The Evolving Role of MeCP2 in Rett Syndrome and Autism

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1 ? The Evolving Role of in Rett Syndrome and Autism Janine LaSalle, Ph.D Medical Microbiology and Immunology Rowe Program in Human Genetics UC Davis School of Medicine

2 Epigenetics and Autism What s the connection? AUTISM? Rett Syndrome MECP2 mutation Angelman, Prader-Willi, 15q syndromes 15q11-13 deficiency or duplication

3 Autism Complex developmental disorder that usually appears 1-3 yr Deficits in social interaction and communication Repetitive stereotyped behavior and abnormal preoccupations Complex genetic etiology influenced by environmental factors

4 Rett Syndrome (RTT) RTT is the only one of the five Pervasive Developmental Disorders with a known genetic cause X-linked dominant, ~80% MECP2 mutation Neurodevelopmental regression around 6 to 18 months of age Mental retardation, ataxia, loss of purposeful hand movements, loss of vocalization skills, seizures

5 Angelman and Prader-willi syndromes Imprinted disorders caused by 15q11-13 deletions or deficiency QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. AS: Maternal 15q11-13 deletion, paternal disomy, maternal UBE3A mutation, imprinting defects QuickTime and a FF (Uncompressed) decompresso are needed to see this picture. PWS: Paternal 15q11-13 deletion, maternal disomy, imprinting defects

6 15q duplication syndrome isodicentric chromosome 15 Cytogenetic abnormality of proximal 15q interstitial duplication or idic 15 Hypotonia, small stature Autistic features, anxiety, seizures Defects in sensory processing, language, and attention QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. idic 15 QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. interstitial duplication

7 Overlap between autism, Rett and 15q11-13 syndromes Phenotypic overlap stereotypic behaviors language impairments seizures (RTT, AS, 20-30% autism) delayed postnatal onset, regression (RTT and autism) mental retardation (RTT, AS, ~80% autism) sleep abnormalities Evidence for genetic overlap MECP2 mutations in rare cases of autism MECP2 mutations in rare cases with AS phenotype 15q11-13 maternal duplications in 1-3% of autism 15q11-13 rearrangements in 3 RTT cases linkage to 15q11-13 loci in autism families

8 Evidence for epigenetic overlap between autism, RTT, and 15q expression is significantly reduced in 79% of autism post-mortem brain samples Methylation of the MECP2 promoter correlates with reduced expression in male autism brain samples GABRB3 expression (15q11-13) is significantly reduced in 56% of autism post-mortem brain samples Biallelic expression levels of GABRB3 are epigenetically dysregulated in Rett and autism postmortem brain Homologous pairing of 15q11-13 in mature neurons is deficient in RTT, autism, and AS Ravi Nagarajan GGG student Amber Hogart GGG student Karen Thatcher GGG student

9 Methyl CpG Binding Protein 2 Binds methylated CpG dinucleotides Transcriptional repressor Xq28 (X chromosome inactivation) Complex developmental regulation (Bird and Wolffe, Cell 1999) ATG ATG AAAA AAAA 5.3 kb 5 69 bp 124 bp 3 76 kb 3 UTR

10 is a marker for mature neurons in the post-natal mammalian brain Sytox Green Anti-

11 Evolving models of come full circle HDAC Sin3a OFF Repressor Model coactivators CREB1 ON Transcriptional activator model? Structural Model ON Splicing YB-1 Chromatin Remodeling ATRX Brahma Active gene modulator model Loop and recruit model LaSalle and Yasui, Epigenomics, in press

12 The majority of bound promoters are on expressed genes Yasui et al, PNAS, 2007

13 is bound to promoters of highly expressed genes Yasui et al, PNAS, 2007

14 Highly methylated promoters are not bound by Yasui et al, PNAS, 2007

15 Example: MeDIP + and Pol2 ChIP at HOXA locus MeDIP Pol2 HOXA1 HOXA2 HOXA3 HOXA4 HOXA5 HOXA6 HOXA7 HOXA9 HOXA10 HOXA11 HOXA13

16 More examples from Nimblegen promoter arrays for MeDIP,, and Pol2 QuickTime and a decompressor are needed to see this picture. POU5F1 (OCT4) QuickTime and a decompressor are needed to see this picture. GAPDH NOL1 CHD4

17 Micrococcal nuclease fractionation of chromatin shows primarily in active fraction QuickTime and a decompressor are needed to see this picture. Western blot DNA dot-blot QuickTime and a decompressor are needed to see this picture. QuickTime and a decompressor are needed to see this picture. Mike Gonzales, unpublished

18 localizes to both inert nuclear heterochromatin as well as nonheterochromatic regions Mecp2 +/y Mecp2 -/y Anti- DAPI Sub-nuclear localization patterns of are consistent with dual functions in repression and activation

19 The majority of binding sites are intergenic or intronic Yasui et al, PNAS, 2007

20 ChIP-chip analysis of established target genes Yasui et al, PNAS, 2007

21 ChIP-chip analysis of imprinted region 15q11-13 chr L3 Sites POTE15 NIPA2 NDN SNRPN C15orf2 NIPA1 OR4M2 MKRN3 SNURF OR4N4 TUBGCP5 CYFIP1 UBE3A ATP10A GABRB3 OCA2 HERC2 NDNL2 APBA2 TJP1 TRPM1 KLF13 SGNE1 GREM1 C15orf16 RYR3 CHRFAM7A CHRNA7 MTMR10 Previously described and validated Validated Genes L3 sites CHRNA7 BC Validated in human CpG islands Yasui et al, PNAS, 2007

22 target genes at closer examination Active genes that represses through intergenic sites JUNB ID1 Active genes that activates through intronic enhancers EGR2 GABRB3

23 ChIP-chip analysis of 19p13.2 and JUNB Yasui et al, PNAS, 2007

24 Methylation of upstream binding site for ID1 with unmethylated promoter Log2 Ratio Site 1 Site 2 ID1 Genomic tiling array Pol2 Promoter tiling array QuickTime and a decompressor are needed to see this picture. Pol binding Site [659 bp,10cpg] -10 Kb Upstream ID1 is upregulated in Mecp2 -/y brain Peddada et al, 2006 Site -1 Site Log2 Ratio Log2 Ratio -1100bp - 666bp - 657bp ID1 Promoter -10.1kb kb kb -109bp +1 ID1 ID1 Promoter * * * * * D -UT UD * * * * * * * * * D -UT UD D -UT UD DNA methylation patterns do not change with differentiation S. Peddada, unpublished

25 Reciprocal regulation of MECP2 and EGR2 intronic enhancer QuickTime and a decompressor are needed to see this picture. QuickTime and a decompressor are needed to see this picture. EGR2 MECP2 EGR2 Neuronal maturation Swanberg et al, 2009

26 15q11-13 GABA A receptor subunit GABRB3 has in intronic enhancer for homologous pairing and biallelic expression QuickTime and a decompressor are needed to see this picture. intronic binding predicted to act as a pairing factor for optimal maternal expression QuickTime and a decompressor are needed to see this picture. Hogart et al, HMG, 2007

27 Dimmer switch model of as long range modulator of gene expression ON OFF HDAC Sin3a OFF Textbook enzymatic model as off switch LO HI ON Structure Location Loop Recruit Modulator rather than silencer

28 Future goals for improved understanding of Characterize and develop reagents for investigating all post-translational modifications Characterize alternative isoforms, particularly e1 in brain Genome-wide ChIP-seq in primary neurons Correlate binding sites with DNA methylation, histone marks, and gene expression genome-wide

29

30 Genomic map of the 15q11-13 deletion/duplication disorders Cen autism PWS/AS BP1 BP2 Weiss et al., 2008 Class 3B idic(15) Class 5A idic(15) Class I PWS/AS deletions/ int dups Class II PWS/AS deletions/ int dups BP3 BP4 BP5 epilesy, autism, schizophrenia, control 15q11.2 microduplication (Stefansson, 2008) 15q13.3 microduplication (Miller, 2008; Christian, 2008) 15q13.3 microdeletion (Sharp, 2008; Pagnamenta, 2008; Miller, 2008; Helbig, 2009; ISRC, 2008; Stefansson 2008))

31 Imprinted and neuronal regulation of 15q11-13 Paternally Expressed MKRN3 MAGEL2 NDN SNRPN PWS- ICR expressed in neurons HBII-13 HBII-85 HBII-52 UBE3A-AS AS CEN TEL UBE3A ATP10A Maternally Expressed DNA FISH Probes AS-ICR 175kb 170kb 161kb

32 Chromatin decondensation of Snrpn to Ube3a is neuron-specific Murine 7qC Mkrn3 Magel2 Ndn IC Snrpn paternal neuronal transcript MBII-13 MBII-85 MBII-52 Ube3a-AS Ube3a Atp10a TEL CEN DNA FISH Probes 207kb 888kb 206kb Brain Snrpn-Ube3a DAPI Snrpn-Ube3a 5 of Snrpn 3 of Atp10a Paternal signal ~5.1µm (average) 266.4µm/5.1µm = 52.2:1 packing ratio 1.3x more compact then a 30nm fiber Maternal signal ~1.1µm (average) 266.4µm/1.1µm = 242.2:1 packing ratio 6x more compact then a 30nm fiber Thymus Snrpn-Ube3a

33 Paternal Snrpn-Ube3a allele decondensation is developmentally regulated and correlates to nucleolar enlargement during neuronal maturation Snrpn-Ube3a signal length (µm) MBII-85 RNA FISH fluorescence (pixel intensity) MBII-85 RNA FISH P21 P P1d P14d Snrpn-Ube3a Length (µm) E15 P1 P21 P70 0 Maternal signal Paternal signal Nuclear radius Nucleolar diameter P-value 3x x x x10-15

34 Transcription is required for paternal Snrpn-Ube3a chromatin decondensation Mouse model 1 Paternal IC deletion PWS model Chamberlain et al, 2004, HMG +/PWS-IC del35kb PWS-IC Hs /+ Mouse model 2 Human IC swap No maternal imprint Johnstone et al, 2005, HMG DAPI Snrpn-Ube3a Transcriptional inhibition in mouse primary neurons

35 Neuron-specific chromatin decondensation is uniquely observed at imprinted snorna clusters and impacts nucleolar size Neuronal FISH with probes from imprinted, ncrna, and highly transcribed loci 2-0-methylation of rrna Nucleolar measurements of Purkinje neurons

36 Multiple copy number variants in multiple neurodevelopmental disorders map to 15q11-13 snornas Cen autism PWS/AS BP1 BP2 Weiss et al., 2008 Class 3B idic(15) Class 5A idic(15) Class I PWS/AS deletions/ int dups Class II PWS/AS deletions/ int dups BP3 BP4 BP5 epilesy, autism, schizophrenia, control 15q11.2 microduplication (Stefansson, 2008) 15q13.3 microduplication (Miller, 2008; Christian, 2008) 15q13.3 microdeletion (Sharp, 2008; Pagnamenta, 2008; Miller, 2008; Helbig, 2009; ISRC, 2008; Stefansson 2008))

37 15q11-13 model for multiple genetic and epigenetic etiologies of autism spectrum disorders BP1-5 Nucleolus UBE3A ICR Paternal 15q11-13 UBE3A ICR BP1-5 Neuronal maturation Maternal 15q11-13

38 15q11-13 model for multiple genetic and epigenetic etiologies of autism spectrum disorders Nucleolus snorna BP1-5 UBE3A snorna rrna size neuronal maturation ICR UBE3A ICR BP1-5 Paternal 15q11-13 Maternal 15q11-13 biallelic piggybacking transcripts GABRB3/A5/G3, APBA2, CHRNA7

39 15q11-13 model for multiple genetic and epigenetic etiologies of autism spectrum disorders Nucleolus Nucleolus PWS Pat del snorna BP1-5 UBE3A ICR UBE3A snorna ICR rrna size neuronal maturation BP1-5 PWS IC del or HBII85-del Paternal 15q11-13 Maternal 15q11-13 biallelic piggybacking transcripts GABRB3/A5/G3, APBA2, CHRNA7

40 LaSalle Lab Dag Yasui Mike Gonzales Diane Schroeder Karen Leung Malaika Singleton Roxanne Vallero Michelle Martin Rima Woods Haley Scoles Amy George Joanne Suarez Keith Dunaway Tiobesta Beyene LaSalle Lab Alumni Rodney Samaco, Baylor Raman Nagarajan, UCSF Sailaja Peddada, UCSF Amber Hogart, NHGRI/NIH Katherine Patzel, NIH GPP Acknowledgements Collaborators, UCD Isaac Pessah Irva Hertz-Picciotto Robert Berman Mari Golub TramAhn Ta Robin Hansen Judy Van de Water Peggy Farnham Mark Bieda Financial Support Collaborators Carolyn Schanen, Nemours, UDE Jim Resnick, University of Florida Amanda DuBose, UFL Wendy Robinson, UBC Paul Kostiniak, SUNY Buffalo Doug Gillard, SUNY Buffalo NIH, 1R01 HD41462, 1R01 HD R01ES NSF predoctoral fellowship (KNL) International Rett Syndrome Foundation Autism Speaks Brain Tissues Jane Pickett Autism Tissue Program NICHD Brain and Tissue Bank