Epigenetic genome control by RNAi and transposon-derived proteins

Transcription

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

2 Transposons and their remnants constitute a significant fraction of eukaryotic genomes Organism Yeast Worm Drosophila Mouse Human Transposon content (% of genome) 3.1% 6.% -12% 38% 48% Heterochromatin / RNAi Transposon-derived proteins

3 Distinct patterns of histone modifications specify discrete downstream regulatory events Heterochromatin Euchromatin Adapted from Zhang and Reinberg 2001

4 Heterochromatin coats extended domains associated with a specific classes of repeat elements in S. pombe Centromere Subtelomeric region pgp1 dg dh cnt dg dh cwf20 dh repeat LTRs ChIP fold enrichment H3K9me Swi6 H3K4me ChIP fold enrichment H3K9me Swi6 H3K4me mat1 mat locus IR-L mat2p cenh mat3m IR-R 2. kb ChIP fold enrichment H3K9me Swi6 H3K4me Grewal and Klar Cell 1996

5 Nucleation and spreading of heterochromatin Nucleation (S phase) Spreading H3K9me Clr4 Dcr1 RdRP Repeat elements Rdp1 Chp1 Ago1 Tas3 RITS Clr4 Rik1 H3K9 HMTase Pol II Clr4 Chp1 Ac STOP sirna = Swi6, Chp2 Boundary element Spreading of heterochromatin requires Clr4 chromodomain binding to methylated H3K9 Zhang et al 2008 NSMB 1:381

6 Heterochromatin serves as a versatile platform for targeting effectors across extended domains Grewal and Jia Nat Rev Genet 8:3-46

7 Post-transcriptional and transcriptional heterochromatic silencing Clr6-C1 Dcr1 sirna Factory SHREC H3K9me Clr6-C2 Repeat elements RDRC Rdp1 Chp1 Ago1 Tas3 Rik1 RITS ClrC sirna PTGS Clr4 Pol II Clr4 Clr4 = Swi6, Chp2 Chp1 SHREC TGS Ac STOP Boundary element Ago1 Clr6-C1 Clr6-C2 SHREC RISC Clr6 Pst1 (Sin3) Pst2 (Sin3) Cph2 Prw1 Clr6 (HDAC) Cph1 Alp13 (MRG1) Png2 TGS effector complexes Clr3 Mit1 (Snf2 ATPase) Clr1 Ccq1 Clr3 (HDAC) Clr2 Sds3 Nicolas et al 2007 NSMB Sugiyama et al 2007 Cell

8 Probe SHREC activities facilitate proper positioning of nucleosomes required for higher-order chromatin assembly Micrococcal nuclease digestion patterns Sugiyama et al 2007 Cell 128:491

9 Heterochromatin mediates recruitment of cohesin that is essential for maintenance of genomic integrity Transcriptional and Recombinational Block Euchromatin Heterochromatin Euchromatin mat1 IR-L mat2p cenh mat3m IR-R 2. kb Swi6 Cohesin K. Noma and S. Grewal

10 WT swi (fold enrichment) Swi Complex M cells (fold enrichment) Swi Complex P cells Heterochromatin mediates cell-type specific spreading of a protein complex essential for mating-type switching mat1-p mat1-m WT swi α-swi WCE α-swi WCE mat2-p cenh mat3-m Enhancer Element 2. kb WT swi6 - α-swi WCE α-swi WCE mat act1 mat act1 mat act1 mat act1 WT swi6 - mat act1 mat act1 mat act1 mat act1

11 Boundaries of a heterochromatic domain Transcriptional and Recombinational Block Euchromatin Heterochromatin Euchromatin 2. kb mat1 IR-L mat2p cenh mat3m IR-R Swi6 Fold Enrichment H3 Lys9-methyl H3 Lys4-methyl

12 TFIIIC binding to the boundary elements blocks spreading of heterochromatin heterochromatin spreading mat3 IR-R kan + ura4 + 1 kb wt IR-R IR-R #1 IR-R #2 IR-R #3 IR-R #4 B-box - FOA + FOA mat3m B-box cons. B-box seq#1 B-box seq#2 B-box seq#3 B-box seq#4 B-box seq# 1 kb IR-R...GTTCGANTC... ***** * * AGCTGTTCGTAGCAGCA ******* GCTCTTTCGATTATAGA ********* CCCGGTTCGAGTCCTAT ******** AGCGGTTCGATTTGCGA **** *** TAAGGTTCTACTTTATC mat3m IR-R B-box ura kb H3 Lys9-methyl (fold enrichment) 20 wt B-box

13 TFIIIC, but not Pol III, localizes to heterochromatin domain boundaries ChIP-chip

14 TFIIIC bind to trna gene clusters at centromere heterochromatin domain boundaries ChIP-chip analyses

15 TFIIIC, but not Pol III, peaks at sites are predominantly associated with divergent gene promoter regions COC3 SPBC336.0c sfc3 fold enrichment TFIIIc Pol III B-box COC4 SPBC139.0 SPBC139.07c fold enrichment COC SPCC63.02c SPCC63.03 fold enrichment 40 20

16 TFIIIC bound loci cluster near the nuclear periphery: implications for genome organization and function TFIIIC/DAPI TFIIIC body

17 Host genome surveillance for retrotransposons and repeats by transposon-derived proteins CENP-Bs are conserved proteins that contain DNA binding and dimerization domains CENP-Bs are derived from transposases of POGO DNA transposons S. pombe CENP-B ABP1 CBH1 CBH2 DNA binding domain DDE Dimerization domain S. pombe genome encodes three CENP-Bs that have redundant roles in centromere heterochromatin formation Mammalian CENP-B POGOK transposase

18 CENP-Bs localize to retrotransposons and their remnants in the S. pombe genome Chromosome I Chromosome II 4.6 Mb Fold enrichment.7 Mb tel1l 14 Chromosome III cen1 tel1r tel2l 3. Mb cen2 mat tel2r Tf2-4 Tf Tf2-3 Tf2- Tf2-2 Tf2-6 Tf2-1 LTRs LTR 8 12 Tf2-11 Tf2-9 LTR IG rip1 IG 8 Tf2- LTRs 12 LTRs Fold enrichment 6 1,000 2,000 3,000 4,000, spo20 IG 1 LTR SPAP32A8.02 Tf2-4 Tf2-3 Tf2-2 Tf2-1 LTRs 1,000 Tf2-6 Tf2-3, ,000 2,000 3,000 4,000,000 Chromosome Position (Kb) 00 rip1 IG Tf F.03 IG LTR Tf IG Tf2- cwf IG LTRs 2 LTRs 3,000 4,000 gut2 IG 1,00 2,000 gut2 IG 20 Tf ,000 1, c IG 1,000 srk1 IG rpc34 IG Tf2-13 LTR rpc34 IGIG rpc34 tel3r ars02 wtf6 4,000 2 Tf ,000 LTR 8 6 Cbh1 rdna Tf cen3 rdna Abp1 tel3l srk1 IG LTR Tf2-13 wtf6 00 1,000 1,00 2,000

19 CENP-Bs displaying distinct peaks at LTRs repress Tf2 retrotransposons Relative fold expression to WT Tf2-LTR Tf2-orf RT-PCR abp1δ cbh1δ cbh2δ abp1δ cbh1δ abp1δ cbh2δ cbh1δ cbh2δ abp1δ cbh1δ cbh2δ

20 CENP-Bs silence LTR-associated genes SPCC11E.07c Relative fold enrichment Abp1 Cbh1 1,462 1,466 1,470 1,474 Chromosome III Position (Kb) 27D7.09c LTR rik1 Tandem LTRs 27D7.11c Relative fold expression to WT E.07c abp1 cbh1 cbh2 abp1 cbh1 abp1 cbh2 cbh1 cbh2 abp1 cbh1 cbh2 Relative fold enrichment Abp1 Cbh1 27D7.c 4,21 4,2 4,29 4,33 Chromosome I Position (Kb) Relative fold expression to WT D7.c abp1 cbh1 cbh2 abp1 cbh1 abp1 cbh2 cbh1 cbh2 abp1 cbh1 cbh2

21 WT CENP-Bs recruit Clr3 and Clr6 histone deacetylases to repress Tf2 retroelements Co-Immunoprecipitation αflag IP Input Clr3-myc Abp1-FLAG IB: αmyc IB: αflag Clr3 ChIP abp Tf2-12 control Clr6 αflag IP - + Input - + Abp1-FLAG IB: αclr6 Input IB: αflag Clr3 = SHREC Clr6 = Clr6 HDAC complexes

22 CENP-Bs and heterochromatin recruit same repressor complexes Clr6 and SHREC repressors 1 CENP-B LTR retrotransposon Repeats Heterochromatic domain Gene

23 CENP-B mechanism silences and immobilizes an extinct retroelement that becomes sequestered into Tf bodies Tf1-neo Tf2 DAPI 7D4.08 DAPI Merged neo - Tf1 7D4.08 Abp1 ChIP Tf1 - + Cbh1 ChIP Tf D4.08 control + Tf1 Tf body Tf LTR CENP-B HDAC?

24 Summary and Implications Pol II transcription of repeats during S-phase facilitates RNAimediated targeting of heterochromatin Heterochromatin is dynamically regulated and serves as a recruiting platform for targeting variety of factors Host genome surveillance for retrotransposons by transposonderived CENP-B proteins - maintenance of genomic integrity - retrotransposon-mediated genome organization CENP-B Tf Bodies retrotransposon surveillance genome organization gene regulatory networks