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
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.,... ......................
.... 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 -·· , ..
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• ·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