Richard M. Myers

Richard M. Myers rmyers@hudsonalpha.org

Whole genome analysis of transcriptional regulation in humans

Myers lab HudsonAlpha Institute

Barbara Wold lab Caltech

Richard M. Myers

Big Goal

To understand how genetic, epigenetic and genomic variation contribute to

human traits, especially diseases and differential responses to the

environment

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Richard M. Myers

TBP

TAFs

TFIIB

TFIIF TFIIE TFIIH

RNA PolII

TATA Inr

1% of genome is protein-coding

~3% of our genome is evolutionarily constrained but non-coding (i.e., likely regulatory)

Identify regulatory sequences, the proteins that control them, their interactions, and the effects of

DNA sequence variation on them 3

Smaller Goal

Richard M. Myers

One transcription factor short story Tim Reddy, Myers Lab, ENCODE

GR binds hormone in cytoplasm, translocates to nucleus

Activates and represses transcription of many genes

The Glucocorticoid Receptor (GR)

Richard M. Myers

Genome-wide view of GR occupancy

Glucocorticoid Response Element

Revised GRE

4,392 sites of GR occupancy identified

Reddy et al. (2010) Genome Research. 19: 2163-2171. PMCID: PMC2792167

Co-occupancy with FosL2

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Richard M. Myers

PER1 (circadian rhythm) gene is upregulated by GR

Richard M. Myers

PER1 (circadian rhythm) gene is upregulated by GR

PER1 gene is activated at 10-20 times lower concentrations of cortisol

Richard M. Myers

pasdfasdf

108 interactomes from our ENCODE group

GM12878 ATF3 BATF BCL3 BCL11A EBF EGR1 GABP IRF4 NRSF P300 (2) PAX5 PBX3 POU2F2 PU.1 RXRA SIN3A SIX5 SRF SP1 TAF1 TCF12 USF1 YY1 ZBTB33 (2) RNA Pol2

K562 BCL3 BCLAF1 EGR1 GABP GATA2 HEY1 NRSF PU.1 SIN3A SIX5 SP1 SRF TAF1 TCF12 USF1 ZBTB33 (2) RNA Pol2

HepG2 BHLHE40 FOSL2 GABP HEB HEY1 JUND NRSF P300 RXRA SIN3A SP1 SRF TAF1 USF1 ZBTB33 (2) RNA Pol2

hESC BCL3 BCL11A EGR1 HEB NRSF RXRA SIN3A SIX5 SP1 SRF TAF1 USF1 (2) RNA Pol II

HeLa GABP NRSF TAF1 RNA Pol2

A549 CTCF (5) GR USF1 RNA Pol2

BE2-C NRSF

Jurkat RNA Pol2

PANC-1 NRSF

PFSK-1 FOXP2 NRSF

SK-N-MC FOXP2

SK-N-SH RA NRSF

U87 NRSF

GM12891 PU.1 POU2F2 TAF1 (2) RNA Pol2

GM12892 PU.1 POU2F2 TAF1 (2) RNA Pol2

Tier 1 Tier 2 Tier 3

ECC1 ER RNA Pol2

36 Transcription factors 16 Cell lines

8 http://genome.ucsc.edu/ENCODE/

Richard M. Myers

DNA methylation Jay Gertz, K-T Varley, Tim Reddy, Flo Pauli, Myers Lab

Cytosine 5‐Methylcytosine

CH3

O

HH H

HOH

P OO‐

O

H

NO

N

NH2

CH2

O‐

DNAMethyltransferase

S‐adenosylmethionine

5’…..CG…..3’3’…..GC…..5’

O

HH H

HOH

P OO‐

O

H

NO

N

NH2

CH2

O‐

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Richard M. Myers

MspI Digest

Ligate Methylated illumina PE Adapters: all C’s in adapter are 5methyl-C =

Gel Extraction/ Size Selection

Sodium Bisulfite Treatment

PCR

Fill in 3’ recessed end and leave 3’ A overhang

……C3 ……GGC5

5CGG…… 3AGCC……

……CCGA3 …..GGC5

T3

T3

40-120 bp + 65bp in Adapters = 105 – 185 bp

5CGG…… 3C……

5CGG…… 3AGCC……

……CCGA3 …..GGC5

5CGG…… 3C……

……C3 …..GGC5

Measure methylation on a wide scale RRBS (Reduced Representation Bisulfite Sequencing; Meissner et al. Nature Aug 2008)

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Richard M. Myers

DNA methylation around transcription start sites

0

10

20

30

40

50

60

70

80

‐5 ‐4 ‐3 ‐2 ‐1 0 1 2 3 4 5

Percen

tMethylaDo

n

DistancefromTSS(kb)

HeLaRep1

HeLaRep2

K562Rep1

K562Rep2

GM12878Rep1

GM12878Rep2

HepG2Rep1

HepG2Rep2

hESCRep1

hESCRep2

GM12892Rep1

GM12892Rep2

HUVECRep1

ECC‐1Rep1

ECC‐1Rep2

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Richard M. Myers

Blood PrimaryTissueandCellLines

LeukemiaCellLines

CancerCellLines

MostV

aryingCpG

s(Top

1.5%)

StandardDeviaDo

n>35

%

0%Methyl

50%Methyl

100%Methyl

Unsupervised clustering of 112 samples

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Richard M. Myers

GOTermEnrichmentP=8.19e‐51• Sequence‐specificDNAbindingtranscripDonfactoracDvity

• 142GeneswithVariableCpGs/658GenesinGOTermCategory• ALX1‐4,DBX1‐2,FOXA‐P,GATA2‐4,HOXA‐D,IRX1‐6,LBX,LHX,NKX2,NKX6,NOTCH,OTX,PAX6‐8,PITX1‐3,POU3F4,POU4F2,POU6F2,RARA,SIX1‐2,TBX1‐3,TLX2,3

1,104genesassociatedwithmostvariableCpGs

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PromoterTransfacMoGfEnrichmentP=6.69E‐36• VitaminDReceptor(VDR)Bindingsite:GGGKNARNRRGGWSA

• 705GeneswithVariableCpGs/9274GeneswithmoDf• NuclearreceptortranscripDonfactoracDvatedbyvitaminD• FormsaheterodimerwiththereDnoid‐Xreceptor• BindstohormoneresponseelementsonDNAresulDnginexpressionortransrepression

Richard M. Myers

HA‐GD‐1 HA‐GD‐3

HA‐GD‐5HA‐GD‐6

HA‐GD‐2 HA‐GD‐4

HA‐GD‐7

HA‐GD‐8

HA‐GD‐9

DNAmethylaDoninathree‐generaDonfamily

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Richard M. Myers

Family members are similar

1

2 4

3

7

8

6 5

78253416

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Richard M. Myers

TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGCGTGGTGTGGTG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGTGGTGGGAGGAGCGTGGTGTGGGG

CpG:

>hg19_dna range=chr6:170403177-170403217 CGGGAGACCCTGCGGTGGGAGGAGCGTGGTGTGGCG >chr6-170403179-F-0-7-8-9-12-24-34 TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG

Genomesequence:

Illuminareads:

Detecting allele-specific methyation

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Richard M. Myers

TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG TGGGAGATTTTGTGGTGGGAGGAGCGTGGTGTGGTG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGCGGTGGGAGGAGCGTGGTGTGGGG CGGGAGATTTTGTGGTGGGAGGAGCGTGGTGTGGGG

CpG:

>hg19_dna range=chr6:170403177-170403217 CGGGAGACCCTGCGGTGGGAGGAGCGTGGTGTGGCG >chr6-170403179-F-0-7-8-9-12-24-34 TGGGAGATTTTGTGGTGGGAGGAGTGTGGTGTGGTG

Genomesequence:

Illuminareads:

SNP:

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Detecting allele-specific methyation

Richard M. Myers

Allele-specific methylation (ASM) is prevalent

5.8%ofSNPsexhibitASMMostASMisnotallornothing 18

Richard M. Myers

Thanks to Caltech Barbara Wold Ali Mortazavi Brian Williams Georgi Marinov Brandon King Ken McCue Diane Trout Katherine Fisher Jost Vielmetter Shirley Pepke

HudsonAlpha

Tim Reddy Jay Gertz K-T Varley Flo Pauli Chris Partridge Kevin Bowling Preti Jain Anita Bansal Mike Muratet Babs Pusey Kim Newberry Jason Dilocker Amy Woodall Stephanie Parker

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National Human Genome Research Institute