Control of Adipogenesis by S6K1-Mediated Epigenetic ...icdm2016.diabetes.or.kr/file/slide/S3-4.pdf · Control of Adipogenesis by S6K1-Mediated Epigenetic Modifications Jeung-Whan

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

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 (Translational Function)

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

S6K1 in Metabolism

Part Ⅰ

S6K1 in Metabolism

S.H.Um et al. nature (2004)

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

Histone Post-translational Modifications

Nature Reviews Neuroscience 8, 355, (2007)

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


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


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


Part Ⅲ

S6K1 in Metabolism

LarissaS.Camevalli et al. Developmental Cell (2010)

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


d-4 d-2 d0 d2 d4 d6 d8

Adipocyte (d8) MSC

(C3H10T1/2)



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


Part Ⅲ


d-4 d-2 d0 d2 d4 d6 d8

Adipocyte (d8) MSC

(C3H10T1/2)



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


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 + +


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

Epigenetic Regulation of Adipogenesis

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)


Transfection of vectors or siRNA Sampling


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


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-/-


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


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


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

Thank You For Listening :-)

Documents

Control of Adipogenesis by S6K1-Mediated Epigenetic ...icdm2016.diabetes.or.kr/file/slide/S3-4.pdf · Control of Adipogenesis by S6K1-Mediated Epigenetic Modifications Jeung-Whan