in these cells (Cd1, Tssc, Phlda, Napl1, Tnfrs, Osbpl1 and Dhcr) (Figure SD-F). We verified the expression of selected genes using RT-PCR (Figure S). Cd1 and Osbpl were both highly expressed in MatDup.dist and PatDup.dist MEFs and in the embryo whereas Phlda and Slca1 expression was very low in MEFs and embryos compared to placentas. When allelespecificity of the low level Slca1 expression was examined in F1 embryos and placentas, a maternal allele-specific bias (1, ) was apparent. Osbpl1 was expressed maternal-allele- - -

12 specifically in the F1 placenta but biallelically in the F1 embryo. In our experience, very little RNA is sufficient to measure allele-specific gene expression. Mapping transcription by microarray-based approaches requires higher overall level of each particular transcript. The KvDMR1 displayed a strong peak of CpG methylation in the maternal allele (Figure ). This coincided with a strong HKme peak and strong HKme signals in MatDup.dist MEFs. HKme was present in both MatDup.dist and PatDup.dist MEFs. Active chromatin marks, HKac, HK1ac, HKme, HKme and HKme were strongly enriched at the KvDMR1 in PatDup.dist MEFs. Our results confirmed and expanded previous observations that maternal-allele-specific HK/1ac and HKme and paternal-allele-specific HKme and HKme mark the KvDMR1 (). Additionally, we revealed that HKme, HKme and HKme showed enrichment not only at the KvDMR1 but also over a larger region along the Kcnq1ot1 transcript in PatDup.dist MEFs. The histone globular domain marks were interesting: HKme and HKme were both enriched along the active allele of the Kcnq1ot1 transcript, but in MatDup.dist cells only HKme but not HKme existed along the silent maternal allele of this gene. We did not detect reproducible CTCF peak the KvDMR1, contrary to ChIP results in normal MEFs (). The difference can be due to different specificities of the antibodies. In the paper above a mixture of anti-ctcf antibodies was used. Alternatively, the amplification step in the ChIP-chip method may introduce a level of uncertainity at low occupancy sites (0). A HKme BLOC stretched along the Cdkn1c/Kcnq1 imprinted domain between Trpm and Phlda in both MatDup.dist and PatDup.dist cells (Figures SD and E). In PatDup.dist cells this stretch was interrupted by a long gap along the transcription of the Kcnq1ot1 non-coding RNA (Figure ). The extent of this paternal allele-specific gap in the HKme BLOC was verified by ChIP real-time PCR (Figure SA). HKme enrichment exhibited reciprocal pattern with gene expression along the domain, it biallelically covered the silent genes but was absent at the expressed genes along this domain. HKme repressive - 1 -

13 marks were biallelic at the Kcnq1 transcript, in agreement with the shift from monoallelic expression toward biallelic repression at 1. dpc (). MIRA peaks in PatDup.dist MEFs indicated paternally biased CpG methylation at the Cdkn1c promoter and upstream Cdkn1c DMR (Figure ) in accordance with the existence of a two paternally methylated somatic DMRs (,, ). The Cdkn1c promoter exhibits maternal allele-specific HK/1ac, HKme and paternal allele-specific HKme marks in the. dpc embryo chromatin (). We found that the Cdkn1c upstream region exhibited more robust chromatin differences than the promoter. These included maternal allele-specific HKac and HK1ac and paternal allele-specific HKme, HKme and HKme marks. We verified these peaks using ChIP-real time PCR (Figure SB). The repressive HKme signal covered both alleles of the Cdkn1c gene but the promoter was free of HKme in MatDup.dist MEFs, (Figure ). We found that CTCF binding was biased to the maternal-allele (Figure and SB) at a previously verified CTCF binding consensus binding site (0). Maternal bias of CTCF is likely due to partial paternal-allelic methylation of the region, also seen by others (). Those imprinted genes of the cluster that were expressed in both MatDup.dist and PatDup.dist MEFs, Cd1, Tssc Phlda, Nap1l, Tnfrs, Osbpl, and Dhcr showed biallelic active histone modification marks (Figure SD-F): MatDup.dist and PatDup.dist MEFs were equally enriched in HKac, HKme, HKme and HKme, whereas repressive marks were absent at these genes in both cell types. These results are in agreement with observations in. dpc embryo regarding the biallelic active HKac and HKme marks at the Osbl, Napl1, Phlda, Tssc and Cd1 genes (), but we did not confirm biallelic repressive marks, except biallelic HKme at Phlda. The difference was likely due to the difference in epigenetic status of these genes between 1. dpc MEFs and. dpc embryos. Tspan, which was not expressed in MEFs, exhibited biallelic active marks, HKac, HK1ac, HKme but not HKme and did not exhibit repressive marks. Slca1, which had very low level of expression in MEFs, exhibited allele-specific chromatin marks in MatDup.dist and PatDup.dist - 1 -

14 MEFs. The low level of expression was maternally biased in normal F1 embryos at 1. dpc (Fig. S). It is likely that the proximity to the ubiquitously maternally expressed Cdkn1c may influence the active chromatin marks at the maternal allele of this transcript in MatDup.dist MEFs. Th, which was not expressed in MatDup.dist and PatDup.dist MEFs, did not exhibit active chromatin marks in either cell type but was marked by repressive HKme in both MEFs. The long stretch of biallelic HKme along Th coincided with biallelic HKme and HKme enrichment. This has to be interpreted with caution, however, because the P1 clone included the first exon of Th and most of the very long first intron (). HKme was biallelic at Cd1 but coincided with biallelic HKme enrichment, not with HKme enrichment (Figure SD) Analysis of scattered imprinted genes along distal Chr Ampd is maternally expressed in the 1. dpc placenta but biallelic in the 1. dpc embryo (). We found a low level biallelic expression using ds cdna hybridization (Figure S). The chromatin composition was largely biallelic except for stronger HKme signals along the gene body and stronger HKme peak downstream of the TSS in MatDup.dist MEFs than in PatDup.dist MEFs. No significant repressive chromatin marks were present. Inppf_v transcript is paternally expressed in the brain (1), but is not expressed in the embryo. Allele-specific chromatin features have not been reported at this imprinted gene before. We did not find expression from the Inppf_v promoter in MEFs based on ds cdna hybridization (Figure ). DNA methylation, HKme, HKme and HKme enrichment, however, was apparent in the maternal allele in MatDup.dist MEFs whereas HKme, HKme and HKac enrichment marked the paternal allele in PatDup.dist MEFs (Figure ). The non-imprinted variant, Inppf was expressed in MatDup.dist and PatDup.dist MEFs equally as measured in RNA microarray and ds cdna hybridization. This variant exhibited biallelic active chromatin marks but lacked repressive marks. The biallelic peaks at the end and - 1 -

15 the strong biallelic ds cdna peaks at the end of the Inppf gene likely represent transcription from the Inppf promoter Global features along distal chromosome After analyzing known imprinted loci along distal Chr, we compared the global chromatin features along this Mb long chromosome segment. Unsupervised hierarchical clustering was used to reveal the degree of similarity between the ChIP-chip samples. Pearson correlation and average linkage analysis found that in general, the MatDup.dist and PatDup.dist MEF samples clustered together according to the epigenetic mark analyzed (Figure S), for example CTCF in MatDup.dist showed the closest relationship with CTCF in PatDup.dist. The exception to this rule was that the distribution of HK1ac and HKme samples clustered closer by cell type rather than by chromatin marks. Importantly, the repressive histone tail modifications HKme and HKme were clustered together and also with the globular domain mark HKme. Active histone tail marks clustered with PolII signals, and also with the globular mark HKme. HKme, HKme and HKme were significantly enriched at gene deserts, whereas CpG methylated regions were associated with gene-rich regions. To determine the pattern of each chromatin mark in relation to the transcription start site, we generated composite profiles (Figure S). Average signals (log ratios) of each chromatin mark were plotted between - kb and kb of the TSS for five equal sized groups of transcripts, classified according to their expression levels. The composite profile of each epigenetic mark was very similar between MatDup.dist and PatDup.dist MEFs (Figure SA and B) and was largely expected. One interesting finding was that whereas both HKme and HKme were present downstream of the TSS of highly expressed genes, HKme exhibited stronger enrichment. Also, interestingly, a peak of HKme but not HKme was present downstream of the TSS of genes displaying low expression or no expression

16 Generating an imprinted gene chromatin signature consensus algorithm The allele-specific chromatin differences between MatDup.dist versus PatDup.dist MEFs were very clear at each known imprinted gene that exhibited imprinted expression in the embryo. We decided to generate a consensus imprinted gene chromatin signature (Figure ) from the allelespecific epigenetic features at these transcripts and test whether this consensus can be used to identify imprinted genes along the duplicated chromosomal regions. The first step was to identify allele-specific (MatDup.dist- or PatDup.dist-specific) peaks (see Methods for details) for each modification mark that falls into the promoter region (defined as -kb to +kb of TSS) of known imprinted transcripts, H1, Igf, Cdkn1c and Kcnq1ot1, on distal Chr (Table S). The second step was to summarize the allele specific peaks as binary indicators (Figure A). The examined modification marks were divided into activation marks (CTCF, HKme, HKme, HKac, HK1ac, HKme, and PolII), and inhibition marks (HKme, HKme, HKme, and DNA methylation). The binary indicator was defined as follows: for the H1 and Cdkn1c transcripts, 1 or 0 in the activation mark category codes true or false for the following statement: MatDup.dist-specific peak exists in the promoter; 1 or 0 in the inhibition mark category codes true or false for the following statement: PatDup.dist-specific peak exists in the promoter. For the Igf and, Kcnq1ot1 transcripts the reciprocal parameters were applied. Based on the modification table, an imprinted gene chromatin signature algorithm was derived. It is a binary predictor: genes that possess chromatin composition containing activation mark HKac and [activation mark CTCF or HKme] and inhibition mark HKme in their promoter region are imprinted, and expressed from the allele associated with the activation marks. Transcript-based prediction The performance of the consensus imprinted gene chromatin signature was tested along the entire duplicated segment of the genome in MatDup.dist and PatDup.dist MEFs. For this analysis we generated additional ChIP on chip data along distal and central Chr and along distal - 1 -

17 Chr1. Each gene in a given genomic segment was considered and its imprinted status in either allele was tested. The results are shown in a heat map (Figure S). Out of genes between,000,00 and 1,1,0 along distal Chr the five known imprinted genes H1, Igf, Igfas, Cdkn1c, and Kcnq1ot1 were predicted (Figure B). In the region between. Mb and Mb on central Chr, genes were tested, and none was recognized as being imprinted. Along distal Chr1, 0 genes were tested and two genes, the paternally expressed Peg1 and Slca were recognized by the algorithm (Figure B). Apart from the four imprinted genes that were considered in deriving the algorithm we predicted additional three imprinted genes in Chr and Chr1 using the algorithm, all with correct allele-specificity. The predictive peaks are provided in Table S Sliding window approach In the previous prediction analysis the detection was limited to annotated genes. We then also performed an unbiased analysis along the entire central and distal Chr and distal Chr1 regions using the algorithm and the sliding window approach. The bimodal predictor criteria were applied to each -kb window tiled on the arrays with 1 kb step size. The hypothesis was that a window containing HKac and [CTCF or HKme] in one allele and HKme in the other allele will be associated with the promoter region of an imprinted gene. We identified 0 such windows at 1 locations and called transcripts in a distance equal or less than kb. 1 out of 1 windows were associated with known transcripts, and were located upstream or within but not downstream of a transcript (Table S). The sliding window approach recognized and correctly predicted the parental expression of each transcript predicted with the gene-based approach, and additionally called the TSS of the known paternally expressed transcript variant, Inppf_v within Inppf (Figure ). Inppf_v was not recognized by the transcript-based approach, because it is not annotated in RefSeq. The windows reported within Trappc were located at the TSS of - 1 -

18 Peg1, in a Trappc intron on Ch1. No novel imprinted genes were found on distal Chr and Chr Allele-specific chromatin at imprinted genes on distal chromosome 1 Allele-specific chromatin features along distal Chr1 have not been reported. We characterized the allele-specific chromatin features of the Peg1 and Slca DMRs (). At both of these DMRs CpG methylation and HKme enrichment was observed in the maternal allele, displayed in PatDup.dist cells whereas HKac and HKme enrichment was found in the paternal allele, displayed in MatDup.dist cells (Figure ). The Peg1 DMR also had a very clear CTCF peak in the paternal allele. Kcnk is maternally expressed in. dpc embryos and in adult brain () and Trappc exhibits imprinted expression in the neonatal brain (1). Kcnk and Trappc were not differentially expressed in MatDup.dist versus PatDup.dist MEFs, and this was in agreement with the absence of allele-specific chromatin pattern

19 DISCUSSION In this study we provide a panoramic map of the allele-specific epigenetic features along distal chromosomes and 1 with high resolution. This is the first study to systematically analyze an over 0 megabase long genomic region in an allele-specific fashion, providing a unique opportunity to determine the extent of allele-specific marking at individual imprinted genes and the extent of imprinted domains. This analysis also allows us to experimentally derive a chromatin-based imprinted gene signature algorithm Reciprocal allele-specific chromatin marks are focused at DMRs and imprinted genes In MatDupdist-PatDup.dist MEFs, five known germ line DMRs, the paternally methylated H1/Igf ICR, and the maternally methylated KvDMR1, Slca, Peg1 and Inppf_v DMRs can be investigated along the duplicated part of the genome (Figure 1). We found that each of these DMRs exhibited reciprocal allele-specific focal chromatin marks, HKme peak at the methylated allele and HKac and at least one more active mark, HKm and/or CTCF insulator peak was present in the unmethylated allele. Allele-specific chromatin was present at genes that were expressed differentially in MatDup.dist versus PatDup.dist MEFs. Promoters of genes exhibiting imprinted expression were always allele-specifically marked by HKac, HKme and HKme and sometimes also by HK1ac and HKme. Marking of imprinted domains by histone acetylation showed differences between the globular domain residue HK1ac and the tail domain residue HKac. HKac enrichment was more focused at the TSS similarly to HKme whereas HK1ac was more spread out along the transcript, closely mimicking HKme, supporting the idea that acetylation at different lysine residues may have different meaning for imprinted gene expression (). The silent promoter allele always contained a distinct HKme peak and sometimes an HKme peak. This result is similar to recent allele-specific chromatin mapping results along the 0 kb T hp deletion on mouse chromosome 1 that shows sharp active and repressive - 1 -

20 chromatin marks in a mutually exclusive fashion in the opposite alleles the Igfr-Airn imprinted domain (). Allele-specific expression was the prerequisite for exhibiting allele-specific reciprocal active and repressive chromatin composition, except at the Inppf_v and Slca1 transcripts. Imprinted genes with placental-specific imprinted expression generally did not exhibit allele-specific chromatin in MEFs. This is expected, because the allele-specific expression of imprinted genes and their epigenetic features in MEFs is highly similar to those of the embryo at 1. dpc (0, ) Broad enrichment of HKme along imprinted domains has an allele-specific component Apart from discreet enrichment foci for reciprocal chromatin marks we also found extended biallelic HKme BLOCs (0, ) along both domains. In the present study we discovered the allele-specific nature of HKme BLOCs. At the H1/Igf domain, HKme spread over a 0 kb long region encompassing H1, Igf, Igfas and Ins imprinted genes and was dominant in the maternal allele. This allele-specific BLOC may facilitate CTCF-s insulation over Igf. Indeed, chromatin organization at the Igf gene depends on CTCF binding in the ICR (0) and is mediated by Polycomb protein, Suz1 binding in the ICR (). The other allele-specific feature of BLOCs was that broad biallelic HKme enrichment was interrupted by allele-specific gaps at transcripts. In MatDup.dist MEFs the H1 promoter region resided in an HKme-free gap. Reciprocally, the ds cdna hybridization signal occupied a window of about kb at the Kcnq1ot1 transcript and the HKme signal exhibited a sharp gap around this transcript in PatDup.dist MEFs (Figure ). The paternal allele-specific HKme gap in the biallelic HKme BLOC is a novel feature of the Kcnq1-Cdkn1c imprinted domain. Previous chromatin analysis by Umlauf () showed that in. dpc embryos HKme is maternal allele-specific at one SNP located at the end of the Kcnq1ot1 RNA and biallelic at another SNP about kb downstream. Pandey and colleagues () mapped the chromatin along this imprinted domain in placenta and liver in a non-allele-specific fashion and found that HKme levels are relatively - 0 -

21 low along the Kcnq1ot1 transcript. They also reported that HKme is paternal-allele-specific at four SNPs (Cd1, Tspan, Kcnq1 and Slca1) in the placenta, but they did not investigate allele-specific HKme in the embryo. It will be interesting to find out whether this Polycombdependent repressing mark is required for monoallelic silencing of the Kcnq1ot1 gene or if the Kcnq1ot1 RNA directly excludes HKme from the paternal allele in the embryo. Interestingly, Kcnq1ot1 has an opposite role in the placenta, it recruits PRC complex members to the paternal allele along the domain () CTCF and DMRs A robust CTCF peak in the H1/Igf ICR in the present study confirmed in vivo CTCF footprints in MatDup.dist versus PatDup.dist MEFs (1) and ChIP-SNuPE results of maternal-allelespecific CTCF binding in normal MEFs (0). The role of CTCF-mediated insulation at this locus in MEFs has been confirmed genetically (0, ). CTCF-mediated insulation-based mechanism has been debated with respect to the KvDMR1 (, ). Although we didn t find reproducible CTCF binding in PatDup.dist MEFs at the KvDMR1 using ChIP-chip, paternal allele-specific in vivo CTCF binding had been detected there previously by ChIP (), suggesting functional importance for these sites. Further genetic analysis will be required to test whether the KvDMR1 regulates imprinted gene expression by an enhancer blocking mechanisms () in specific organs () perhaps at low-occupancy CTCF sites (0). We found that CTCF binding is maternal allele-specific in the paternally methylated upstream somatic DMR of Cdkn1c (,, ) at a consensus binding site (0) (Figure ). In addition we identified a very clear novel CTCF peak in the Peg1 DMR in the paternal allele. Allele-specific CTCF binding may thus play a role in regulating imprinted expression of the Cdkn1c and the Peg1 genes. Indeed, CTCF may be a more common theme in genomic imprinting than previously appreciated. Allele-specificity of histone globular domain modifications - 1 -

22 We reported recently that the methylated alleles of DMRs are distinguished by HKme whereas the unmethylated alleles are enriched in HKme (). The present panoramic analyses confirmed that HKme is paternal allele-specific at the H1/Igf ICR whereas HKme is paternal allele-specific at the Igf gene; and also that HKme is paternal-allelespecific whereas HKme was biallelic at the KvDMR1 (). We now revealed that HKme is also maternal allele-specific at the maternally methylated Inppf_v DMR and paternal-allele-specific at the paternally methylated Cdkn1c upstream somatic DMR. The allelespecific difference between HKme and HKme was not always present at regions outside of DMRs (HKme overlapped sometimes with either HKme or with HKme at specific regions). Globally, however, cluster analysis placed HKme together with active chromatin marks, histone acetylation, HK methylation, CTCF and gene density whereas HKme clustered with gene poor regions and repressive histone marks, HKme and HKme. Additionally, in the composite profile of genes displaying low expression or no expression, a peak of HKme but not HKme was present downstream of the TSS (Figure S). It will be interesting to find out the role of the different methylated forms of HK in imprinted gene regulation Highly efficient prediction of imprinted genes based on allele-specific chromatin signature Recently, large-scale studies aimed at detecting novel imprinted genes (). The predictions were based on RNA expression differences (1,,,, 1), DNA-sequence based computational predictions (0, 1, ), DNA methylation differences (, ) and epigenetic signature (, 1,,, ). The chromatin-based studies successfully predicted and identified novel imprinted genes, but with variable specificity. RNA polymerase II (PolII) binding was allele-specifically measured in high throughput ChIP-SNP assay (), but the predicted imprinted genes have not been verified. Using ChIP with deep sequencing, 1/0 of the top sites enriched in HKme and HKme marks in the oppositely methylated alleles identified imprinted regions in ES cells (). - -

23 Overlapping patterns of HKme and HKme in somatic cells together with a sperm DMR verified known DMRs in a high density custom imprinting array and predicted novel imprinted features, but these haven t been confirmed (1). Overlapping HKme and DNA methylation double hits were enriched at imprinted regions and overlapping HKme, DNA methylation and CTCF binding triple hits was even more enriched () but the prediction was not absolutely precise. The comprehensive allele-specific chromatin data obtained from our study allowed us to generate and test a consensus imprinted gene chromatin signature. This empirically derived definition, HKac and [CTCF or HKme] in one allele and HKme in the other allele, with the sliding window approach recognized imprinted genes with an unprecedented, 0% precision. It identified only imprinted genes along the duplicated genomic segments, H1, Igf, Igfas, Cdkn1c, Kcnq1ot1 and Inppf_v on distal Chr, and Peg1, Slca on distal Chr1. The proof for the algorithm s utility is that based on the chromatin of distal Chr imprinted genes it correctly predicted imprinted genes using a similar data set from an independent duplicated chromosome, distal Chr1. Additionally, the approach correctly determined the expressed allele for each gene. The regions having predictive power were located at the proximity of imprinted genes themselves and at their DMRs. A limitation of this method seems to be that it did not predict novel imprinted genes in the genomic region analyzed in the given cell type. The likely explanation can be that there are no more differentially expressed genes in MEFs along the duplicated genomic region. Our study can be considered as proof-of-principle analysis, based on two well-studied chromosome regions in one cell type. Indeed, the predictive power of the algorithm for the given genomic region was better than if predictions were based on DNA methylation only or on monoallelic gene expression in the given cell type. The predictor recognized imprinted genes in the absence of a DNA methylation mark and in the absence of expression. Igf and Igfas are not associated with a typical DMR. Methylation of the Igf DMR1 is biased toward the expressed paternal allele (1, ). It would not be possible to predict their - -

24 imprinting status based on DNA methylation, but the chromatin-based predictor algorithm correctly identified Igf and Igfas as paternally expressed imprinted genes. Inppf_v is imprinted in brain with paternal allele-specific expression, but is not expressed in other organs (1). The imprinting signature algorithm in combination with the sliding window approach recognized Inppf_v as imprinted based on allele-specific chromatin profile even though it was not annotated and did not show expression in MEFs according to ds cdna hybridization (Figure ). Because the prediction of Inppf_v was an exception, our results suggest that imprinted gene predictions that rely on epigenetic features should be done in a cell type/organ-specific fashion. Our analysis provides evidence that reciprocal translocations along the mouse genome () may aid the discovery of novel imprinted genes with high specificity. Our predictor algorithm can be also applied to deep sequencing of ChIP-ed DNA fractions from F1 of different mouse strains/subspecies. The advantage of this method is that duplication of mouse chromosomes is not required and the two parental alleles can be directly compared in a single sample along the entire genome (). The disadvantage will be that allele-specific information will be limited to peaks where SNP is available between the mouse strains and the analysis will not allow a panoramic display of chromatin composition Allele-specific chromatin is autonomous to the maternally and paternally inherited chromosomes The role of allelic trans-sensing () between parental chromosomes has been suggested in genomic imprinting (, -, 0, 1,, 0). Using pronuclear transplantation experiments we showed earlier that allelic trans-sensing and counting is not required for allele-specific expression of imprinted genes (). At least at the Uaf1-rs imprinted locus, trans-sensing is not required for allele-specific chromatin configuration, assessed by nuclease hypersensitity (1). In MatDup.dist and PatDup.dist MEFs the paternally and maternally inherited chromosomes along the distal Chr regions never had a chance to physically interact. Yet, the paternally - -

25 inherited Chr correctly established and maintained paternal-allele specific chromatin features in the absence of maternally inherited Chr in PatDup.dist MEFs. The same was true for the maternally inherited Chr in MatDup.dist MEFs and for the paternally or maternally inherited alleles of distal Chr1 in MatDup.dist and PatDup.dist MEFs, respectively. The chromatin composition along the entire H1/Igf domain was identical in MatDup.dist versus PatDup.dist with those of the maternal allele and paternal allele, respectively, in normal MEFs (0, ). This proves that the maternally and paternally inherited chromosomes act independently from each other in establishing and maintaining the parental allele-specific chromatin features along these imprinted domains ACKNOWLEDGEMENTS We thank Diana Tran and Guillermo Rivas for performing the allele-specific RNA quantitation and Mai Dang, summer student, for technical assistance. This work was supported by a Public Health Service grant (GM0) from the National Institute of General Medicine to P.E.S

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31 BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome. Genome Res. 1. Paulsen, M., K. R. Davies, L. M. Bowden, A. J. Villar, O. Franck, M. Fuermann, W. L. Dean, T. F. Moore, N. Rodrigues, K. E. Davies, R. J. Hu, A. P. Feinberg, E. R. Maher, W. Reik, and J. Walter. 1. Syntenic organization of the mouse distal chromosome imprinting cluster and the Beckwith- Wiedemann syndrome region in chromosome p1.. Hum Mol Genet :-.. Paulsen, M., O. El-Maarri, S. Engemann, M. Strodicke, O. Franck, K. Davies, R. Reinhardt, W. Reik, and J. Walter Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse. Hum Mol Genet :1-1.. Peters, J., and C. Beechey. 00. Identification and characterisation of imprinted genes in the mouse. Brief Funct Genomic Proteomic :0-.. Qian, N., D. Frank, D. O'Keefe, D. Dao, L. Zhao, L. Yuan, Q. Wang, M. Keating, C. Walsh, and B. Tycko. 1. The IPL gene on chromosome p1. is imprinted in humans and mice and is similar to TDAG1, implicated in Fas expression and apoptosis. Hum Mol Genet :01-.. Rauch, T., and G. P. Pfeifer. 00. Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-cpg islands in cancer. Lab Invest :-0.. Rauch, T. A., and G. P. Pfeifer. 0. DNA methylation profiling using the methylated-cpg island recovery assay (MIRA). Methods :1-.. Regha, K., M. A. Sloane, R. Huang, F. M. Pauler, K. E. Warczok, B. Melikant, M. Radolf, J. H. Martens, G. Schotta, T. Jenuwein, and D. P. Barlow. 00. Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome. Mol Cell :-.. Rentsendorj, A., S. Mohan, P. Szabó, and J. R. Mann. 0. A genomic imprinting defect in mice traced to a single gene. Genetics 1:1-.. Ruf, N., S. Bahring, D. Galetzka, G. Pliushch, F. C. Luft, P. Nurnberg, T. Haaf, G. Kelsey, and U. Zechner. 00. Sequence-based bioinformatic prediction and QUASEP identify genomic imprinting of the KCNK potassium channel gene in mouse and human. Hum Mol Genet 1: Sandhu, K. S., C. Shi, M. Sjolinder, Z. Zhao, A. Gondor, L. Liu, V. K. Tiwari, S. Guibert, L. Emilsson, M. P. Imreh, and R. Ohlsson. 00. Nonallelic transvection of multiple imprinted loci is organized by the H1 imprinting control region during germline development. Genes Dev : Sanz, L. A., S. Chamberlain, J. C. Sabourin, A. Henckel, T. Magnuson, J. P. Hugnot, R. Feil, and P. Arnaud. 00. A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb. Embo J :-.. Schoenherr, C. J., J. M. Levorse, and S. M. Tilghman. 00. CTCF maintains differential methylation at the Igf/H1 locus. Nat Genet :-.. Schulz, R., T. R. Menheniott, K. Woodfine, A. J. Wood, J. D. Choi, and R. J. Oakey. 00. Chromosome-wide identification of novel imprinted genes using microarrays and uniparental disomies. Nucleic Acids Res :e

32 Schulz, R., K. Woodfine, T. R. Menheniott, D. Bourc'his, T. Bestor, and R. J. Oakey. 00. WAMIDEX: a web atlas of murine genomic imprinting and differential expression. Epigenetics :-.. Shin, J. Y., G. V. Fitzpatrick, and M. J. Higgins. 00. Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. Embo J :1-.. Singh, P., J. Cho, S. Y. Tsai, G. E. Rivas, G. P. Larson, and P. E. Szabó. 0. Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes. Nucleic Acids Res (in press).. Singh, P., L. Han, G. E. Rivas, D. H. Lee, T. B. Nicholson, G. P. Larson, T. Chen, and P. E. Szabó. 0. Allele-specific HK Di-versus Trimethylation Distinguishes Opposite Parental Alleles at Imprinted Regions. Mol Cell Biol 0:-0.. Smilinich, N. J., C. D. Day, G. V. Fitzpatrick, G. M. Caldwell, A. C. Lossie, P. R. Cooper, A. C. Smallwood, J. A. Joyce, P. N. Schofield, W. Reik, R. D. Nicholls, R. Weksberg, D. J. Driscoll, E. R. Maher, T. B. Shows, and M. J. Higgins. 1. A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith- Wiedemann syndrome. Proc Natl Acad Sci U S A :0-.. Smith, R. J., W. Dean, G. Konfortova, and G. Kelsey. 00. Identification of novel imprinted genes in a genome-wide screen for maternal methylation. Genome Res 1:-. 0. Srivastava, M., S. Hsieh, A. Grinberg, L. Williams-Simons, S. P. Huang, and K. Pfeifer H1 and Igf monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H1. Genes Dev 1:-. 1. Szabó, P., S. H. Tang, A. Rentsendorj, G. P. Pfeifer, and J. R. Mann Maternal-specific footprints at putative CTCF sites in the H1 imprinting control region give evidence for insulator function. Curr Biol :0-.. Szabó, P. E., and J. R. Mann. 1. Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igfr: no evidence for allelic trans-sensing and counting mechanisms. Embo J 1:01-.. Szabó, P. E., S. H. Tang, F. J. Silva, W. M. Tsark, and J. R. Mann. 00. Role of CTCF binding sites in the Igf/H1 imprinting control region. Mol Cell Biol : Tartof, K. D., and S. Henikoff.. Trans-sensing effects from Drosophila to humans. Cell :01-.. Terranova, R., S. Yokobayashi, M. B. Stadler, A. P. Otte, M. van Lohuizen, S. H. Orkin, and A. H. Peters. 00. Polycomb group proteins Ezh and Rnf direct genomic contraction and imprinted repression in early mouse embryos. Dev Cell 1:-.. Thorvaldsen, J. L., K. L. Duran, and M. S. Bartolomei. 1. Deletion of the H1 differentially methylated domain results in loss of imprinted expression of H1 and Igf. Genes Dev 1:

33 Tremblay, K. D., J. R. Saam, R. S. Ingram, S. M. Tilghman, and M. S. Bartolomei. 1. A paternal-specific methylation imprint marks the alleles of the mouse H1 gene. Nat Genet :0-1.. Umlauf, D., Y. Goto, R. Cao, F. Cerqueira, A. Wagschal, Y. Zhang, and R. Feil. 00. Imprinting along the Kcnq1 domain on mouse chromosome involves repressive histone methylation and recruitment of Polycomb group complexes. Nat Genet : Verona, R. I., J. L. Thorvaldsen, K. J. Reese, and M. S. Bartolomei. 00. The transcriptional status but not the imprinting control region determines allelespecific histone modifications at the imprinted H1 locus. Mol Cell Biol : Wagschal, A., H. G. Sutherland, K. Woodfine, A. Henckel, K. Chebli, R. Schulz, R. J. Oakey, W. A. Bickmore, and R. Feil. 00. Ga histone methyltransferase contributes to imprinting in the mouse placenta. Mol Cell Biol : Wang, X., Q. Sun, S. D. McGrath, E. R. Mardis, P. D. Soloway, and A. G. Clark. 00. Transcriptome-wide identification of novel imprinted genes in neonatal mouse brain. PLoS One :e.. Wang, Z., C. Zang, J. A. Rosenfeld, D. E. Schones, A. Barski, S. Cuddapah, K. Cui, T. Y. Roh, W. Peng, M. Q. Zhang, and K. Zhao. 00. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 0:-0.. Wen, B., H. Wu, H. Bjornsson, R. D. Green, R. Irizarry, and A. P. Feinberg. 00. Overlapping euchromatin/heterochromatin- associated marks are enriched in imprinted gene regions and predict allele-specific modification. Genome Res 1:-1.. Williamson, C. M., A. Blake, S. Thomas, C. V. Beechey, J. Hanckok, and e. a. M. Harwell. World Wide Web Site -Mouse Imprinting Data and Referenceshttp://har.mrc.ac.uk/reserach/genomic_imprinting/.. Wood, A. J., R. G. Roberts, D. Monk, G. E. Moore, R. Schulz, and R. J. Oakey. 00. A screen for retrotransposed imprinted genes reveals an association between X chromosome homology and maternal germ-line methylation. PLoS Genet :e0.. Yatsuki, H., K. Joh, K. Higashimoto, H. Soejima, Y. Arai, Y. Wang, I. Hatada, Y. Obata, H. Morisaki, Z. Zhang, T. Nakagawachi, Y. Satoh, and T. Mukai. 00. Domain regulation of imprinting cluster in Kip/Lit1 subdomain on mouse chromosome F/F: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region. Genome Res 1:

34 FIGURE LEGENDS Figure 1. Uniparental duplication of distal chromosome allows chromosome-wide analysis of allele-specific epigenetic features of two imprinted domains. (A) Normal embryos inherit one set of chromosomes from each parent. Maternally and paternally inherited alleles of chromosomes (red and blue) and Chr1 (pink and light blue) are shown. (B) In MatDup.dist embryos two copies of the distal Chr segment, telomeric to the TH translocation breakpoint are inherited from the mother and two copies of distal Chr1 are inherited from the father. (C) In PatDup.dist embryos two copies of distal Chr are inherited from the father and two copies of distal Chr1 are inherited from the mother. Hyper- and hypomethylated alleles of known DMRs are marked by closed and open lollipops, respectively. (D) Two clusters of imprinted genes in distal chromosome are regulated by reciprocal germ line methylation and different imprinting mechanisms. Imprinted expression of the H1/Igf imprinted domain (to the left) is regulated by a paternally (P) methylated (closed lollipops) DMR. The unmethylated (open lollipops) allele is specifically bound by CTCF insulator protein (yellow ovals) that blocks Igf activation by the shared enhancers (grey circles) in the maternal chromosome (M). H1 is expressed from the maternal chromosome (red). Igfas and Ins are paternally expressed (blue), the latter exhibiting imprinted expression in the yolk sac. (B) The Cdkn1c1/Kcnq1 imprinted domain (to the right) is telomeric to the H1/Igf domain. It is under the control of the maternally methylated KvDMR1, which overlaps the promoter of the paternally expressed non-coding RNA, Kcnq1ot1 (orange line). Cdkn1c is expressed from the maternal chromosome. Kcnq1ot1 is expressed from the paternal chromosome. Transcription of a set of imprinted genes, Th, Ascl, Tspan, Cd1, Tssc, Slca1, Phlda, Napl1, Tnfrs, Osbpl and Dhcr in the placenta is repressed in the paternal chromosome by Kcnq1ot1 expression from the paternal allele. Imprinted expression of Kcnq and Slca1 is developmentally regulated. The embryonic lethality phenotype of - -

35 PatDup.dist embryos was rescued by an Ascl transgene, integrated at another chromosome. The overlap of the P1 clone with the endogenous locus is indicated. Map is not to scale Figure. High resolution allele-specific analysis of the H1, Igf and Igfas imprinted genes. The chromatin (with the antibodies indicated to the right) and methylation (MIRA) signals are plotted along the chromosome as log P value scores for the maternal allele in MatDup.dist (red bars) and for the paternal allele in PatDup.dist (blue bars) MEFs. The P value score was obtained by NimbleScan software and is derived from the Kolmogorov-Smirnov test comparing the log ratios (ChIP or MIRA vs. input) within a 0-bp window centered at each probe and the rest of the data on the array. Transcripts are marked by rectangles, arrowhead indicating the direction of transcription. The H1/Igf ICR and the Igf DMRs are labeled with yellow rectangle. Significant allele-specific peaks located at - kb to +kb from the transcription start sites (TSS) are marked by asterisk. Additionally, significant maternal HKme peaks are marked along the imprinted domain in this Figure and in Figure SB. Genomic coordinates are indicated on the top according to UCSC Genome Browser mouse genome version mm Figure. High resolution allele-specific chromatin analysis along the Kcnq1ot1 and Cdkn1c/Slca1 imprinted genes. Kcnq1ot1 ncrna and Cdkn1c are paternally and maternally expressed in MEFs, respectively. The KvDMR1, the Cdkn1c DMR and the Slca1 DMR () (yellow rectangles) are very clearly marked by allele-specific chromatin. The HKme peaks in the bracketed area and peak 1 in the Cdkn1c upstream area were confirmed using ChIPrealtime PCR (Figure S). Slca1 exhibited some allele-specific chromatin marks in the absence of high level transcription in MEFs (Figure S). Other details are as in Figure. Figure. Chromatin analysis of Inppf_v. Inppf_v, is paternally expressed in the brain, but is not expressed in MEFs. Allele-specific marks can be discerned at Inppf_v but not at the non- - -

36 imprinted Inppf in MEFs, including maternal DNA methylation in the Inppf_v DMR. Other details are as in Figure Figure. Derivation and testing an imprinted gene predictor algorithm. (A) Allele-specific peaks of epigenetic marks were tabulated at four annotated transcripts according to allele-specific expression profile of the transcript. Allele-specific peaks were identified for activation and repressive epigenetic marks in the promoter region (- kb to + kb from the TSS) of four known imprinted genes, located on distal Chr, in MatDup.dist and PatDup.dist MEFs (Table S). From these the activation mark peaks in the expressed allele (green) and the repression mark peaks in the silent allele (orange) were considered for maternally expressed and paternally expressed transcripts. From this table a consensus imprinted gene signature was derived: HKac and [HKme or CTCF] in the expressed allele and HKme in the silent allele. These are colored: maternal allele (red) and the paternal allele (blue). (B) The consensus was then tested as bimodal predictor using chromatin data of annotated transcripts along distal Chr, central Chr and distal Chr1 (Table S and Figure S). The predictor was further tested by a sliding-window approach, which did not depend on transcript annotations (Table S) Figure. Chromatin analysis of imprinted genes on distal Chr1. A map depicting the imprinting status of the Peg1 and the Slca imprinted regions along distal Chr1 is shown at the top. ChIP on chip results are shown for the maternal allele in PatDup.dist (red bars) and for the paternal allele in MatDup.dist (blue bars) MEFs with the antibodies indicated to the right. The last two rows depict MIRA methylation analysis. - -

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