Introduction to epigenetics: chromatin modifications, DNA methylation and the CpG Island landscape

Héctor Corrada BravoCMSC702 Spring 2013

(many slides courtesy of Rafael Irizarry)

Genetics: the alphabet of life

• Letters of DNA sequence carry the information

• How is this information read and parsed.

• We need grammar!

Differentiation

Different genes are expressed during different stages and in different tissues

(3.4x10-10 meters/bp) x (6x109 bp/genome) = ~2 meters/genome

Radius of the nucleus is ~ 10 µM !!!

Klug and Cummings, 1997

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

http://www.albany.edu/~achm110/solenoidchriomatin.html

Conformation is dynamic! (we’ll discuss methods to assay this conformation later on…)

We’ll study methods to assay a number of mechanisms of epigenetic regulation

DNA methylation Nucleosome positioning andhistone modifications

In eukaryotes, DNA methylation usually occur at CpG dinucleotides

Transcriptional regulation by nucleosome and histone modification

Nucleosome positioning is mainly repressive

Histone modification can be either active or repressive

TF

TF

TF target site

TF

TF

Ace

TFH3K27me3

H3K9ac

TF

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)

DNA Methylation is sometimes repressive

Robertson and Wolffe, Nat Rev Genet, 2000

Unmethylated CpG dinucleotides

Methylated CpG dinucleotides

Transcription repressors bound to methyl-group

DNA methylation in cancer

DNA methylation, histone modifications andnucleosome positioning are coordinated!

New technologies are allowing us to now assay this coordination

[Brinkman, et al., Genome Research 2012]

Epigenetics: the grammar of life

Epigenetics literally means above the genome

One more thing….

How does a cell retain epigenetic state?

Methylation

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

What happens during cell replication?

TTCGATTACGA

AAGCTAATGCT

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

What happens during cell replication?

TTCGATTACGA

AAGCTAATGCT

TTCGATTACGA

AAGCTAATGCT

CH3 CH3CH3 CH3

What happens during cell replication? DNA methylation is replicated!

TTCGATTACGA

AAGCTAATGCT

TTCGATTACGA

AAGCTAATGCT

Liver

Brain

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

Liver

Brain

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

Liver

Brain

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

CpG Islands

We said DNA methylation occurs at CpG dinucleotides.

Where are they in the genome?

CpGs are depleted

Remaining ones cluster

Remaining ones cluster

Proportion of CpG’s stratified by CG content.

Two modes:high CpG ratelow CpG rate

The clusters refered to as CpG Islands

CpGs are depletedRemaining CpGs cluster into islands enriched near promoters

New CGI definition: Irizarry et al. (2009) Mammalian Genome

CpG Island definition

Gardiner-Garden and Frommer • 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

Observed versus expected

Obs

erve

d di

nucl

eotid

es

Expected (%G x %C)

Takai and Jones PNAS 2002 use a stricter definitionHMM based definition

GpC

CpG

How do we measure DNA methylation?

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

Liver Brain

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3TTCGATTACGA

AAGCTAATGCT

CH3

CH3

TTCGATTACGA

AAGCTAATGCT

CH3

CH3

85% Methylationchr3:44,031,616-44,031,626

Bisulfite Treatment

Bisulfite Treatment

GGGGAGCAGCATGGAGGAGCCTTCGGCTGACT

GGGGAGCAGTATGGAGGAGTTTTCGGTTGATT

BS-seq

GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTCGTAGTATCTGTC TATGTCGTAGTATTTG TATATCGTAGTATTTT TATATCGTAGTATTTG NATATCGTAGTATNTG TTTTATATCGCAGTAT ATATTTTATGTCGTA ATATTTTATCTCGTA ATATTTTATGTCGTA GA-TATTTTATGTCGTGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTAC

GTTCAATATT

Coverage: 13Methylation Evidence: 13Methylation Percentage: 100%

BS-seq

GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTCGTAGTATCTGTC TATGTCGTAGTATTTG TATATTGTAGTATTTT TATATCGTAGTATTTG NATATTGTAGTATNTG TTTTATATTGCAGTAT ATATTTTATGTCGTA ATATTTTATCTTGTA ATATTTTATGTCGTA GA-TATTTTATGTCGTGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTAC

GTTCAATATT

Coverage: 13Methylation Evidence: 9Methylation Percentage: 69%

BS-seq

GTCGTAGTATTTGTCT GTCGTAGTATTTGTNN TGTTGTAGTATCTGTC TATGTTGTAGTATTTG TATATTGTAGTATTTT TATATTGTAGTATTTG NATATTGTAGTATNTG TTTTATATTGCAGTAT ATATTTTATGTCGTA ATATTTTATCTTGTA ATATTTTATGTTGTA GA-TATTTTATGTCGTGATCACAGGTCTATCACCCTATTAACCACTCACGGGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTATTATTTATCGCACCTAC

GTTCAATATT

Coverage: 13Methylation Evidence: 4Methylation Percentage: 31%

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

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.

Measuring DNA Methylation

• Estimating percentages• Use “local-likelihood”

method– Based on loess

(Plot courtesy of Kasper Hansen)

Crosslink YLyse & Sonicate

IP Reverse crosslinks

Total Reverse crosslinks Amplify

Amplify

Sequence

Sequence

MeDIP (like ChIPchip)

Other controls for IP(e.g., no antibody, non-

specific antibody)

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