shiv grewal

24
Epigenetic genome control by RNAi and transposon-derived proteins Shiv Grewal Center for Cancer Research, NCI National Institutes of Health, Bethesda

Upload: halien

Post on 04-Jan-2017

253 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: Shiv Grewal

Epigenetic genome control by RNAiand transposon-derived proteins

Shiv Grewal

Center for Cancer Research, NCINational Institutes of Health, Bethesda

Page 2: Shiv Grewal

48%Human38%Mouse

10-12%Drosophila

6.5%Worm

3.1%Yeast

Transposon content(% of genome) Organism

Transposons and their remnants constitute a significant fraction of eukaryotic genomes

Heterochromatin / RNAiTransposon-derived proteins

Page 3: Shiv Grewal

Distinct patterns of histone modifications specify discrete downstream regulatory events

Heterochromatin Euchromatin

Adapted from Zhang and Reinberg 2001

Page 4: Shiv Grewal

cnt dhdg cwf20pgp1 dg dh

H3K4meSwi6H3K9me

ChI

P fo

ld e

nrich

men

t

10

20

30

40

Centromere

ChI

P fo

ld e

nrich

men

t

LTRsdh repeat

10

20

30

40

Subtelomeric region

H3K4meSwi6H3K9me

Heterochromatin coats extended domains associated with aspecific classes of repeat elements in S. pombe

ChI

P fo

ld e

nrich

men

t

10

20

30

40 H3K4me Swi6 H3K9me

aa

IR-L IR-Rmat2P mat3McenH2.5 kbmat1

mat locus

Grewal and Klar Cell 1996

Page 5: Shiv Grewal

Clr4HP HP

RdRP

Nucleation(S phase)

Spreading

siRNA

RITSRik1Clr4

Dcr1

Repeat elements

H3K9me

Chp1Ago1 Tas3

HPHP

HP

HPClr4

HP

HP = Swi6, Chp2

Boundaryelement

Pol II

Nucleation and spreading of heterochromatin

STOP

Spreading of heterochromatin requires Clr4 chromodomain binding to methylated H3K9

Chp1

AcHP

Rdp1 H3K9HMTase

Zhang et al 2008 NSMB 15:381

Page 6: Shiv Grewal

Grewal and JiaNat Rev Genet

8:35-46

Heterochromatin serves as a versatile platform for targetingeffectors across extended domains

Page 7: Shiv Grewal

HP HP

RDRC

siRNA

RITSRik1Clr4

Dcr1

Repeat elements

H3K9me

Chp1Ago1 Tas3

Clr4HP

HPHP

HP

HPClr4

STOP

HP = Swi6, Chp2

Boundaryelement

PTGS

Clr6-C1 Clr6-C2

Pol II Ac

a

Mit1 (Snf2 ATPase)Clr1Ccq1

Clr2Clr3 (HDAC)

SHREC

Clr3

Sugiyama et al 2007 Cell

Clr6

aaa

Clr6-C1 Clr6-C2

Pst1 (Sin3)Pst2 (Sin3)

Cph2Prw1Clr6 (HDAC)Cph1Alp13 (MRG15)Png2

Sds3

Nicolas et al 2007 NSMB

TGS effector complexes

SHRECTGS

SHREC

Post-transcriptional and transcriptional heterochromaticsilencing

ClrC

Chp1

Rdp1

siRNA Factory

Ago1

RISC

Page 8: Shiv Grewal

SHREC activities facilitate proper positioning of nucleosomesrequired for higher-order chromatin assembly

Micrococcal nuclease digestion patterns

Prob

e

Sugiyama et al 2007 Cell 128:491

Page 9: Shiv Grewal

Heterochromatin mediates recruitment of cohesin that isessential for maintenance of genomic integrity

aa

10

5

Cohesin

aa

IR-L IR-Rmat2P mat3McenHmat1

10

5

Swi6

2.5 kb

Transcriptional and RecombinationalBlock

Euchromatin Heterochromatin Euchromatin

K. Noma and S. Grewal

Page 10: Shiv Grewal

WT

P ce

lls

α-Swi

1 2 3 4 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 54 55 56 57 58 59 60 62 63 65 66 67 6 68 69 70 71 72 73 74 75 76 77 78 79 80

WCE

0123456

Swi C

ompl

ex(fo

ld e

nric

hmen

t)

WT

matact1

matact1

matact1

matact1

α-Swi

WCE-swi6

-swi6

mat2-P mat3-M cenH 2.5 kb

Swi C

ompl

ex(fo

ld e

nric

hmen

t)

WT

1 2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 54 55 56 57 58 59 60 62 63 65 66 67 6 68 69 70 71 72 73 74 75 76 77 78 79 80

matact1

M c

ells

0123456

WT

matact1matact1matact1

WCE

α-Swi

WCE-swi6

-swi6

α-Swi

Enhancer Element

mat1-P

Heterochromatin mediates cell-type specific spreading of aprotein complex essential for mating-type switching

mat1-M

Page 11: Shiv Grewal

aaa

10

5

IR-L IR-Rmat2P mat3McenHmat1

2.5 kb

10

5

15

1.0

0.5

1.5

Swi6

H3 Lys9-methyl

H3 Lys4-methyl

Transcriptional and RecombinationalBlock

Euchromatin Heterochromatin Euchromatin

Boundaries of a heterochromatic domain

Fold

Enr

ichm

ent

Page 12: Shiv Grewal

aaaaa

B-box cons. ....GTTCGANTC.... ***** * *B-box seq#1 AGCTGTTCGTAGCAGCA *******B-box seq#2 GCTCTTTCGATTATAGA *********B-box seq#3 CCCGGTTCGAGTCCTAT ********B-box seq#4 AGCGGTTCGATTTGCGA **** ***B-box seq#5 TAAGGTTCTACTTTATC

mat3M IR-R1 kb

aaa

wt

IR-R∆

IR-R∆ #1

IR-R∆ #2

IR-R∆ #3

IR-R∆ #4

- FOA + FOA

kan+

B-box∆

mat3 IR-R

heterochromatin spreading

ura4+

1 kb

aaa

10

20

30

mat3M

2.5 kbura4+IR-RB-box∆

wt

B-box∆

H3

Lys

9-m

eth

yl(f

old

en

rich

me

nt)

TFIIIC binding to the boundary elements blocks spreading ofheterochromatin

Page 13: Shiv Grewal

TFIIIC, but not Pol III, localizes to heterochromatin domainboundaries

ChIP

-chi

p

Page 14: Shiv Grewal

TFIIIC bind to tRNA gene clusters at centromereheterochromatin domain boundaries

ChIP-chip analyses

Page 15: Shiv Grewal

aa

COC5

SPCC63.02c SPCC63.03

COC4

SPBC1539.05 SPBC1539.07c

COC3

sfc3SPBC336.05c

10

20

30

40

10

20

30

40

10

20

30

40

TFIIIcPol III

fold

enr

ichm

ent

fold

enr

ichm

ent

fold

enr

ichm

ent

TFIIIC, but not Pol III, peaks at sites are predominantly associatedwith divergent gene promoter regions

B-box

Page 16: Shiv Grewal

TFIIIC bound loci cluster near the nuclear periphery: implicationsfor genome organization and function

TFIIIC body

TFIIIC/DAPI

Page 17: Shiv Grewal

Host genome surveillance for retrotransposons andrepeats by transposon-derived proteins

CENP-Bs are conserved proteinsthat contain DNA binding anddimerization domains

CENP-Bs are derived fromtransposases of POGO DNAtransposons

S. pombe genome encodes threeCENP-Bs that have redundantroles in centromereheterochromatin formation

ABP1

DNA binding domain DDE

Dimerizationdomain

CBH1

CBH2

MammalianCENP-B

POGOKtransposase

S. pombeCENP-B

Page 18: Shiv Grewal

Chromosome III3.5 Mb

cen3 rDNArDNAtel3L tel3R

500 1,000 1,500 2,000

Tf2-12 Tf2-13

4

10

14

2

6

12

8LTR

ars3002wtf6

LTRs rpc34 IGLTR

gut2 IGsrk1 IG

Chromosome II4.6 Mb

cen2 mattel2L tel2R

1,000 2,000 3,000 4,000

Tf2-10

Tf2-11Tf2-9

4

10

14

2

6

12

8

LTR rip1 IG725.03 IG LTRs

Chromosome I5.7 Mb

cen1tel1L tel1R

1,000 2,000 3,000 4,000 5,000

Tf2-7-8Abp1

Fold

enr

ichm

ent

4

10

14

2

6

12

8

Tf2-5Tf2-1

Tf2-3Tf2-2

Tf2-4

Tf2-6LTR LTRs

1,000 2,000 3,000 4,000

10

20

30

5

15

25 Tf2-11

Tf2-9Tf2-10

LTRrip1 IG

20F10.03 IG

cwf10 IG725.03 IGLTRs

Chromosome Position (Kb)

Tf2-5Tf2-1Tf2-2

Tf2-7-8Tf2-3Tf2-4

SPAP32A8.02

Tf2-6spo20 IG

Fold

enr

ichm

ent

1,000 2,000 3,000 4,000 5,000

10

20

30

5

15

25

LTRsLTR

Cbh1rpc34 IG

500 1,000 1,500 2,000

10

20

30

5

15

25

Tf2-12 Tf2-13

63.12c IG

wtf6

LTRs

LTR

gut2 IG

srk1 IG

rpc34 IG

CENP-Bs localize to retrotransposons and their remnants inthe S. pombe genome

Page 19: Shiv Grewal

CENP-Bs displaying distinct peaks at LTRs repress Tf2retrotransposons

abp1Δcbh1Δcbh2Δ

Tf2-LTRTf2-orf

abp1Δ cbh1Δ cbh2Δ abp1Δcbh1Δ

abp1Δcbh2Δ

cbh1Δcbh2Δ

Rela

tive

fold

exp

ress

ion

to W

T

10

20

5

15

25

30RT-PCR

Page 20: Shiv Grewal

CENP-Bs silence LTR-associated genes

Chromosome I Position (Kb)4,521 4,525 4,529 4,533

Rela

tive

fold

enr

ichm

ent

4

10

14

2

6

12

8

1618

Tandem LTRs

27D7.10c

27D7.09c 27D7.11c

Abp1Cbh1

Chromosome III Position (Kb)1,462 1,466 1,470 1,474

Rela

tive

fold

enr

ichm

ent

4

10

14

2

6

12

8

Abp1Cbh1

SPCC11E10.07c rik1LTR

Rela

tive

fold

exp

ress

ion

to W

T

1

2

3

4

511E10.07c

abp1∆ cbh1∆ cbh2∆ abp1∆cbh1∆

abp1∆cbh2∆

cbh1∆cbh2∆

abp1∆cbh1∆cbh2∆

Rela

tive

fold

exp

ress

ion

to W

T

2

45

1

3

67

27D7.10c

abp1∆ cbh1∆ cbh2∆ abp1∆cbh1∆

abp1∆cbh2∆

cbh1∆cbh2∆

abp1∆cbh1∆cbh2∆

Page 21: Shiv Grewal

CENP-Bs recruit Clr3 and Clr6 histone deacetylasesto repress Tf2 retroelements

Clr3 = SHREC Clr6 = Clr6 HDAC complexes

InputαFLAG IPClr3-mycAbp1-FLAG

+ +- +

IB: αmyc

IB: αFLAG

+ +- +

InputαFLAG IPAbp1-FLAG- +IB: αClr6

IB: αFLAG

- +

Co-Immunoprecipitation

Clr3

3.8 1.3

WT

abp1∆

4.5 0.8

controlTf2-12

Clr6

Input

ChIP

Page 22: Shiv Grewal

CENP-B

Clr6 and SHREC repressors

HP1

Heterochromatic domainLTR retrotransposon Gene

CENP-Bs and heterochromatin recruit same repressorcomplexes

Repeats

Page 23: Shiv Grewal

CENP-B mechanism silences and immobilizes an “extinct”retroelement that becomes sequestered into “Tf” bodies

Cbh1 ChIP

Tf1- +

control7D4.08 Abp1

ChIP

- +Tf1

7D4.08

Tf1-neo

+ Tf1

Tf2DAPI

- Tf1

7D4.08DAPI Merged

Tf body

Tf LTRCENP-BHDAC?

neo

Page 24: Shiv Grewal

Summary and Implications

Pol II transcription of repeats during S-phase facilitates RNAi-mediated targeting of heterochromatin

Heterochromatin is dynamically regulated and serves as a recruitingplatform for targeting variety of factors

Host genome surveillance for retrotransposons by transposon-derived CENP-B proteins - maintenance of genomic integrity - retrotransposon-mediated genome organization

CENP-BTf Bodies

retrotransposonsurveillance

gene regulatorynetworks

genomeorganization