Epigenetic regulation of gene expression Anne Peaston PhD Research Scientist The Jackson Laboratory anne.peaston@jax.org 1 Definition of the problem Mechanisms DNA methylation chromatin-based non-coding RNA 2 B-1/ B-1 B′/ B′ B′/B-1 B-1/ B-1 B′/B-1 B-1/B-1 3 F1 4 Epigenetics: explaining the unexpected Altered phenotype e.g. coat color, gene expression No change in underlying DNA sequence Mitotically and/or meiotically heritable Responsive to environment i.e. reversible How to get many different sets of gene expression from a single genome 5 Mosaic expression in a single individual, and variation between isogenic individuals Avy/Avy x a/a 6 Morgan et al, Nature Genetics 1999 Development of an entire animal from a single cell 7 Packing vs Access DNA Nucleosomes ATCGGGATC 30 nm fiber Loop domains 300X More Folding Mitotic chrom. 1000X 8 (Alberts, Molecular Biology of the Cell) (Alberts, Molecular Biology of the Cell) DNA methylation -- 5'CpG3' cytosine guanine H CH3 DNMT N H O N H N N C 1' N N N O H N C 1' H Major groove of the double helix 9 DNA methylation in eukaryotes DNA methylation — — + — (+) + + DNA methylation binding proteins — + + + + + + Imprinting — — — — (+) + + 10 DNA methylation and cell memory Self-complementary CpG dinucleotides DNA replication Maintenance methylation Holliday and Pugh 1975 Science 11 Riggs 1975 Cytogenet Cell Genet Relative methylation at CpG sites Establishment of DNA methylation ~ 70% 0 5 10 15 Days post conception 20 12 Yoder, Walsh & Bestor, 1997 Trends Genet Relative methylation at CpG sites Establishment of DNA methylation: imprinting Autosomes Inactive X & active X CpG islands Paternal allele in female germ cells H19 Imprinted gene Maternal allele in male germ cells 0 5 10 15 Days post conception 20 13 DNA methyl transferases Regulatory domain PCNA Catalytic domain RFT Dnmt1 NLS CXXC BAH Dnmt2 PWWP ATRX Dnmt3a PWWP ATRX Dnmt3b (Mammals) 14 Goll & Bestor 2005, AnnRevBiochem DNA methyltransferase functions Species Activity Loss-of-function Phenotype Dnmt1 mouse maintenance E9.5 lethal, genome-wide loss MeCpG Abnormal gene activation (imprinting, X, TE) Dnmt2 mouse Cytoplasmic tRNA None fish “ Development defects retina, brain, liver fly “ None S. pombe unknown “ Dnmt3a mouse De novo Death 4-8 wk, germ cell imprint failure, no TE MeCpG Dnmt3b Mouse Human De novo E14.5 lethal, no TE MeCpG Immunodeficiency, centromeric instability, Facial abnormalities (ICF) syndrome15 Rai et al. 2007 Genes Dev DNA demethylation -- CpG islands ON OFF ? CpG methylation default, islands exclude global activity ? 16 How does DNA methylation inhibit transcription? • Interference with transcription factor or other protein binding • Biochemical experiments using • artificial CpG methylation & transfection • re-expression of X-linked genes after 5-azacytidine treatment • e.g. imprinted expression of Igf2 gene in mice Mat Igf2 CTCF CTCF ICE H19 E H19 E me Pat Igf2 ICE 17 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 Sin3A HDAC MeCP2 me 18 Chromatin and DNA methylation Neurospora and Arabidopsis – methylation depends on H3K9me Plants/fungi – RNAi targets chromatin modification, gene silencing & meC Mouse reduced 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 Chromatin-based mechanisms DNA Nucleosomes: Fundamental repeating unit of chromatin H1 147 bp 20 Histone modifications (≥ 56) 4 9 5 8 14 12 18 16 23 27 36 79 20 UB UB Allis, Jenuwein, Reinberg. Epigenetics. 2007 21 How many distinct combinations? 4 9 5 8 14 12 18 16 23 27 36 79 20 Is there a histone code? 22 Histone modifying enzymes 23 Chromatin remodelling – Polycomb and Trithorax 24 Chromatin remodelling – Polycomb and Trithorax 25 Epigenetic regulation of retrotransposons 26 Martens et al, EMBO2005 27 Kloc et al Current Biology 2008 DNA methylation and phenotype • DNA methylation and mutation • Genomic instability • Cancer • Complex disease • Environmental effects on DNA methylation 29 Extra Reading 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 Assigned Reading (Jin et al. GADD45A Does Not Promote DNA Demethylation PLoS Genetics 2008) See over page for suggestions for 2010 30 Chromatin state has a profound effect on biology 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 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