Slides - ENCODE

Players and Models of
Transcription Regulation in 3D genome
YijunRuan([email protected])
TheJacksonLaboratoryforGenomicMedicine
DepartmentofGene=csandGenomeSciences,UConnHealthCenter
at
ENCODEusersmee=ng,StanfordUniversity
June10,2016
Players and Models of
Transcription Regulation in 3D genome
Theplayers:DNA,Protein,RNA
Protein-mediatedchroma@ninterac@ons
RNA-mediatedchroma@ninterac@ons
3DNucleome
RinnandGu3manScience2014
ChIA-PETfor3Dgenomemapping
(mul@plexdatasets)
Protein
binding
Interaction
complex
Self-ligation
PETs
Inter-ligation
PETs
Binding peaks
Higher-order topological proximity
Clusters
Singletons
Inclusive:
High specificity and resolution:
•  Protein bindings,
•  Enriched chromatin interaction,
•  Non-enriched contacts (Hi-C like data)
•  Functional element specific,
•  Haplotype resolved,
•  Single nucleotide resolution
Fullwoodetal2009Nature;Lietal2012Cell;Tangetaletal2015Cell
InsituHi-C
vs.CTCFChIA-PET(GM12878)
Chr.7
10
PET=7190
Miseq
8
6
4
2
Chromosomescale
PET=57355
Hiseq
0
PET=329
6Mb
Miseq
Megabasescale
PET=2381
PET=66
Hiseq
0.6Mb
Miseq
Sub-megabasescale
PET=437
PET=27
Hiseq
0.2Mb
Miseq
kilo-basescale
Freq.
PET=196
Den.
Totalcontact
reads=4.9Billion
Raoetal2014Cell
TotalPETreads:1Miseq=6.4Million;1Hiseq=30.8Million
Tangetal2015Cell
Hiseq
Connec=ngloopstoformCTCFContactDomains(CCD)
Doubleknot
chr4:109556994-113054287 (3.5Mb)
Freq.
1000
CTCF loops
CCD
CTCF motifs
Den.
CTCF peaks
600
Hi-C TAD
in situ Hi-C loops
ChromHMM
100
RNA-Seq
100
Gene
Size distribution of loop and domain
2,267 3,852
Inward
motifs
159
Boundaries
4,177
(97%)
166
63
56
In situ Hi-C loop
CCD
0.50
0.50
Hi-C TAD
0.25
0.25
0.00
0.00
6
Outward
motifs
Indiv. CTCF loop
0.75
0.75
ecdf
CCD
Cumulative density
1.00
1.00
125
(3%)
1e+04
10kb
CCD=TAD
1e+05
100kb
1e+06
1e+07
1Mb
10Mb
span
Genomic span
1e+08
100Mb
RNAPIIassociatedchroma=ninterac=ons
Extra-genic enhancer-promoter interaction (EP)
Single gene complex
e
p
g
chr2:225000000-228000000
Inter-genic promoter-promoter interaction (PP)
Multi-gene complex
e
p
g
p
g
chr2:220000000-220180000
Lietal2012Cell
CCD
2Dmappingdata
CTCF
RNAPII
RNA-Seq
3Dvisualiza=on
3Dsimula=on
E
5 µm
CTCF RNAPII DNA
PrzemyslawSzala,DariuszPlewczynski
0.5 µm
Colocalization
PawelTrzaskoma,GrzegorzM.Wilczynski,
Common&cell-typespecific
chroma=nloopingstructures
CTCF
0
2
RNAPII
4
0
2
4
4
4
878
GM12
8
GM12
2
78
0
2
0
4
4
2
2
R = 0.61
Head
MCF7
HeLa
K562
4e-04
4e-04
0
Head
Tail
2e-04
2e-04
Tail
MCF7
K562
Density
Density
Density
6e-04
6e-04
R = 0.42
0
1e-04
1e-04
2e-04
2e-04
0e+000
0e+000
-10000
-10000-5000 0
0 500010000
10K-5000 00 500010000
10K
10K
±2.5K Distance ±2.5K
-10000
0 500010000
-10000-5000 0
0 500010000
10K-5000 0
10K
10K
±2.5K Distance ±2.5K
Common&celltype-specificCCDstructure
B-cell
Erythroid
Cervical
Breast
Gene=cvaria=onsalterchroma=ndomains
chr6:31426075-31930740 (504 kb)
GM12878
T|A
All loop
CCD
All peak
T
M loop
M peak
A
GM12878
Motifs
P loop
P peak
Biased SNP
HeLa
(169|37)* T|A
Loop
Peak
A/A
Loop
CCD
Peak
CTCFbindingmo@f
MCF7
MCF7
HeLa
A/A
Haplotype-resolvedinterac=onslinkedtogene=cs
CTCF ChIA-PET
SNP rs12936231 G|C
1000
All loop
All peak
1000
G
100
M loop
M peak
300
P loop
C
D’ with low LOD
D’ with high LOD
0.1
1.0
P peak
CEU
300
Biased SNP
Gene
(270 | 4)
IKZF3
GSDMB
ZPBP2
GSDMA
0.5
5.0
chr17:37911048-38179492
0
0.0
ORMDL3
M:AGTTACTTACATTAGCCCCCAGATGGAGTGAAACCATCAAGTA
P:AGTTACTTACATTAGCCCCCAGATGCAGTGAAACCATCAAGTA
High-riskSNPsforasthmaandautoimmunediseasealter
domain-widetranscrip@onofcertaingenes(Verlaan2009)
SNP-basedvalida=onofCTCFbindingandlooping
Individual 1
Heterozygous
G
G
T
?
T
Phased
Loop
Peak
Individual 2
Homozygous
Individual 3
G
G
G
G
Homozygous
T
T
T
T
3DGenomeStructureàGenomeFunc=on
3D chromatin architecture
Haplotype chromatin interaction
Individual 1
Topological domains
Heterozygous functional
G
T
G
T
Individual 2
CTCF-mediated contact domain
CTCF
Homozygous functional
G
G
G
G
RNAPII
Individual 3
Homozygous dysfunctional
T
T
T
Open
Active
Closed
Inactive
T
Topological
Gene=cvaria=onsààTraits/diseases
mechanism
Players and Models of
Transcription Regulation in 3D genome
Theplayers:DNA,Protein,RNA
Protein-mediatedchroma@ninterac@ons
RNA-mediatedchroma@ninterac@ons
3DNucleome
RinnandGu3manScience2014
Genome-wideapproachforRNA-chroma=ninterac=ons
RNAInterac@onwithChroma@nbyPairedEndTagSequencing,RICh-PET
RNA-chromatin
contacts
A
RNAtags
chr3R:7047557-7050982
Me18S-C1096
Me18S-A425
Psi28S-291
Me28S-G2017
(snoRNAs)
RpL3
(protein-coding)
Crosslinking
fragmentation
Linker annealing
cDNA synthesis
DNA ligation
RNA-Seq
DNA linker
RNA linker
RICh-PET
RNA tag
NNNNNN
RNA linker anneal
RT to make 1st cDNA
DNA linker ligation
NNNNNN
Proximity ligation
XIST target distribution in human
DNAtags
Chromosome
Chromosome
GM12878
HeLa
GM12878 XIST
HeLa XIST
15%
10%
2nd strand cDNA
5%
0%
chr 1
PET template
Amplification
Sequencing
2
4
5
7
6
8
9
10
11
12
13
14
15
17 19 21 X
16 18 20 22
roX2 target distribution in Drosophila
80%
Chromosome
roX2
60%
PET mapping
3
40%
Intra-chromosome
Inter-chromosome
20%
0%
chr 2L
2R
3L
3R
4
X
MeizhenZheng,OscarLuo
MostncRNAstargetac=veopenchroma=nloci
chr2L:16200000-18200000
30
RICh-PET
DNA tag
RICh-PET
DNA peak
n = 6599
DNA peak loci
DHS
ChromHMM
100
100
75
75
573
1656
V4
10000
RNAPII binding
peak loci
3819
551
RNA-Seq
588
H3K27ac
25
25
RNAPII
peak
n = 5770
100
100
60
615
0
0
Supported
Unsupported
3-way
Not
group
Shared
Shared
DHS
site
n = 5573
10
H3K27me3
50
50
763
chr2L:17300000-17550000
50
RICh-PET
DNA tag
DNA peak loci
DHS
ChromHMM
10000
RNAPII binding
peak loci
RNA-Seq
40
Density
30
30
20
10
0
P
T
E
O
A
R
H
HL
S
60
H3K27ac
Ø  Enrichedinpromoter&enhancerregions
10
H3K27me3
RNAPIIChIP/RICh-PETfordetec=ng
transcrip=onalRNA-chroma=ninterac=ons
chr2R:14474361-14615784
ChromHMM
DNase HS
15
RICh-PET
DNA tag
Peak call
Pol2-ab
500
Rep1
DNAtag
Intra-chromosome
400
Rep2
Inter-chromosome
30
coding
non-coding
Rep3
RNAPII/RICh-PET
RNAtag
RNA-Seq(RPKM)
6000
RNAPII
ChIP-Seq
100
RNA-Seq
100
60
H3K27ac
H3K27me3
10
RNAPIIRICh-PET(RPKM)
ncRNAcontactsenrichedatTADboundaryregions
chrX:11600000-13000000 (1.4 Mb)
RNA-Seq
Dnase HS
2000
RNAPII peak
1000
RNAPII loop
500
RNAPII RICh-PET
DNA peaks
5
§ 
roX2
§ 
7SK
§ 
Hrsomega
15
5
60
H3K27ac
H3K27me3
15
5kb resolution
7SK
roX2
Hsromega
Combinatorybindingbymul=plexproteinandRNAfactors
3819ncRNAtargetsites
chr3R:26198479-26310882
n=1207
n=1451
n=1023
TFChIP-Seq(modENCODE)
RNA-seq
DHS
RNAPII RICh-PET
DNA tag
7SK
Hsr omega
RNA
factors
H3K4me1
H3K4me3
RNA-Seq
Protein
factors
(modENCODE)
H3K27ac
H3K27me3
snRNA:7SK
Hsr omega
Modulatory
Cons@tu@ve
Others
TotesttheroleofncRNAs,…
x
x
x x x
Immuno-stain
RNase A -
ATAC-Seq
RNase A +
x
x
x
RNAPIIChIA-PET
chr2L:13196092-13314472
RNaseA+
(100µg/mL)
DAPI
RNA-seq
Chromatin state
DHS
20
Control, RNaseA-
(200µg/mL)
ATAC-seq
RNAPII
20
100µg/mL RNaseA
20
200µg/mL RNaseA
20
(400µg/mL)
15
2 µM
3 µM
RNaseAcontrol
RNAPII-loop
Merge
15
15
400µg/mL RNaseA
PET=14
Control, RNaseA-
PET=6
200µg/mL RNAseA
PET=6
400µg/mL RNaseA
Acknowledgements
Lab members
Collaborators
MeizhenZheng
WangPing
ZhonghuiTang
OscarLuo
XingwangLi
JianHuaCao
DanjuanWang
EmalyPiecuch
JufenZhu
GuoliangLi
JAX
CZZhang
XiaoanRuan
ZhengqingOuyang
DuyguUcar
MichaelS=tzel
JeffChuang
PaulMichalski
KrishnaKaruturi
KarolinaPalucka
JacquesBanchereau
KenPaigen
CharlesLee
EdLiu
RouxFamily
UConn
BrentGraveley
MarcLalande
JGI
Chia-LinWei
NIH
RafaelCasellas
JohnO’Shea
WarrantLeonard
UniversityofWarsaw,Poland
DariuszPlewczynski
PrzemekSzałaj
GrzegorzM.Wilczynski
MNHN,Paris
LaurentSachs