Rb targets histone 3 methylation

and HP1 to promotors

S.J. Nielsen, R. Schneider, U. Bauer, A. J. Bannister, A. Morrison, D.

O‘Carrol, R. Firesteink, M. Clearyk, T. Jenuwein R.E. Herrera, T. Kouzarides

2001, letters to nature

MBE

18. September 2007

BONIFERT, Tobias

YEDIKAPU, Mine

Overview

• Introduction

• Experiments

• Conclusions

• Comment

2

• Comment

Introduction

Retinoblastoma-(Rb)-Protein was

discovered in context with

retinoblastoma disease

3

Introduction

Rb regulates cellcycle by

binding and repression of

transcription-regulating

proteins

In presence of mitogen

activity the Rb-protein stops

4

activity the Rb-protein stops

its inhibiting function which

leads to cell proliferation

�cancer

Some of the Rb-associated

proteins necessary for

repression are situated on

histones.

Introduction

Deacetylated histones have a negative effect on the

transcription rate

Rb recruits this deacetylase activity to repress

5

� Does the Rb-protein influence

histone-methylase activity?

transcription

Histone-methylase activity also leads to a diminished

transcription rate

Pull down• Prey protein = histone methylase

+ Rb-GST

• GST fusion proteins

– Immobilized on glutathione-

sepharose columne

– H3-derived peptides bound to

Sulfonic beads

GST

GST

Rb

Incubation

Chromatograpy

6

• Rb captures prey protein while

Incubation

• Isolation of Tagged particles by

Chromatography

• SDS-Page gelelectrophoresis

separation of captured proteins

GST

• GST-Rb fusion can purify histone

methylase activity

• samples purified with GST and

GST-Rb are added to

– H3, H4 and GAR

• For visualization of methylase

activity at H3, H4 or GAR,

radioactive marked CH3 is added

7

radioactive marked CH3 is added

• --> autoradiography

• Rb associated methylase activity

is not a GAR dependent arginine

methylase --> it must be a lysine

methylase.

H3

DNA

Nucleosome

Met

Methylase

H3

8

Where at H3 happens the methylation?

Rb

Methylation site of H3

�Edman degradation of radioactively methylated histone H3

9

�methylation at lysine 9 of histone H3

H3

DNA

Nucleosome

Met

Methylase

H3

Lys 9

10

Which is the methylase associated with Rb?

Rb

SUV39H1 - The Rb associated methylase

Hints

– possesses methylase activity

conserved in its SET domain

– has specifity for lysine 9 of

Histone H3

11

Results

– GST-Rb fusion can bind

haemagglutinin(HA)-tagged

SUV39H1

Methods

– transfection of HEK-293 cells

with HA-SUV39H1 or empty

expression vector (Mock)

– extracts incubated with GST or

GST-Rb --> Pull-down

– Bound SUV39H1 western

blotted using anti-HA antibody

H3

DNA

Nucleosome

Met

SUV39H1

H3

Haemagglutinin (HA)

Rb

GST

Methylase

12

Rb

Acts SUV39H1 as a co-repressor with Rb?

Lys 9

SUV39H1 - An Rb co-repressor

Methods

– HeLa cells transfected with

reporter, containing the cyclin E

promotor driving luciferase

together with combinations of

expression vectors for E2F1, Rb,

SUV39H1 and SUV39H1∆SET

Results

13

Results

– cyclin E promoter is

stimulated by E2F

– SUV39H1 is a co-repressor

– Rb represses E2F activity slightly

– SUV39H1 + Rb leads to further

repression of E2F activity

– SUV39H1 with deleted SET-motif

(SUV∆SET) does not lead to

repression

Does a lack of the co-repressor SUV39H1 lead to

elevated amount of mRNA?

14

elevated amount of mRNA?

Repressive functions of SUV39H1

Methods

– Double-knockout of genes for SUV39H1

and related SUV39H2 in mice fibroblasts

– RT-PCR

Results

15

Results

– Rb repressor needs SUV39H1 to repress

cyclin E and cyclin A genes

– In double knockout cells cyclin E and A

mRNA amounts are elevated

– Genes Cdc25, HPRT, GAPDH are not

regulated by Rb and show no differences

compared to wild-type

Is there another factor in the Rb-SUV39H1

interaction involved?

16

interaction involved?

The role of HP1

Hints

• SUV39H1 is known to be in a complex with the HP1 protein

• HP1 function downstream of SUV39H1

• HP1 recognizes methylated Lys9 on histone H3

17

Methods

• Co-immunoprecipitation

• Westernblott

Co-immunoprecipitation

Rb/HP1-

Complex (?)

solution

Anti-RbAnti-HP1

Western-blotting

+ Anti Rb + Anti HP1

Precipitation

18

Precipitation of

HP1-X +?

Precipitation of

Rb-X + ?

HP1-X

Rb-X

Rb and HP1 form a complex?

HP1 associates with Rb

Results

• co-immunoprecipitation proves that HP1

and Rb form a complex

HP1 contains an LXCXE motif

– competes for the methylase binding to Rb

19

– competes for the methylase binding to Rb

– but does not effect its own binding to the

methylase

Recognizing Met and Rb

In a further step, the

scientists proved by

using recombinant

Rb as well as

recombinant HP1, H

DNA

Nucleosome

Met

HP1

20

recombinant HP1,

that HP1 can

recognize the

methylated Lys9 of

H3 and Rb at the

same time.

H

3

Rb

H3SUV39H1

Lys 9

Are Rb regulated Promoters associated with

21

Are Rb regulated Promoters associated with

HP1?

DNA

Nucleosome

Chromatin-Immunoprecipitation

22

H

3H3

Antibody to recognize

Lys 9 methylated H3

Cyclin Epr

HP1

in the presence of

Rb, methylase

activity and HP1 are

targeted to the cyclin

E promoter.

E2F

Conclusion

Rb binds to transcription factors which drive specific genes.

When Rb deacetylates lysine 9 of histone H3, the methylase SUV39H1

is able to methylate this amino acid.

The Rb/SUV39H1-complex is able to increase Rb-induced repression

23

The Rb/SUV39H1-complex is able to increase Rb-induced repression

activity.

After the Rb/SUV39H1-induced Methylation of Lysine 9, the Protein

HP1 can Bind to the complex.

Although the role of HP1 is not sufficiently clear, the experiments

showed that HP1, linked to the Rb/SUV39H1 -complex is involved in

Gene-repression.

Comment

Introduction is missing.

Unsufficient explanation of used methods

24

No significance tests

references

B. Alberts et al. (2002): Molecular biology of the cell, 4. edition, Garland

Science, New York

M.A. Becky Golat et al. (2004): In Vitro His-Tag Pull-Down Assay MagZ

25

M.A. Becky Golat et al. (2004): In Vitro His-Tag Pull-Down Assay MagZ

Particles. Promega Notes, 88

http://www.eyecancer.com/Doctor/Gallery.aspx?nID=4&Category=Retinal

+Tumors

http://www.ncbi.nlm.nih.gov/SCIENCE96/chr.cgi?13

Thank you for listening.