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