Control of Adipogenesis by S6K1-Mediated
Epigenetic Modifications
Jeung-Whan Han
Research Center for Epigenome Regulation (RCER)
School of Pharmacy
Sungkyunkwan University
Conflict of interest disclosure
Committee of Scientific Affairs
None
Committee of Scientific Affairs
Contents
Part Ⅰ. Nuclear translocation of S6K1.
Part Ⅱ. S6K1 interacts with, and phosphorylates H2B at S36.
Part Ⅲ. S6K1-mediated H2BS36 phosphorylation regulates
H3K27me3.
Part Ⅳ. S6K1-mediated H2BS36 phosphorylation promotes
adipogenic commitment.
Part Ⅰ
mTOR-S6K Signaling Pathway
NTD: Nterminal domain; CTD: C-terminal domain; NLS: nuclear localization signal; Pro: proline-rich domain; P: phosphorylated residue.
S6K1 / S6K2 isoforms : From two different genes P85 S6K1 / p70 S6K1 / p31 S6K1 : Alternative splicing variants
S6K1
S6K2
Part Ⅰ
Substrates of S6K1 (Pre-translational Function)
rDNA Transcription
Pyrimidine Synthesis Processing
Nucleolus
Pre-60S
Pre-40s
CAD P
Ribosome Biogenesis
Potential Target Genes in Breast Cancer Cells
S6K1
Transcriptional Program
Ben-Sahra et al., 2013 Robitaille et al., 2013
Chauvin et al., 2014 Mitchell et al., 2015
Heinonen et al., 2008 Düvel et al., 2010
Purine Synthesis mTOR
Ben-Sahra et al., 2016
Metabolic Gene Network
Part Ⅰ
Molecular Roles of p70 S6K in Cytoplasm
Russell R C. Development (2011) Sung Hee Um. Nature (2004)
Protein Synthesis Phosphorylation of IRS1
Question. 1. What is the mechanism underlying physiological functions of S6K1?
2. Can S6K1 act as a transcriptional regulator?
Part Ⅰ
1. Nuclear translocation of p70 S6K1.
p70
S6
K
pp
70
S6
K
10T1/2 Fibroblasts
Merg
e
DA
PI
0 min 15 min 30 min 5 min 10% FBS: 60 min 15 min (Rapa.)
Rela
tive
p7
0S
6k s
ign
al
in
nu
cle
us
(a
rbit
rary
un
its
)
0 5 15 30 60 15-Rapa. FBS (min)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5p70 S6 kinase
phospho-p70 S6 kinaseR
ela
tive
p7
0S
6k s
ign
al
in
cyto
so
l (a
rbit
rary
un
its
)
0 5 15 30 60 15-Rapa. FBS (min)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Part Ⅰ
1. Nuclear translocation of p70 S6K1.
HeLa
10T1/2 Fibroblast
Cytoplasm
α-tubulin
Lamin A/C
S6K1
Rapamycin - + - +
Nucleus
RNA PolⅡ
S6K1
α-tubulin
P-S6K1
Whole cell lysate Cytoplasm Nucleus
Rapamycin - - - + + - - - + + - - - + +
Time of stimulation (min) 0 15 30 15 30 0 15 30 15 30 0 15 30 15 30
Yi et al., Mol. Cell, 2016
Part Ⅱ
Kinases and Histone PTMs
Sung Hee Baek. Molecular Cell (2011)
Fig 1. Kinases that Modify Histones H2B and H3 and Residues Modified
Part Ⅱ
Phosphorylation Sites in Histones
Sequence: http://www.uniprot.org/uniprot/Q16778 Sequence: http://www.uniprot.org/uniprot/Q64524
Homo sapiens Mus musculus
R S R K E S Y K R K K H2B 36 32
Part Ⅱ
2. S6K1 phosphorylates H2B in vitro.
S6K
Autorad.
- - - - + + + +
Coomassie
H2A H2B H3 H4 H2A H2B H3 H4
PF-4708671 - 10 50 100nM
Coomassie
Autorad.
Autorad.
Coomassie
S6K + + - -
27-46 7-26 Peptides 27-46 7-26
H2B 27 32 36 38 46
27-46 KKRKRSRKESYSIYVYKVLK
S32A KKRKRARKESYSIYVYKVLK
S36A KKRKRSRKEAYSIYVYKVLK
S38A KKRKRSRKESYAIYVYKVLK
S6K + + + +
27-46 S32A S36A S38A Peptides
Coomassie
Autorad.
- - - -
27-46 S32A S36A S38A
Recombinant Histones
H2B peptides
Yi et al., Mol. Cell, 2016
Part Ⅱ
3. S6K1 interacts with, and phosphorylates H2B in vivo.
Western blot
Immunoprecipitation
IgG WCL S6K
IP
Input IgG GFP
IB: S6K
IB: GFP IB: S6K
IB: H2B
IgG WCL S6K
IP IP
Endogenous H2B Ectopic GFP-H2B
HeLa 10T1/2
- + shS6K - #03 #04
S6K
si-S6K
H2B
H2BS36p
HeLa 10T1/2 Fibroblast
S6K
H2B
H2BS36p
Yi et al., Mol. Cell, 2016
Part Ⅱ
Summary 1
mTORC1
S6K1
Nucleus
Cytoplasm
Nuclear S6K1 directly interacts with, and phosphorylates H2B at S36.
Question:
Does S6K1 affect other histone modificaitons?
Serum stimulation
gain of H2BS36Ph
S6K1
?
S6K1
mTORC1
P
P
P
P
Part Ⅲ
4. S6K1 is required for H3K27 trimethylation.
P-S6K
S6K
H3K4Me3
H3K36Me3
Actin
P-S6
S6
PF-4708671 - +
H3K27Me3
H3Ac
H2BS36P
- +
S6K
si-S6K
Actin
H3K27Me3
FBS
P-S6
S6
Starvation
H3
H3K27Me3
PF-4708671
+ + +
- + +
- - +
04
S6K
si-s6k
NC NC
H3
P-S6
03
si-s6k
03 04
H3K27Me3
10T1/2 HeLa
Yi et al., Mol. Cell, 2016
Part Ⅲ
Adipogenesis from Mesenchymal Stem Cell line
10T1/2 cells (Mesenchymal Stem Cells)
Preadipocytes Adipocytes
Commitment Terminal Differentiation
day0 day1 day2 day3 day4 day5 day6 day7 day8
BMP4 MDI Insulin Insulin Insulin
Adipocytes
day-4
D (Dexamethasone) Binds and activates glucocorticoid receptor (GR)
M (IBMX) Increases intracellular cAMP level to activate protein kinase A
I (Insulin) Stimulates insulin like growth factor 1 (IGF1) pathway
Predipocytes MSCs
4 days 8 days
Part Ⅲ
4. S6K1 is required for H3K27 trimethylation.
d-4 d-2 d0 d2 d4 d6 d8
Adipocyte (d8) MSC
(C3H10T1/2)
Commitment Terminal Differentiation
BMP4 MDI Insulin Insulin Insulin
H3K27me3
DAPI
Merge
BMP4 0 15’ 30’ 1h 2h 1d
S6K1
DAPI
Merge
BMP4 0 15’ 30’ 1h 2h 1d
BMP4
Nucleus
α-tubulin
Lamin A/C
S6K1
BMP4 0 30’ 2h 1d 0 30’ 2h 1d
Cytoplasm
0
1
2
3
4
5
6
0 30' 2h 1d
nuc S6K1
0
1
2
3
0 30' 2h 1d
cyt S6K1
BMP4
Yi et al., Mol. Cell, 2016
Part Ⅲ
4. S6K1 is required for H3K27 trimethylation.
d-4 d-2 d0 d2 d4 d6 d8
Adipocyte (d8) MSC
(C3H10T1/2)
Commitment Terminal Differentiation
BMP4 MDI Insulin Insulin Insulin
P-S6K
PF-4708671 - 1h
S6K
H2BS36P
H3K27Me3
H2B
P-S6
S6
BMP4 2h 2h
Short
treatment Long
treatment
4d 4d
- 4d
H3
P-S6K1
0 30’ 2h 1d
S6K1
BMP4
H2BS36p
H3K27me3
H3
H2B
- +
S6K1
BMP4
H2B
H2B
S6K1
P-S6K1
S6K1
P-S6
S6
H2BS36p
H2B
IP :
S6K1
IP :
IgG
Input
Yi et al., Mol. Cell, 2016
Part Ⅲ
Histone Phosphorylation and Crosstalk
Crosstalk between Histone Phosphorylation and Other Modifications
Part Ⅲ
7. S6K1-mediated H2BS36Ph is required for EZH2 effects.
H2B-WT : Normal H2B
H2B-S36D : Phospho-mimic
H2B-S36A : Phospho-resistance
0
5
10
15
20
IgG H2BS36Ph
Con
Rapa
ChIP : H2BS36Ph
PCR : HOXA9 primer
**
En
rich
me
nt re
lative
to
in
pu
t
ChIP : EZH2, H3K27Me3
PCR : HOXA9 promoter
***
***
***
0
5
10
15
20
25
EZH2 H3K27Me3
WT
36D
36A
*
En
rich
me
nt re
lative
to
in
pu
t
P-S6
S6
Actin
H3K27me3
H2B-S36D
- + +
- - +
PF-4708671
GFP EZH2
GFP
EZH2
GFP
IP :
GFP
IP :
IgG
36D 36A GFP-H2B
EZH2
GFP Input
BMP4 + +
Yi et al., Mol. Cell, 2016
Part Ⅲ
Summary 2
S6K1-mediated H2BS36 phosphorylation promotes H3K27me3 during adipogenic commitment.
What genes are suppressed by S6K1-H2Bp-H3K27me3 axis during adipogenic commitment?
mTORC1
S6K1
Nucleus
Cytoplasm
Serum stimulation
gain of H3K27me3
S6K1
S6K1
mTORC1
P
P
P
EZH2
M
Gene silencing
?
P
Part Ⅳ
8. S6K1-mediated H2BS36Ph suppresses Wnt genes expression.
RT-qPCR
d-4 d-2 d0 d2 d4 d6 d8
Adipocyte (d8) MSC
(C3H10T1/2)
Commitment Terminal Differentiation
Transfection of vectors or siRNA Sampling
Yi et al., Mol. Cell, 2016
Rela
tive
mR
NA
le
ve
l
***
***
***
***
*
*
0
9
18
Wnt6 Wnt10a Wnt10b
NC
siS6K1 #03
siS6K1 #04
Rela
tive
mR
NA
le
ve
l
***
**
**
*
**
*
0
2
4
Wnt6 Wnt10a Wnt10b
S6K1-WT
S6K1-CA
S6K1-DN
0
2
4
6
Wnt6 Wnt10a Wnt10b
WT36D36A
0
5
10
Wnt6 Wnt10a Wnt10b
Con
PF
PF+36DR
ela
tive
mR
NA
le
ve
l R
ela
tive
mR
NA
le
ve
l
*** ***
*** *** ** *
*
*
**
*
*
*
Part Ⅳ
8. S6K1-mediated H2BS36Ph suppresses Wnt genes expression.
ChIP
Yi et al., Mol. Cell, 2016
0
5
10
Wnt6 Wnt10a Wnt10b
Con
BMP4
BMP4+Rapa
En
rich
me
nt re
lative
to
in
pu
t
ChIP :
S6K1
0
2
4
6
Wnt6 Wnt10a Wnt10b
ChIP :
H2BS36p
* *
* ***
* ***
* * * *
* *
0
4
8
12
16
Wnt6 Wnt10a Wnt10b
ConBMP4BMP4+Rapa
0
4
8
12
16
Wnt6 Wnt10a Wnt10b
En
rich
me
nt re
lative
to
in
pu
t
** ***
* *
* **
* *
* * *** **
ChIP :
EZH2
ChIP :
H3K27me3
0
10
20
Wnt6 Wnt10a Wnt10b
WT
36D
36A
ChIP :
EZH2
ChIP :
H3K27me3
**
**
**
** **
**
**
* **
*** **
*
0
2
4
6
Wnt6 Wnt10a Wnt10b
Part Ⅳ
11. Expression of adipogenic genes in S6K1 knock-out mice.
Rela
tive
mR
NA
le
ve
l
Re
lative
mR
NA
le
ve
l **
**
***
*
*
**
0
2
4
6
8
Wnt6 Wnt10a Wnt10b
WT
S6K1-/-
0.0
0.5
1.0
1.5
adipsin aP2 PPARγ
WT
S6K1 -/-
WT S6K1-/-
Yi et al., Mol. Cell, 2016
Mouse Epididymal WAT (eWAT)
Part Ⅳ
12. Histone modifications in S6K-deleted mice
S6K-/- WT
S6K K.O. mouse Obesity-induced mice
1 2 3 4 5 1 2 3 4 5
NCD HFD
P-S6K1
H3K27me3
H2BS36p
S6K1
H2B
H3
S6K1
Actin
H3K27me3
H2BS36p
H3
H2B
Yi et al., Mol. Cell, 2016
Part Ⅳ
13. P-S6K & histone modifications in human WAT
# Height Weight BMI Gfoup
1 161 75 28.93 Overweight
2 155 64.9 27.01 Overweight
3 153 56 23.92 Overweight
4 154 53 22.35 Normal
5 165 66 20.20 Normal
6 165 54.7 20.09 Normal
𝐵𝑀𝐼 𝑏𝑜𝑑𝑦 𝑚𝑎𝑠𝑠 𝑖𝑛𝑑𝑒𝑥 = (𝑊𝑒𝑖𝑔ℎ𝑡)
(𝐻𝑒𝑖𝑔ℎ𝑡)2
Underweight < 18.5
Normal 18.5 ~ 22.9
Overweight (At Risk) 23.0 ~ 24.9
Overweight (Obese) ≥ 25.0
Part Ⅳ
13. P-S6K & histone modifications in human WAT
*
BMI
H2B
S36P
h
R2=0.63
20
25
30
0 20 40
20
25
30
0 20 40
BMI
P-S
6K
1
R2=0.81
0
20
40
Overwt Normal
0
20
40
Overwt Normal
H2B
S36P
h
P-S
6K
1
*
20
25
30
0 5 10
H3K
27m
e3
R2=0.55
P-S6K1
H2B
H3K27me3
H2BS36Ph
Overweight Normal
S6K1
1 2 3 4 5 6
28.9 27.0 23.9 22.4 20.2 20.1 BMI
H3
Yi et al., Mol. Cell, 2016
Summary & Conclusion
- Promotion of adipogenesis by S6K1 is mediated through the regulation of a complex epigenetic network, triggered by S6K1 nuclear translocation and H3K27 trimethylation by EZH2.
- S6K1 binds to and phosphorylates H2B at serine 36, promoting H3K27me3 by EZH2.
- H3K27me3 acts at the Wnt6, Wnt10a, and Wnt10b gene promoters to block their expression.
- In turn, inhibition of Wnt gene expression leads to upregulation of PPARγ and C/EBPα, inducing increased adipogenesis.
- Consistent with these findings white adipose tissue from S6K1-deficient mice has no detectable H2BS36p or H3K27me3, while exhibiting enhanced Wnt gene expression.
- Critically, the extent of H2BS36p and H3K27me3 from adipose tissue of mice maintained on a high fat diet or from obese humans, correlates with their body mass index.
- These findings place S6K1-dependent H2BS36p as a key regulator of H3K27me3 during the commitment of mesenchymal stem cells to the adipogenic lineage.
mTORC1
S6K1
Nucleus
Cytoplasm
Adipogenic stimulation
H2BS36p H3K27me3
S6K1
S6K1
mTORC1
P
P
P
EZH2
M
Wnt Gene silencing
P
Biochemical and Molecular Biology Laboratory
P.I.
Prof. Jeung-Whan Han
Graduate Students
Jae Cheol Lee (Stanford Univ)
Ye Ji Jeon
Sora Ahn
Sang Ah Yi (Sungkyunkwan Univ)
Shin Won Ha (Dgist)
So Young Bang (Sungkyunkwan Univ)
Eun Kyeong Park (Sungkyunkwan Univ)
Min Gyu Lee (Sungkyunkwan Univ)
Ki Hong Nam (Sungkyunkwan Univ)
Jee Hee You (Sungkyunkwan Univ)
Ji Hoon Park (Sungkyunkwan Univ)
Bo Kyung Kim (Sungkyunkwan Univ)
Hwa Mok Oh (Sungkyunkwan Univ)
Funding
Medical Research Center
Brain Korea 21
Collaborators
Catalan Institute of Oncology, Bellvitge Biomedical Research Institute, IDIBELL, Hospitalet de Llobregat, Barcelona, Spain Prof. George Thomas
College of Medicine, Catholic University of Korea Prof. Jong Won Rhie
School of medicine, Sungkyunkwan University Prof. Sung-Hee Um
Acknowledgement