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S. pombe genome contains several different classes of repeat elements

retrotransposon. S. pombe genome contains several different classes of repeat elements. Transposon-derived proteins Heterochromatin. The organization of genome into higher-order structures has important biological implications.

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S. pombe genome contains several different classes of repeat elements

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  1. retrotransposon S. pombe genome contains several different classes of repeat elements • Transposon-derived proteins • Heterochromatin

  2. The organization of genome into higher-order structures has important biological implications • Stable maintenance of gene expression patterns during development • Maintenance of genomic integrity and prohibition of inter- or intrachromosomal recombination in repetitive DNA sequences • Lineage-specific control of long-range chromatin interactions • Proper segregation of chromosomes • Cancer and other human diseases

  3. DNA binding domain Dimerization domain DDE ABP1 S. pombe CENP-B CBH1 CBH2 Mammalian CENP-B POGOK transposase 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 genome encodes three CENP-Bs that have redundant roles in centromere chromatin assembly

  4. Tf2-4 rpc34 IG LTRs Tf2-7-8 Tf2-7-8 Tf2-3 LTR LTR Tf2-12 Tf2-5 Tf2-2 725.03 IG Tf2-6 Tf2-10 Tf2-10 LTRs LTR ars3002 LTRs LTR LTR wtf6 14 14 14 Tf2-9 Tf2-11 rip1 IG 12 12 12 Tf2-13 725.03 IG 10 10 srk1 IG 10 LTR 8 8 8 6 6 6 4 4 4 SPAP32A8.02 LTRs rip1 IG 2 2 2 Tf2-4 spo20 IG Tf2-3 Tf2-9 1,000 1,000 2,000 3,000 4,000 5,000 2,000 3,000 4,000 63.12c IG rpc34 IG gut2 IG Tf2-2 Tf2-1 Tf2-1 LTRs LTRs rpc34 IG wtf6 30 30 gut2 IG 25 25 Tf2-11 20F10.03 IG 20 20 Tf2-6 Tf2-13 cwf10 IG Tf2-12 15 15 srk1 IG LTR 10 10 5 5 500 1,000 1,500 2,000 CENP-Bs localize to retrotransposons and their remnants in the S. pombe genome Chromosome I Chromosome II Chromosome III 4.6 Mb 5.7 Mb 3.5 Mb tel1L cen1 tel1R tel2L cen2 mat tel2R tel3L cen3 tel3R rDNA rDNA Abp1 Fold enrichment 500 1,000 1,500 2,000 Cbh1 30 25 Fold enrichment 20 15 Tf2-5 10 5 1,000 2,000 3,000 4,000 5,000 1,000 2,000 3,000 4,000 Chromosome Position (Kb)

  5. LTR rik1 SPCC11E10.07c 5 14 11E10.07c 12 Abp1 4 10 Cbh1 8 30 3 6 Tf2-LTR 4 Relative fold enrichment Tf2-orf 2 25 2 1,462 1,466 1,470 1,474 20 1 15 Relative fold expression to WT Chromosome III Position (Kb) 10 5 abp1 cbh1 cbh2 abp1 cbh1 abp1 cbh2 cbh1 cbh2 CENP-Bs silence LTR-associated genes and Tf retroelements RT-PCR Relative fold expression to WT cbh1∆ cbh2∆ abp1∆ cbh1∆ abp1∆ cbh2∆ abp1∆ cbh1∆ cbh2∆ abp1∆ cbh2∆ cbh1∆ abp1 cbh1 cbh2

  6. Transcriptional gene silencing effector protein complexes Clr6 HDAC Clr3 HDAC

  7. Co-Immunoprecipitation Input FLAG IP Clr3-myc + + + + - + - + Abp1-FLAG IB: myc IB: FLAG FLAG IP Input - + - + Abp1-FLAG IB: Clr6 IB: FLAG CENP-Bs recruit Clr3 and Clr6 histone deacetylases to repress Tf2 retroelements ChIP abp1∆ WT Tf2-12 Clr3 control 3.8 1.3 Clr6 4.5 0.8 Input Clr3 = SHREC Clr6 = Clr6 HDAC

  8. Probe SHREC activities facilitate positioning of nucleosomes required for higher-order chromatin assembly Silencing and recombination suppression Micrococcal nuclease digestion patterns

  9. CENP-Bs and their associated HDACs are required for clustering of retrotransposon elements FISH using Tf2 retroelement probe Tf2 Tf body Tf LTR CENP-B DAPI HDAC ? H2O2 treatment abp1∆ clr3clr6 (HDAC) WT Silencing and recombination suppression

  10. Subtelomeric region LTRs dh repeat H3K9me H3K9me H3K9me dg dg dh dh cnt pgp1 cwf20 Swi6 Swi6 H3K4me H3K4me H3K4me ChIP fold enrichment mat locus 40 40 40 30 30 30 Swi6 20 20 20 Grewal and Klar Cell 1996 ChIP fold enrichment 10 10 10 Heterochromatin coats extended domains associated with a specific classes of repeat elements in S. pombe Centromere ChIP fold enrichment

  11. Clr4 Chp1 Spreading Nucleation HP Atf1/Pcr1: mat Taz1/Ccq1: tel Clr4 HP HP Unknown: cen Clr4 Rik1 Nucleation and spreading of heterochromatin H3K9me HP HP Repeat elements Ac Pol II HP HP RdRP Chp1 Clr4 Ago1 Tas3 Antisense transcript HP TFIIIC Rdp1 H3K9 HMTase Dcr1 RITS Boundary element siRNA HP = Swi6, Chp2 Spreading of heterochromatin requires Clr4 chromodomain binding to methylated H3K9

  12. RNAi HDACs Effectors Cohesin Clr4 Chp1 SMCs Transcriptional silencing Inhibit “spurious” transcription Recombination suppression Centromere and telomere function Clr4 Rik1 Heterochromatin serves as versatile recruiting platform to regulate diverse chromosomal processes H3K9me HP HP HP HP Repeat elements Pol II HP Ac HP HP RDRC Chp1 Clr4 Ago1 Tas3 HP TFIIIC Dcr1 RITS siRNA Boundary element HP = Swi6, Chp2 Grewal and Jia Nat Rev Genet 8:35-46

  13. CENP-B HP1 CENP-Bs and heterochromatin recruit same repressor complexes Clr6 and SHREC repressors Repeats LTR retrotransposon Heterochromatic domain Gene

  14. Cascade of events at heterochromatin during the cell cycle condensin HP = chromodomain proteins Swi6 and Chp2

  15. Rik1, a component of Clr4 methyltransferase, is recruited to Pol II transcribed repeats during S-phase cen repeat Ago1 Antisense Clr4 Rik1 cen repeat Rik1 control ChIP Pol II Clr4 complex

  16. H3-K9 methylation Chp1 ago1 Rik1 Rdp1 Clr4-Flag IP WT clr3∆ cul4-1 9 Rik1 ChIP Flag-Clr4 chp1∆ Flag-Clr4 Flag-Clr4 tas3∆ Untagged 6 3 Anti- Chp1 Clr4 Anti- Flag Chp1 12 * WT clr3∆ cul4-1 RNAPIIChIP 8 IP-Western 4 Rik1 binding correlates with RNAPII transcription of cen repeat elements Relative fold enrichment

  17. Transcription coupled loading of heterochromatin factors during S-phase HP = chromodomain proteins Swi6 and Chp2

  18. Raf2 Rik1 Clr4 Clr4 complex Clr4 complex components are distributed across euchromatic regions 2784391 2776391 2790391 0.5 ChIP enrichment (log2 ) Rik1-myc -0.5 0.5 Raf2-myc ChIP enrichment (log2 ) -1

  19. Raf2 Rik1 Clr4 Clr4 complex Clr4 complex subunits show similar distribution profiles at euchromatic genes 0.2 Rik1 profile ChIP enrichment (log2 ) -1000 0 1000 2000 -0.2 0.2 ChIP enrichment (log2 ) Raf2 profile -1000 0 1000 2000 -0.2

  20. Exploring RNAi connections to other nuclear functions RNAi mutants Mutants defective in nuclear functions double mutants yeast colony size positive interaction no interaction negative interaction

  21. RNAi and heterochromatin machineries positively interact with factors involved RNA Pol II transcription (JmJc) (HDAC)

  22. RNAi and heterochromatin factors show negative genetic interactions with DNA repair machinery

  23. Re-wiring of conserved functional modules in different organisms

  24. Acknowledgments Grewal Lab Hugh Cam Ee Sin Chen Martin Zofall Ke Zhang Tamas Fischer Bowen Cui Natalia Kommissarova Ken-ichi Yamane Chanan Rubin Takeshi Mizuguchi Nazanin Ashourian Tomo Sugiyama Former Lab Members Tomoyasu Sugiyama (Tsukuba Univ) Estelle Nicolas (CNRS, Toulouse) Ken-ichi Noma (Wistar Institute) Songtao Jia (Columbia, NY) Ira Hall (Univ Virginia) Jun-ichi Nakayama (Riken, Kobe) Takatomi Yamada (Riken, Tokyo) Collaborators Henry Levin (NICHD) Wolfgang Fischle (Max Planck) Peter FitzGerald (NCI) Danesh Moazed (Harvard) Nevan Krogan and Assen Roguev (UCSF)

  25. SHREC TGS Clr4 SHREC Clr6-C2 Clr6-C1 PTGS Clr4 Rik1 TGS effector complexes Clr6 Clr3 Nicolas et al 2007 NSMB Sugiyama et al 2007 Cell Post-transcriptional and transcriptional heterochromatic silencing HP HP HP HP Repeat Pol II HP Ac HP Chp1 Chp1 Clr4 Ago1 Tas3 HP TFIIIC Rdp1 ClrC Dcr1 RITS RDRC Boundary element siRNA HP = Swi6, Chp2

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