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Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression

Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression. Genetic Variation is a Change in the DNA Sequence. SNPs Indels Rearrangements Mendelian Inheritance. X. Epigenetics relies on DNA methylation.

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Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression

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  1. Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression

  2. Genetic Variation is a Change in the DNA Sequence SNPs Indels Rearrangements Mendelian Inheritance

  3. X

  4. Epigenetics relies on DNA methylation Epigenetics - heritable change in phenotype without a change in DNA sequence Organismal or cell generations A mechanism involved in epigenetic phenotyperegulation DNA methylation is a transcription (OFF) switch Histone methylation is a transcription (OFF) switch Histone acetylation is a transcription (ON) switch Will also involve other molecular mechanism

  5. DNA methylation in different species S. cerevisiae Arabidopsis Vertebrates Drosophila C. elegans S. pombe N. crassa DNA methylation + (+)‏ + + — — — DNA methylation binding proteins + + + + + + — (+)‏ + + Imprinting — — — — A. Peaston TJL

  6. cytosine guanine H CH3 N H O N DNMT N N H N N N C1' O H N C1' H DNA methylation -- 5'CpG3' A. Peaston TJL

  7. 7 Establishment of DNA methylation Relative methylation at CpG sites ~ 70% 0 5 10 15 20 Days post conception Yoder, Walsh & Bestor, 1997Trends Genet

  8. 8 Relative methylation at CpG sites Autosomes & active X CpG islands Inactive X Paternal allele in female germ cells H19 Imprinted gene Maternal allele in male germ cells 0 5 10 15 20 Days post conception Establishment of DNA methylation: imprinting

  9. Law & Jacobsen Nat Rev. Genet 2010

  10. DNA demethylation Passive Active e.g. zygotic paternal genome, IL2 promoter A. Peaston TJL

  11. ON OFF DNA demethylation -- CpG islands ? CpG methylation default, islands exclude global activity ? A. Peaston TJL

  12. Law & Jacobsen Nat Rev. Genet 2010

  13. DNA Demethylation and 5hydroxymethyl cytosine 5-methylcytosine oxidized to 5 hydroxymethylcytosine Tet family (ten-eleven translocation)

  14. Wu & Zhang 2011 Cell Cycle

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

  16. CTCF CTCF Mat H19 E Igf2 ICE me Pat Igf2 ICE H19 E How does DNA methylation inhibit transcription? • Biochemical experiments using • artificial CpG methylation & transfection • re-expression of X-linked genes after 5-azacytidine treatment • Interference with transcription factor or other protein binding • e.g. CTCF binding A. Peaston TJL

  17. Assessing DNA methylation Avy/a Morgan et al, Nature Genetics 1999

  18. LTR CpG methylated IAP OFF pseudoagouti LTR CpG unmethylated IAP ON yellow DNA methylation at the agouti locus Avy allele Hypothesis

  19. B BamH1 M Msp1 H HpaII Isoschizomers, C/CGG Methylation-sensitive restriction enzymes Avy allele A. Peaston TJL

  20. 5MeCpG CpG bisulfite DNA UG CG PCR CG TG Bisulfite sequencing Locus-specific detailed single-molecule analysis Frommer et al, PNAS 1992

  21. Sperm 95% Oocytes 98% Zygotes 90% Blastocyst 62% Bisulfite Sequencing Lane et al, Genesis 2003

  22. Higher throughput Pyrosequencing of clones (You still have to clone) SAGE

  23. Higher throughput Next Generation Sequencing High throughput, non-biological cloning

  24. And permutations..... Murrell, Rakyan & Beck, Hum Mol Genet 2005

  25. Chromatin and DNA methylation Neurospora and Arabidopsis – methylation depends on H3K9me Plants – RNAi targets chromatin modification, gene silencing & DNAme 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

  26. The nucleosome core. Site of action. Tails that get modified are evident in this image.

  27. http://atlasgeneticsoncology.org/Deep/Images/ChromatinFig2Engl.gifhttp://atlasgeneticsoncology.org/Deep/Images/ChromatinFig2Engl.gif

  28. Histone modifying enzymes 29

  29. Chromatin remodelling – Polycomb and Trithorax 30

  30. 31 Kloc et al Current Biology 2008

  31. Epigenetic changes and phenotype • DNA methylation and mutation • Genomic instability • Cancer • Complex disease • Environmental effects on DNA methylation A. Peaston TJL

  32. Epigenetics/Epigenomics Modification of histones Mostly Acetylation (+) and Methylation (-) Histone acetyl transferases Histone deacetylases Modification of DNA In eukaryotes, mostly 5-methylcytosine (-) Dnmt (DNA methyl transferases) Removal? A sixth base; 5-hydroxymethylcytosine

  33. Epigenetics/Epigenomics Methylated DNA recruits HDACs Increased condensation of chromatin decreased expression Methylated chromatin may recruit Dnmts Transcription factors may recruit Dnmts, HDACs or HMTs or HATs depending on function Signals can propagate along the chromosome using bi-functional enzymes (chromodomain and HMT activity)

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