Epigenetic genome control by RNAiand transposon-derived proteins
Shiv Grewal
Center for Cancer Research, NCINational Institutes of Health, Bethesda
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
Distinct patterns of histone modifications specify discrete downstream regulatory events
Heterochromatin Euchromatin
Adapted from Zhang and Reinberg 2001
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
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
Grewal and JiaNat Rev Genet
8:35-46
Heterochromatin serves as a versatile platform for targetingeffectors across extended domains
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
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
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
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
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
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
TFIIIC, but not Pol III, localizes to heterochromatin domainboundaries
ChIP
-chi
p
TFIIIC bind to tRNA gene clusters at centromereheterochromatin domain boundaries
ChIP-chip analyses
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
TFIIIC bound loci cluster near the nuclear periphery: implicationsfor genome organization and function
TFIIIC body
TFIIIC/DAPI
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
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
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
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∆
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
CENP-B
Clr6 and SHREC repressors
HP1
Heterochromatic domainLTR retrotransposon Gene
CENP-Bs and heterochromatin recruit same repressorcomplexes
Repeats
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
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
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