An Epigenomic and Transcriptional Basis for Insulin Resistance · ftfH ll MIR4268 SNORD114-29 CSF3R S100A8 Pre-ad i pocyte Macrophage . Histone profiles suggest the presence of novel

An Epigenomic and Transcriptional Basis for Insulin Resistance

Evan Rosen

ENCODE Research Applications and Users Meeting 2015

1990

No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30%

2009 1995 2000 2005

Behavioral Risk Factor Surveillance System, CDC

Obesity

2009 1995 2000 2005

<6.3% 6.4%–7.5% 7.6%-8.8% 8.9%–10.5% >10.6%

Diabetes

Obesity and diabetes trends among US adults

What are the critical transcriptional pathways that underlie key transitions or distinctions in adipose biology?

adipogenesis

insulin resistance

thermogenesis

insulin insulin

Find candidate TFs Identify target genes Function

Function Identify cognate TFs Find cis motifs

Covalent histone modifications

The epigenome

DNA methylation

Noncoding RNA

-2 0 3 9

Human adipose stromal cells (Lipoaspirate explants)

-2 0 2 7

Mouse 3T3-L1 cells (Clonal cell line)

Induction

Day

Day

Comparative epigenomic analysis of L1 and hASC adipogenesis

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

hASC (day -2)

L1 (day -2)

Pparg

PPARG

Comparative epigenomic analysis of L1 and hASC adipogenesis

Cell, 2010 143:156

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

L1 (day 0)

hASC (day 0)

Pparg

PPARG

Comparative epigenomic analysis of L1 and hASC adipogenesis

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

L1 (day 2)

hASC (day 3)

Pparg

PPARG

Comparative epigenomic analysis of L1 and hASC adipogenesis

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

H3K27ac

H3K4me1

H3K4me2

H3K4me3

H3K27me3

H3K36me3

L1 (day 7)

hASC (day -2) hASC (day 0) hASC (day 3) hASC (day 9)

Pparg

PPARG

Comparative epigenomic analysis of L1 and hASC adipogenesis

Strategy for identification of sequence-specific regulators

CAGCCATGCCAGCCT AGTCACTGACCATAA CAAAACTTCTGTTTT TTACCTAGAGAACCC

CCTAGAGAACCCTGC TTGGACATGTTTGTA CATGTTTCCTCCTGA GGGTGTTAGTCCCTC

GTTAGTCCCTCGGGG AGGCAGAGGTCACTG CCTGGACTTGCTGAG TCACCCTGTCAGCCT

Ranked list of enriched TF motifs

Database of TF motifs

Cell type-specific enhancer sequences

Cell, 2010 143:156

Motif ranks from adipogenesis recover many known regulators

Most enriched in pre-adipocyte- specific enhancers

Most enriched in adipocyte- specific enhancers

?

?

Cell, 2010 143:156

Knockdown of PLZF or SRF enhances adipogenesis

Cell, 2010 143:156

<( z 0:: E Qj 0::

shluc

l::]shl uc -shPLZF l::lshSRF

shPLZF shSRF

'"

Zbtb16 Srt

Insulin resistance: is there a common molecular denominator?

Infection/sepsis Burn injury Starvation

Also:

Many molecular mediators have been proposed:

–  Cortisol –  TNF-α –  IL-6 –  Growth hormone –  Insulin –  Glucose –  Free Fatty Acids –  Glucosamine

Insulin resistance: is there a common molecular denominator?

To what extent are molecular pathways shared in these conditions?

Insulin resistance

glucose growth hormone

cortisol TNF glucosamine

Insulin resistance

glucose growth

hormone cortisol TNF glucosamine

Common mediator?

Cellular models of insulin resistance: TNF, dexamethasone

Insulin Resistance

Insulin

WT

Treated Glucose uptake

3T3-L1 Adipocytes

+ TNF, Dex

TNF Dex

–  Both GCs and TNF are elevated in multiple insulin resistant states

–  Exogenous GCs/TNF induce insulin resistance in vivo –  TNF-/- mice are protected from diet-induced insulin

resistance –  Glucocorticoid antagonists block diet-induced insulin

resistance in mice

Why Dex and TNF?

Dex and TNF are very different

-  Dex is the prototypical anti-inflammatory agent; acts through a nuclear receptor

-  TNF is the prototypical pro-inflammatory agent;

acts through a cell-surface receptor

Cellular models of insulin resistance: TNF and Dex

Virtually all mechanisms proposed for insulin resistance involve signal transduction or mitochondrial pathways Yet…. -Thiazolidinedione class of insulin-sensitizing drugs work by binding and activating the transcription factor PPARγ -Cellular models of insulin resistance develop slowly over the course of many days -There is a wealth of data linking chromatin state to obesity and its complications

Nuclear mechanisms of insulin resistance?

Glu

up

take

(c

pm

)

* *

0 1000 2000 3000 4000 5000

vehicle Dex TNF

basal insulin

Establishment of the comparative IR model

% o

f Glu

up

take

02hr

6hr12hr 1D 2D 4D 6D

0

30

60

90

120

TNFDex

Control

pAkt Akt

C D T C D T

Basal +Insulin

Control Dex TNF

Pparg

Cebpa

Fabp4

Adipoq

Slc2A4

Dlk1

0.0

0.5

1.0

1.5

2.0 Cont Dex TNF

Relative expression

Dex and TNF do not cause de-differentiation

The overlapping gene set affected by Dex and TNF is altered in obesity

* Dex TNF ·0. D Invariant

* - Dex_UP ·Q) - TNF_UP .C) 0.1 c: • Over lap_UP '"' -"= ·(,)

1075 ::!:! 0. ·0 -N .C)

·0 ·0. 1 _I

Up-regulated genes ·0.

Dex-only

Cont .. .. .. r-Dex ' .. .. L TNF .. .. .. r-

PR 3 (p:$0.0063) 3

TNF

Dex-TNF-overlapping

.. .. .. c .. .. i L

... i L

Up-regulated H3K27ac peaks within+/-200kb of TSS of Dex/TNF-induced genes

TNF-only

.. .. .. L

.. .. .. L

• ' • L

Dex-only

Cont .. .. .. C Dex ' .. .. L TNF .. .. .. c+

TNF

Dex-TNF-overlapping

.. .. .. ~

.. .. ' ~ .. .. ' ~

Up-regulated H3K27ac peaks within+/-200kb of TSS of Dex/TNF-induced genes

TNF-only

. . .

. . . L

. ' . Octamer 3 AL

(p~0.0013) 3"' JIIGC. TBP 3 n (p~o.oo31) 3 I A1._

KAISO 3 .../lfltGC {p~0.017) 3 .J.\A/11 j_

Dex-only

Cont .. .. .. C Dex ' .. .. L TNF .. .. .. c+

TNF

Dex-TNF-overlapping

GR (p:S0.0013)

CdxA (p:S0.0064)

VCR (p:S0.0067)

.. .. ..

.. .. ..

..

' ' I

HFH1(FOXQ1)3 .rftmL ~ (p:so.oo7a) LW.\3 t1l.x.!.

c L

L

(p:S~~24) ~~A&I A!GTrCr

Up-regulated H3K27ac peaks within+/-200kb of TSS of Dex/TNF-induced genes

TNF-only

.. .. .. r-

.. .. .. r

• ' • r-

Dex TNF

GR NF-κB

Insulin Resistance

?

Is the GR required for TNF to induce insulin resistance?

TNF causes GR binding to predicted motifs

IgG GR0

10

20

30ChowHFD

IgG GR0

5

10

15

20

25 ContDexTNF

3T3-L1 mSVF Primary

*

*

*

- 2h 24h 6D 2h 24h 6D - 2h 24h 6D 2h 24h 6D

Flag-GR

TBP

Tubulin

Dex TNF Dex TNF

Cytosolic Nuclear

TNF induces nuclear translocation of the GR

TNF induces genome-wide GR binding

• Cont • Dex • TNF

Dex alliQ.~ .•. ~.~ TNF all •k'4_,~j~

Commoo ~~---~l~~ TNF -specific ; J T A T

--~~I-.. ~~~~

GR is required for TNF to fully induce insulin resistance

c 1. Nr3c1 ~ 100 * • Dex 2 * TiNF ct)

~ 1. 80 (.) a.

60 ~ .... 4) 0 40 0. * ~ > ·-10 (.) 20 - ~ G)

~ 0. 0 ~"' ~"J ~

0

~6 g./ ~ ~ '-~( ~ 0

f:J~/ f:J~/ f:J~/ ~ (:) (:) ~~ ~ . ~~ • • • • • • ~ ~ ~

Dex TNF

GR NF-κB

Insulin Resistance

GR is required for TNF to fully induce insulin resistance

Dex-only

Cont .. .. .. r-Dex ' .. .. L TNF .. .. .. r-

TNF

Dex-TNF-overlapping

GR (p:S0.0013)

Cd xA (p:S0.0064)

VCR (p:S0.0067)

AR (p:S0.024)

.. .. .. c .. .. i L

... i L

l ~~~6.~!GT~l~ ~~~

A-w. ~~A&I x!GTrCJ

Up-regulated H3K27ac peaks within+/-200kb of TSS of Dex/TNF-induced genes

TNF-only

.. .. .. L

.. .. .. L

• ' • L

Dex TNF

GR NF-κB

VDR

Insulin Resistance

Is the VDR a mediator of insulin resistance?

Dex and TNF increase Vdr binding to predicted motifs

*

IgG VDR

0

5

10

15ChowHFD

VDR causes insulin resistance

*

Vdr expression is elevated in obesity

c: 0 ., ., ! c. 1ll .. >

"' .. a; a:

Vdr

D Chow • - HFD

•

c: 0 ., ., ! c. >< .. .. >

"' .. 1 ~

•

Vdr

D ob/+

• oblob D ob/+ (• ) Rosi • oblob (• ) Rosi

Dex and TNF increase Vdr expression

IgG GR0

10

20

30

L1 Primary

Chow

HFD

Dex

I

J

TNF

I ' I I I I I I I •

What about humans?

DNA for Methylation

RNA for RNA-seq

Crosslinked nuclei for ChIP-seq

Also: - Serum - Buffy Coat Layer - SVF pellets -  Whole Fat

Adipocytes

SVF

Oil

Collagenase Low-speed centrifugation

Adipocyte Nuclei

Oil

Our isolated adipocytes yield excellent ChIP-seq profiles free from evidence of stroma or immune cells

Human Adipocyte Nuclei: Subject 7

ENCODE Human Whole Adipose Tissue

ENCODE CD14 + Monocytes

hASC derived pre-adipocytes

hASC derived adipocytes

d ,,,,., 1 _._ ........... -..... ........... _ __...... .. ~ • I I •• L.. .. • ~

.. . ....... _ .............. _ ........ • 0M. A,y l,, ...... •·· - ~-- ------ ----- _ __j,__~ - ~-- --- ---- A .. Al•,.a

- - _j_ --~--- - -

ttll GAPDH

Ubiquitous

..,..l. ...

~ H7177~

PPARG ADIPOQ FABP4

Adipocyte

~ CFD

ftfH ll MIR4268 SNORD114-29 CSF3R S100A8

Pre-ad i pocyte Macrophage

Histone profiles suggest the presence of novel transcripts and alternative promoters in human adipocytes

H3K27ac

H3K4me3

ENCODE Adipose RNA-seq

RefSeq Gene Annotation

- ,l_ - A ..l I l __ j__ J I II _. - -- 1 1_ L

) ) ) ) >I ) ~ I< I~ ( ( ( ( H ~ SASH1 DST PIK3C2B UTRN

lntragenic 5' region

.... &1 ..

j_ J~

- --- - - -

annotation desert

We can identify cis-elements that differ between IR and IS subjects

A IS1

IS2

IS3

IS4

ISS

IS6

IS?

IR1

IR2

IR3

IR4

IRS

IR6

_.,_,.M ... t* •• h.,ol&- ...... , ..... "

... - '_..__ __ J. • --o-"- .dU

_. ._ ...... 'ftt .. h ,, _____ ... L_ s1 Mt 4

h '&' 1 •• I 0 '0 .. .

. .... - ...... - a

• b ... Let • .. ·-.. ..... . .. • j.. ...

.,... ......................

.... 0 ...... ...... -· , ..... ,,. '0' ·e 4·ece .. A ....... ~--'=- a b 0 4 ...

f'IElM DOK2

.. . .... _.- . tt.

DOK3 SLC2A4

ett* N

.... &

HK2 ADIPOQ

B • , Ht ......... 'sf d +.e+ A trfrtt b ••d-... dtrt .........

. .. ... . .. ,. -~ .... ,.,, ... __ ~ ., .. __

... . --- ....... ..__,. _ __, .. ...__ ------ .& ........ - '+r > -

.... __ ... _,_ ....... -.. .,., .......... ..... __ .... ... tdt

• rAz ........... , ·---... .___ .. -... -......

... ,1_ .1:. _ .... ___ .... _Prr4Prr4 ... _.........-- .. • • • ....... 4 • .A ... .. . .... ,. •• d • ... .. ..... L ,. .. -· dnit · e• .,,,. .It• •=· •, t· ++ - . ·"-·-- .. ,.., ,.j. .. .. in'* . ,. L ,a, • 0 -·· , ..

I t -

• ·t 't ... .. 7 =""• - + .. • Or

~- ... ... .•. , • lu I ...... , .... I. 1111 ... ~~~~~~ •• .. _ .. .. .... ....... .... 111111 .... --+-- te c&d b+ 'z'itf+ ..... • I ·t ft --- ...... ,_ ... h-- .. I- b ----......... ___ ...,, -~-

........ ++ I> > > >

< < < < <I EVL CD84

.. ... __ _ ... .. -~ &I) )1. ~ ft-7-7 I) ) ) ) )

ALDH1L 1-AS2

IL 10RA FAM47E < < < G STON1-GTF2A1L ALDH1 L 1

1.  Dex and TNF causes discrete changes in epigenome of L1 cells that associate with IR.

2.  Motif finding in differentially regulated regions can identify novel pathways leading to IR.

3.  TNF causes IR, in part, through ligand-independent activation of the GR. 4.  The VDR is a GR target that further induces downstream IR genes.

5.  Tmem176a, Colq, Lcn2 and Serpina3n are part of an IR-inducing gene network downstream of GR and VDR.

6.  Human studies are underway to confirm and extend these results.

Summary

BIDMC Sona Kang Linus Tsai Yiming Zhou Xingxing Kong Michael Griffin Hyun Cheol Roh Manju Kumari Eleanna DeFilippis Erin Merkel Su Xu Zhao Xu

Broad Institute Tarjei Mikkelsen Chuck Epstein Noam Shoresh Robbyn Issner Holly Whitton Xiaolan Zhang

MGH Chad Cowan Ray Camahort

Acknowledgements

Funding from the NIH and ADA

Penn/Princeton Adam Evertts Ben Garcia