© 2013 american society of plant biologists methods for studying epigenetic modifications

© 2013 American Society of Plant Biologists

Methods for studying epigenetic modifications

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


Methods for studying epigenetic modifications

TTCGCCGACTAA TTCGCCGAuTAA

•DNA methylation– bisulfite sequencing

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

•siRNA production – deep sequencing


Bisulfite treatment differentiates cytosine and methylcytosine

Bisulfite treatment

TTCGCCGACTAA

No treatment

TTCGCCGACTAA

TTCGCCGAuTAA

Methyl-cytosine

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

O N

NH2

N

~O N

NH2

N

~

CH3

cytosine 5-methylcytosine

O N

NH2

N

~

CH3

O N

O

N

~uracil 5-methylcytosine

Bisulfite treatment


Bisulfite treatment differentiates cytosine and methylcytosine

Bisulfite treatment

TTCGCCGACTAA

No treatment

TTCGCCGACTAA

TTCGCCGAuTAA

TTCGCCGACTAA TTCGCCGATTAA

Methyl-cytosine

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.


Chromatin Immunoprecipitation (ChiP)

Modified histone (e.g. H3K4me)

Cross-link DNA to histone



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





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)

GATC

Dam methylase from E. coli

Protein of interest (e.g. LHP1)

A fusion protein is made of Dam and the protein of interest

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

Dam is an adenine methyltransferase with specificity for the sequence GATC


Methylation can be detected by methylation sensitive enzymes

Region near binding site

PRODUCT FORMED

GATC

DpnII restriction endonuclease digestion (methylation sensitive)

GATC GATC

PCR amplification

GATCGATC

NO PRODUCT

Region not near binding

site

GATC


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

Fluorescently-labeled ddNTPs

Primer 5’CCGGTTAATemplate strand 3’GGCCAATTAAGCGGCTGATT

Base

Four standard dNTPs

+

+

+ DNA Polymerase




Primer 5’CCGGTTAATTemplate strand 3’GGCCAATTAAGCGGCTGATT

Primer 5’CCGGTTAATTCTemplate strand 3’GGCCAATTAAGCGGCTGATTT

DNA polymerase incorporates unlabeled dNTPs (that allows normal extension of the strand) or labeled ddNPTs (that do not allow the strand to extend any longer),

resulting in a mixture of differently sized labeled molecules.



Detector

Laser

Separate DNA molecules by size using electrophoresis

TTCGCCGACTAA


A method of next-generation sequencing (NGS): Pyrosequencing

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


Primer 5’CCGGTTAATTemplate strand 3’GGCCAATTAAGCGGCTGATT

T


Pyrosequencing - chemistry

PYROPHOSPHATE

+ Adenosine 5’ phosphate sulphate ATP

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

Sulfurylase



+PYROPHOSPHATE

Adenosine 5’ phosphate sulphate ATP

ATP promotes light production by luciferase

+ATP

Luciferin

LUCIFERASE

Light production

ATP Sulfurylase



T

T


Pyrosequencing – data acquisition

dTTP

dTTP

No Reaction

5’CCGGTTAA3’GGCCAATTGAGC....

5’CCGGTTAAT3’GGCCAATTACGA...

PPi ATPdNTPs are added sequentially and additions monitored by light production.

CYCLE ONE



dCTP

dCTP

5’CCGGTTAAC3’GGCCAATTGAGC....

5’CCGGTTAAT3’GGCCAATTACGA...

CYCLE TWO

PPi ATP

No ReactiondNTPs are added sequentially and additions monitored by light production.



dTTP dGTP dCTP dATP dTTP dGTP dCTP dATP

TIME

AG

AC

The sequence of light production at each spot (called a 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.


Another “next-gen” sequencing method: Illumina

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.

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.


Comparison of next-generation sequencing platforms

Reprinted by permission from Macmillan Publishers Ltd: Quail, M.A., Smith, M., Coupland, P., Otto, T.D., Harris, S.R., Connor, T.R., Bertoni, A., Swerdlow, H.P., and Gu, Y. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 13: 341.


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.

Abundance of siRNAs

GREEN = H3K27me3PURPLE =

methylcytosine

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

EPIGENOMICS and METHYLOMICS


Reprinted by permission from Macmillan Publishers Ltd: Zhong, S., Fei, Z., Chen, Y.R., Zheng, Y., Huang, M., Vrebalov, J., McQuinn, R., Gapper, N., Liu, B., Xiang, J., Shao, Y., and Giovannoni, J.J. (2013). Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol. [in press].

Density of methylated DNA and other features in chromosomes of the tomato fruit

The tomato methylome

© 2013 american society of plant biologists methods for studying epigenetic modifications

Documents

histone slide

histone shear dna

h3k4me slide

sequence gatc slide

h3k4me crosslink dna

ch3 o n o n

histone affinity

near binding site gatc