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Genome-wide analysis of enhancer-RNA transcription reveals regulatory mechanisms by an anti-diabetic drugs (TZD) in adipocytes
Kyoung-Jae Won, Ph.D.Sep 8 2014
Genomics 2014
Non-coding RNA Annotation in ENSEMBL
tRNAMt-tRNArRNAscRNAsnRNAsnoRNAmiRNAMisc_RNAlincRNA
eRNA(enhancer driven RNA)
DiscussionseRNA: Transcripts at enhancers
Kim et al (2010) Nature
Enhancer-driven RNAs (eRNAs) are an important factor for gene regulation
Lam et al (2014) Trends in Biochemical Sci.
Function of eRNA : noise ? : activating role ? : scaffold ?
At least eRNAs show enhancer activity
Transcription: An Understudied Step in Gene Regulation
Gene Regulation TranscriptionSteady-state
mRNAs
GRO-seq• Global Run On – sequencing • Isolate all newly synthesized RNAs in a cell• Measures active transcription rather than steady state mRNA levels• Maps the position and orientation of new transcripts• Earliest changes can identity primary target genes rather than secondary targets• Unbiased detection of novel transcripts, including non-coding RNAs• detects enhancer RNAs
Core et al, Science, 2008
Thiazolidindiones (TZDs)
Activates PPARs (peroxisome proliferator-activated receptor)Effective anti-diabetic drugs: increase insulin sensitivitySide effect: weight gain, cardiac loads, peripheral edema
Still widely used for the treatment of diabetes despite its notorious side-effects
+ strand
+ strand
Before Rosi
Rosi + 10m
Rosi + 30m
Rosi + 1h
Rosi + 3h
PPARg
Up Regulation of Transcription Due to Rosi
CEBPa
Pre-Adipocyte
Fabp4
Ppargc1b: ↑
Adipoq: ↑
Log2-FoldChangein Gene Body
Up and Down Regulation of Transcription Due to Rosi
Cited2: ↓
Cav1: ↓
567
1384
GROseq Profiles at TSS in Adipocyte
Log2-FoldChangein Gene Body
NascentGene Transcript
Steady-statemRNA
Nascent Transcription Changes Precedes mRNA Regulation
GROseq vs Steady-state mRNALog2 Fold Change Over Rosi + 0
10m 30m 1h 3h
30m
GROseq
Ste
ady-
stat
e m
RN
A
Log2-FoldChangein Gene Body
NascentGene Transcript
Steady-statemRNA
Nascent Transcription Changes Precedes mRNA Regulation
GROseq vs Steady-state mRNALog2 Fold Change Over Rosi + 0
10m 30m 1h 3h
30m
GROseq
1h
2h
6h
12h
24h
36h
48h
Ste
ady-
stat
e m
RN
A
Log2-FoldChangein Gene Body
Nascent Transcription Change Precedes mRNA Regulation
10m 30m 1h 3h
30m
1h
2h
6h
12h
24h
36h
48hS
tead
y-st
ate
mR
NA
Gene Body Transcript
Ste
ad
y-st
ate
mR
NA
1h
30m
2h
6h
12h
24h
36h
48h
3h10m 30m 1h
Log2-FoldChangein Gene Body
Enhancer RNAS (eRNAs)
+ strand
- strand
PPARg
CEBPa
Before Rosi
Rosi + 10m
Rosi + 30m
Rosi + 1h
Rosi + 3h
eRNA
Rosi-regulated eRNAs
Regulation of eRNAs and Nascent Transcription by Rosi
log2FC(eRNA)
log
2FC
(Gen
e Tr
ansc
rip
t)
eRNA3h10m 30m 1h
3−3 0 3−3 0 3−3 0 3−3 0
2
−2
0
2
−2
0
2
−2
0
2
−2
0
Gen
e tr
ansc
ript
Correlation between eRNA and gene regulation
3h10m 30m 1h
eRNA
Ge
ne
tra
nscr
ipt
3h
10m
30m
1h
eRNA Changes Precede Nascent Gene Transcript
EnhancerRNA
NascentGene Transcript
Steady-statemRNA
0 10 20 30 40 50 600
1
2
3
4Pdk4
GeneeRNA
Rosi treatment (minutes)
Rela
tive
expr
essio
n
eRNA induction preceeds gene induction
eRNAs up-regulation is associated with PPARγ
eRNAs up-regulation is associated with PPARγ
eRNAs down-regulation is associated with multiple-factors including C/EBP, Fosl2,JunD and ATF2
: Repression is not directly mediated by PPARγ
1 2 3 4 5 Arbp Ins0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 Fosl2 ChIP
Per
cent
Inp
ut
1 2 3 4 5 Arbp Ins0
0.5
1
1.5 ATF-2 ChIP
Per
cent
Inp
ut
1 2 3 4 5 Arbp Ins0
0.5
1
1.5JunD ChIP
Ad
Ad + 1h rosi
Per
cent
Inp
utStrength of binding did not change upon rosi treatment
The majority of eRNA are upregulated by 10 minutes: downregulated eRNAs can be secondary
eRNA changes are associated with Med1 change
log
2(ta
g c
ou
nts
[ro
si/
un
tre
ate
d])
Med1 ChIP-seq
Down Unchanged Up
** **
eRNA regulation by rosi
-2
-1
0
1
2
3
-3
Upon rosi treatment Med1 recruitment significantly decreased at downregulated eRNAs and increased at upregulated eRNAs relative to unregulated eRNAs
Squelchingis a biological phenomenon in which a transcriptional activator acts to inhibit the expression of another gene.
GROseq measures the level of eRNAs and gene transcript
Discussions
Integrative studies using GROseq revealed the rosi-associated regulatory mechanisms• Gene transcription levels needs time until they reach to steady
state levels• eRNA fires before transcriptional initiation• up-regulation is mediated by PPARγ• Multiple TFs are associated with down-regulation• down-regulation is mediated by co-activator redistribution
Step et al. (2014) Genes and Development
Supported by • NIH/NIDDK, • Institute for Diabetes Obesity and Metabolism
at the University of Pennsylvania• Pharma Foundation
UPENNMitchell Lazar
Sonia StemPatrick SealeDavid StegerKlaus Kaestner
Rinho Kim
Thanks to
Nha NguyenHee-Woong LimShuo LiInchan Choi
Metabolic BiologyMitchell Lazar, Klaus KeastnerPatrick Seale, David StegerStem CellKennett Zaret, Montserrat AgueraChromatin remodelers, histone variantsBenjamin Garcia, Hua-YingEpigenetic landscapesWei Wang, Bing Ren (UCSD)Cancer biologyCathryn Wellen, Mariuz Wasik
In the lab..
Integrative studiesRNAseq, GROseq
ChIPseq, ChIP-exoCLIP-seq
mC, 5hmC,Histone modification