1. Epigenetic regulation of gene expression Anne Peaston PhD Research Scientist The Jackson Laboratory anne.peaston@jax.org 1

2. Definition of the problem MechanismsDNA methylation chromatin-based non-coding RNA 2

3. B-1/B-1 B ′/ B ′ B-1/ B-1 B ′ /B-1 B-1/ B-1 B ′ /B -1 3

4. F1 4

5. Mitotically and/or meiotically heritable Altered phenotype e.g. coat color, gene expression No change in underlying DNA sequence Epigenetics: explaining the unexpected How to get many different sets of gene expression from a single genome Responsive to environment i.e. reversible 5

6. Morgan et al, Nature Genetics 1999 Mosaic expression in a single individual, and variation between isogenic individuals A vy /A vy xa/a 6

7. Development of an entire animal from a single cell 7

8. Packing vs Access (Alberts, Molecular Biology of the Cell) 30 nm fiber Loop domains More Folding Mitotic chrom. (Alberts, Molecular Biology of the Cell) Nucleo- somes ATCGGGATC 300X1000X DNA 8

9. DNA methylation -- 5'CpG3' H H H N O N H N N N C1'C1' N OC1'C1' N N H cytosineguanine CH 3 DNMT Major groove of the double helix 9

10. S. cerevisiae S. pombe N. crassa C. elegans Drosophila Vertebrates Arabidopsis DNA methylation binding proteins — ++++++ DNA methylation — + —— (+)++ Imprinting ———— (+)+ + DNA methylation in eukaryotes 10

11. DNA methylation and cell memory Holliday and Pugh 1975 Science Riggs 1975 Cytogenet Cell Genet DNA replication Maintenance methylation Self-complementary CpG dinucleotides 11

12. 05101520 Days post conception Relative methylation at CpG sites Establishment of DNA methylation ~ 70% Yoder, Walsh & Bestor, 1997 Trends Genet 12

13. Establishment of DNA methylation: imprinting 05101520 Days post conception Relative methylation at CpG sites H19 Imprinted gene Maternal allele in male germ cells Paternal allele in female germ cells Inactive X Autosomes & active X CpG islands 13

14. DNA methyl transferases Dnmt1 Regulatory domainCatalytic domain PCNA NLS RFT CXXCBAH Dnmt2 Dnmt3a PWWPATRX Dnmt3b PWWPATRX (Mammals) Goll & Bestor 2005, AnnRevBiochem 14

15. DNA methyltransferase functions Dnmt2mouseCytoplasmic tRNA None fish “Development defects retina, brain, liver “S. pombeunknown fly“None Dnmt3bMouse Human E14.5 lethal, no TE Me CpG Immunodeficiency, centromeric instability, Facial abnormalities (ICF) syndrome De novo Rai et al. 2007 Genes Dev Dnmt3amouseDe novoDeath 4-8 wk, germ cell imprint failure, no TE Me CpG SpeciesActivityLoss-of-function Phenotype Dnmt1mousemaintenanceE9.5 lethal, genome-wide loss Me CpG Abnormal gene activation (imprinting, X, TE) 15

16. DNA demethylation -- CpG islands ON OFF ? CpG methylation default, islands exclude global activity ? 16

17. How does DNA methylation inhibit transcription? Biochemical experiments using artificial CpG methylation & transfection re-expression of X-linked genes after 5-azacytidine treatment Mat Igf2ICE CTCF EH19 CTCF Pat Igf2ICEEH19 me Interference with transcription factor or other protein binding e.g. imprinted expression of Igf2 gene in mice 17

18. MeCP2 How does DNA methylation inhibit transcription? MethylCpG-binding proteins recruit different co-repressor complexes Titratable transcriptional inhibition DNA-protein complex specific for methylated DNA MeCP2, MBD1-4, Kaiso me Sin3A HDAC HKMT Me 18

19. Chromatin and DNA methylation Neurospora and Arabidopsis – methylation depends on H3K9me Plants/fungi – RNAi targets chromatin modification, gene silencing & me C Mousereduced CpG methylation with : deficiency of H3K9 methyltransferases deficiency of EZH2 (H3K27 methyltransferase) increased CpG methylation with : antisense transcription through promoters unsettled role of RNAi Many species – link to SWI/SNF-like remodeling proteins 19

20. Chromatin-based mechanisms Nucleosomes: Fundamental repeating unit of chromatin 147 bp DNA H1 20

21. 49141823273679 5 8121620 UB Allis, Jenuwein, Reinberg. Epigenetics. 2007 Histone modifications ( ≥ 56) 21

22. How many distinct combinations? 49141823273679 5 8121620 Is there a histone code? 22

23. Histone modifying enzymes 23

24. 24 Chromatin remodelling – Polycomb and Trithorax

25. 25 Chromatin remodelling – Polycomb and Trithorax

26. 26 Epigenetic regulation of retrotransposons Martens et al, EMBO2005

27. Kloc et al Current Biology 2008 27

29. DNA methylation and phenotype DNA methylation and mutation Genomic instability Cancer Complex disease Environmental effects on DNA methylation 29

30. Multiple reviews – scholar google, pub med, other. Epigenetics. 2007. Eds Allis, Jenuwein, Reinberg. Cold Spring Harbor Press Cold Spring Harbor Symposia on Quantitative Biology - Epigenetics Extra Reading Assigned Reading (Jin et al. GADD45A Does Not Promote DNA Demethylation PLoS Genetics 2008) See over page for suggestions for 2010 30

31. http://www.sciencemag.org.ezproxy.jax.org/cgi/reprint/323/5912/373.pdf http://www.sciencemag.org.ezproxy.jax.org/cgi/rapidpdf/science.1181495v1.pdf Chromatin state has a profound effect on biology

32. Assigned Reading for 2010 possibilities (from 2009) An Epigenetic Role for maternally inherited piRNAs in Transposon Silencing. Brennecke et al 2008 Science 322:1387 A mouse speciation gene encodes a meiotic histone H3 methyltransferase. Ondrej Mihola et al 2009 Science 323:373 (commentary p350). See also next article on gene for Drosophila hybrid sterility Chromatin-associated periodicity in genetic variation downstream of transcriptional start sites. Sasaki et al 2009 Science 323:401 Science 323:220-222 commentary on stem/pluripotent maintenance/resistance to differentiation via chromatin modifications/histone ubiquitination