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Héctor Corrada Bravo CMSC702 Spring 2013 (many slides courtesy of Rafael Irizarry)

Introduction to epigenetics: chromatin modifications, DNA methylation and the CpG Island landscape. Héctor Corrada Bravo CMSC702 Spring 2013 (many slides courtesy of Rafael Irizarry). Genetics: the alphabet of life. Letters of DNA sequence carry the information

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Héctor Corrada Bravo CMSC702 Spring 2013 (many slides courtesy of Rafael Irizarry)

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  1. Introduction to epigenetics: chromatin modifications, DNA methylation and the CpG Island landscape Héctor Corrada Bravo CMSC702 Spring 2013 (many slides courtesy of Rafael Irizarry)

  2. Genetics: the alphabet of life • Letters of DNA sequence carry the information • How is this information read and parsed. • We need grammar!

  3. Differentiation Different genes are expressed during different stages and in different tissues

  4. (3.4x10-10 meters/bp) x (6x109 bp/genome) = ~2 meters/genome Radius of the nucleus is ~ 10 µM !!! Klug and Cummings, 1997

  5. [(6 x 109 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 106 nucleosomes/genome ~ 5 % of nuclear volume

  6. Conformation is dynamic! (we’ll discuss methods to assay this conformation later on…) http://www.albany.edu/~achm110/solenoidchriomatin.html

  7. We’ll study methods to assay a number of mechanisms of epigenetic regulation Nucleosome positioning and histone modifications DNA methylation In eukaryotes, DNA methylation usually occur at CpGdinucleotides

  8. TF TF TF TF TF TF Transcriptional regulation by nucleosome and histone modification Nucleosome positioning is mainly repressive Histone modification can be either active or repressive H3K9ac Ace H3K27me3 TF target site

  9. Histone code hypothesis “… multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions …” ― Strahl and Allis, Nature, (2000)

  10. DNA Methylation is sometimes repressive UnmethylatedCpGdinucleotides Methylated CpGdinucleotides Transcription repressors bound to methyl-group Robertson and Wolffe, Nat Rev Genet, 2000

  11. DNA methylation in cancer

  12. DNA methylation, histone modifications and nucleosome positioning are coordinated! New technologies are allowing us to now assay this coordination [Brinkman, et al., Genome Research 2012]

  13. Epigenetics: the grammar of life Epigenetics literally means above the genome

  14. One more thing…. How does a cell retain epigenetic state?

  15. Methylation A A G C T A A T G C T T T C G A T T A C G A CH3 CH3

  16. T T C G A T T A C G A A A G C T A A T G C T CH3 CH3 What happens during cell replication?

  17. T T C G A T T A C G A A A G C T A A T G C T T T C G A T T A C G A A A G C T A A T G C T CH3 CH3 What happens during cell replication?

  18. A A G C T A A T G C T A A G C T A A T G C T T T C G A T T A C G A T T C G A T T A C G A CH3 CH3 CH3 CH3 What happens during cell replication? DNA methylation is replicated!

  19. A A G C T A A T G C T T T C G A T T A C G A Liver A A G C T A A T G C T T T C G A T T A C G A Brain

  20. A A G C T A A T G C T T T C G A T T A C G A CH3 CH3 Liver A A G C T A A T G C T T T C G A T T A C G A Brain CH3 CH3

  21. A A G C T A A T G C T T T C G A T T A C G A A A G C T A A T G C T T T C G A T T A C G A CH3 CH3 CH3 CH3 Liver A A G C T A A T G C T A A G C T A A T G C T T T C G A T T A C G A T T C G A T T A C G A Brain CH3 CH3 CH3 CH3

  22. CpG Islands We said DNA methylation occurs at CpGdinucleotides. Where are they in the genome?

  23. CpGs are depleted

  24. Remaining ones cluster

  25. Remaining ones cluster Two modes: high CpG rate low CpG rate Proportion of CpG’s stratified by CG content.

  26. The clusters refered to as CpG Islands CpGs are depleted Remaining CpGs cluster into islands enriched near promoters New CGI definition: Irizarry et al. (2009) Mammalian Genome

  27. CpG Island definition Gardiner-Garden and Frommer Observed versus expected • N > 200 • GC-content > 50% • obs/exp > 0.6 • Lists contain 20,000 CGI • Irizarry et al. (2009) Mammalian Genome • Wu et al (2010) Biostatistics • Lists contain 100,000 CGI GpC Observed dinucleotides Takai and Jones PNAS 2002 use a stricter definition HMMbased definition CpG Expected (%G x %C)

  28. How do we measure DNA methylation?

  29. Liver Brain A A G C T A A T G C T T T C G A T T A C G A A A G C T A A T G C T T T C G A T T A C G A CH3 CH3 CH3 CH3

  30. CH3 CH3 T T C G A T T A C G A T T C G A T T A C G A T T C G A T T A C G A T T C G A T T A C G A T T C G A T T A C G A T T C G A T T A C G A A A G C T A A T G C T A A G C T A A T G C T A A G C T A A T G C T A A G C T A A T G C T A A G C T A A T G C T A A G C T A A T G C T A A G C T A A T G C T chr3:44,031,616-44,031,626 T T C G A T T A C G A CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 85% Methylation

  31. Bisulfite Treatment

  32. Bisulfite Treatment GGGGAGCAGCATGGAGGAGCCTTCGGCTGACT GGGGAGCAGTATGGAGGAGTTTTCGGTTGATT

  33. BS-seq GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTCGTAGTATCTGTC TATGTCGTAGTATTTG TATATCGTAGTATTTT TATATCGTAGTATTTG NATATCGTAGTATNTG TTTTATATCGCAGTAT ATATTTTATGTCGTA ATATTTTATCTCGTA ATATTTTATGTCGTA GA-TATTTTATGTCGT Coverage: 13 Methylation Evidence: 13 Methylation Percentage: 100% GATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTCAATATT

  34. BS-seq GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTCGTAGTATCTGTC TATGTCGTAGTATTTG TATATTGTAGTATTTT TATATCGTAGTATTTG NATATTGTAGTATNTG TTTTATATTGCAGTAT ATATTTTATGTCGTA ATATTTTATCTTGTA ATATTTTATGTCGTA GA-TATTTTATGTCGT Coverage: 13 Methylation Evidence: 9 Methylation Percentage: 69% GATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTCAATATT

  35. BS-seq GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTTGTAGTATCTGTC TATGTTGTAGTATTTG TATATTGTAGTATTTT TATATTGTAGTATTTG NATATTGTAGTATNTG TTTTATATTGCAGTAT ATATTTTATGTCGTA ATATTTTATCTTGTA ATATTTTATGTTGTA GA-TATTTTATGTCGT Coverage: 13 Methylation Evidence: 4 Methylation Percentage: 31% GATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTACGTTCAATATT

  36. BS-seq • Alignment is much trickier: • Naïve strategy: do nothing, hope not many CpG in a single read • Smarter strategy: “bisulfite convert” reference: turn all Cs to Ts • Smartest strategy: be unbiased and try all combinations of methylated/un-methylated CpGs in each read

  37. BS-seq • There are similarities to SNP calling • EXCEPT: we want to measure percentages • Use a binomial model to estimate p, percentage of methylation • Allow for sequencing errors, coverage differences, etc.

  38. Measuring DNA Methylation • Estimating percentages • Use “local-likelihood” method • Based on loess (Plot courtesy of Kasper Hansen)

  39. Y MeDIP (like ChIPchip) Total Reverse crosslinks Amplify Sequence Crosslink Lyse & Sonicate Reverse crosslinks Other controls for IP (e.g., no antibody, non-specific antibody) IP Amplify Sequence

  40. Next few lectures • Measuring DNA methylation • How to find genomic regions that are differentially methylated in two groups (say, cancer and normal) • Measuring nucleosome occupancy and histone modifications • First stabs at decoding the histone code • Determining genomic 3d structure

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