Methods for studying epigenetic modifications

www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0110b

Methods for studying epigenetic modifications

DNA methylation bisulfite sequencing
TTCGCCGACTAA TTCGCCGAuTAA

Histone modification
chromatin immunoprecipitation (ChIP) DNA adenosine methylation identification (DamID)

siRNA production deep sequencing

Bisulfite treatment differentiates cytosine and methylcytosine

NH2 N N NH2 CH3

Methylcytosine

TTCGCCGACTAA No treatment Bisulphite treatment TTCGCCGAuTAA

~
cytosine

~
5-methylcytosine

Bisulfite treatment
O N N NH2 CH3

TTCGCCGACTAA When DNA is bisulfite treated, unmethylated cytosine is converted to uracil. Methylcytosine is not affected.

~
uracil

~
5-methylcytosine

Bisulfite treatment differentiates cytosine and methylcytosine

Methylcytosine

TTCGCCGACTAA No treatment Bisulphite treatment TTCGCCGAuTAA TTCGCCGATTAA

After bisulfite treatment, unmethylated Cs are read as T and so differ in the treated and untreated samples. By contrast, methyl-C is read as C and is the same as the reference sequence.

TTCGCCGACTAA TTCGCCGACTAA

Chromatin Immunoprecipitation (ChiP) Modified histone

(e.g. H3K4me) Cross-link DNA to histone

Chromatin Immunoprecipitation (ChiP)

Cross-link DNA to histone

Shear DNA to smaller pieces; add specific antibody to modified histone; affinity purify antibody and bound histone/DNA complex. `

Chromatin Immunoprecipitation (ChiP)

Cross-link DNA to histone

Remove crosslink, purify DNA, examine by PCR, sequencing or microarray

Shear DNA to smaller pieces; add specific antibody to modified histone; affinity purify antibody and bound histone/DNA complex. `

DNA adenine methylation ID (DamID)

A fusion protein is made of Dam and the protein of interest Dam is an adenine methyltransferase with specificity for the sequence GATC

Dam methylase from E. coli

Protein of interest (e.g. LHP1)

The fusion protein binds to selected regions of chromatin (e.g. H3K27me3) and methylates adenines at nearby GATC sites

Methylation can be detected by methylation sensitive enzymes

Region not near binding site GATC GATC
DpnII restriction endonuclease digestion (methylation sensitive)

Region near binding site

GATC
PCR amplification

GATC

GATC NO PRODUCT

GATC PRODUCT FORMED

Deep sequencing by next generation DNA sequencing methods

Classical DNA sequencing one molecule examined at a time

Next generation DNA sequencing one million molecules examined at a time

Reprinted by permission from Macmillan Publishers, Ltd: NATURE copyright 2005. Margulies, M., et al., (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.

Conventional DNA sequencing

Base G A

+
Four standard dNTPs

Fluorescently-labeled ddNTPs

Primer Template strand

5CCGGTTAA 3GGCCAATTAAGCGGCTGATT

DNA Polymerase

Conventional DNA sequencing

Primer Template strand 5CCGGTTAA 3GGCCAATTAAGCGGCTGATT

Primer Template strand

5CCGGTTAAT 3GGCCAATTAAGCGGCTGATT

Primer Template strand

5CCGGTTAATTC 3GGCCAATTAAGCGGCTGATTT

DNA polymerase incorporates unlabeled dNTPs or labeled ddNPTs, resulting in a mixture of differently sized labeled molecules.

Conventional DNA sequencing

Separate DNA molecules by size using electrophoresis
Detector Laser

TTCGCCGACTAA

One method of next generation sequencing: Pyrosequencing

Primer Template strand 5CCGGTTAA 3GGCCAATTAAGCGGCTGATT

In pyrophosphate sequencing, no labeled nucleotides or ddNTPs are used. The pyrophosphate liberated after a nucleotide addition acts as a substrate.....

Primer Template strand

5CCGGTTAAT 3GGCCAATTAAGCGGCTGATT

Pyrosequencing - chemistry

PYROPHOSPHATE

Adenosine 5 phosphate sulphate

ATP Sulfurylase

....for ATP sulfurylase, to produce ATP.....

ATP

Pyrosequencing - chemistry
ATP promotes light production by luciferase

PYROPHOSPHATE

Adenosine 5 phosphate sulphate

ATP Sulfurylase

LUCIFERASE

ATP

+
Luciferin

Light production

Pyrosequencing - chemistry

T T

Pyrosequencing data acquisition

5CCGGTTAA 3GGCCAATTGAGC....

dTTP No Reaction

CYCLE ONE

dTTP dNTPs are added sequentially and additions monitored by light production.
5CCGGTTAAT 3GGCCAATTACGA...

PPi

ATP

Pyrosequencing data acquisition

5CCGGTTAAC 3GGCCAATTGAGC....

dCTP

CYCLE TWO

PPi

ATP

dCTP dNTPs are added sequentially and additions monitored by light production.
5CCGGTTAAT 3GGCCAATTACGA...

No Reaction

Pyrosequencing data acquisition

dTTP dGTP dCTP dATP dTTP dGTP dCTP dATP

TIME

AG

The sequence of light production at each spot (called a AC flowgram) reveals the DNA sequence.

A pyrosequencing flowgram

Reprinted by permission from Macmillan Publishers, Ltd: NATURE copyright 2005. Margulies, M., et al., (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.

Other next generation sequencing methods

Other types of next generation sequencing have been developed that share the features of high throughput, and massive parallelism to reduce labor and reagent costs.

Reprinted by permission from Macmillan Publishers, Ltd: NATURE Biotechnology Copyright 2008. Shendure, J., and Ji, H. (2008) Next-generation DNA sequencing. Nature Biotech. 26: 1135-1145.

Using next-generation sequencing, epigenetic modifications can be identified genome-wide: EPIGENOMICS

GREEN = H3K27me3 PURPLE = methylcytosine

Abundance of siRNAs

Kasschau KD, Fahlgren N, Chapman EJ, Sullivan CM, Cumbie JS, et al. 2007 Genome-Wide Profiling and Analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57. Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., Jacobsen, S.E. (2007) Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 5: e129.