Heterochromatin and RNAi Are Required to Establish CENP-A Chromatin at Centromeres

1. Heterochromatin and RNAi AreRequired to Establish CENP-AChromatin at Centromeres Hernan Diego Folco, Alison L. Pidoux, Takeshi Urano, Robin C. Allshire Hsiang Ho

2. otr : outer repeats imr : innermost repeats cnt/cc : central core otr otr Background • Centromere nucleosome differs from conventional nucleosome by the presence of a centromere-specific histone H3 variant (CENP-A, centromere protein A, in place of canonical H3) • CENP-A cnp-1 = fission yeast homologue of CENP-A • In fission yeast, kinetochore establishment requires flanking heterochromatin providing assembling signal. These could include RNA-induced transcriptional silencing (RITS), H3K9Me, HP1, cohesin. • siRNA originating from otr serves as platforms for the recruitment of modifying enzs, direct H3K9Me to initiate heterochromatin formation However, the primary signals specifying the site of CENP-A chromatin, and thus kinetochore assembly, are unknown…

3. Hypothesis Heterochromatin marks sites for CENP-A chromatin assembly Experimental system Endogenous chromosome are tethered by cohesin, so to study the mitotic stability of centromere, small circular minichromosomes were used. Clr4: H3K9 methyltransferase CENP-A chromatin formation in clr4Δ is dependent on the entering state: preassembled (cross) or naked DNA (transform)

4. Fig 1. Clr4 H3K9 methyltransferase and otr heterochromatin are required to establish CENP-ACnp1 chromatin

5. Fig 2. • CENP-CCnp3 and Sim4 (Kinetochore component) were retained in endogenous cc1/3, cc of pH-icc3i-H and when it is crossed into clr4Δ, but not when transformed into clr4Δ. • Small pH-cc2 or pHH-cc2 minichromosomes may be particularly sensitive to the absence of heterochromatin • ∴minichromosome containing two nearly complete otr repeats flanking an entire cen3 central domain were used. • Same conclusion were reached as in pH-cc2 or pHH-cc2 . ∴kinetochore proteins association depends on initial propagation • Once CENP-ACnp1 chromatin and kinetochore proteins assemble on centromeric DNA, they are propagated without flanking heterochromatin. Clr4 is required to establish, but not to maintain CENP-ACnp1 and kinetochore proteins.

6. Fig 3. clr4+ reintroduction triggers CENP-ACnp1 recruitment to minichromosome (A) and minichromosome stability (B). • Not sensitive to TBZ (white)= fully restore centromere function. Red =lost • Clr4 reintroduce restore full centromere-kinetochore function Clr4 reintroduce bring back CENP-ACnp1 of clr4Δ-T

7. Fig 4. Swi6 and RNAi components are required for CENP-A establishment. RNAi-mediated otr heterochromatin assembly =HP1 siRNA originating from otr serves as platforms for the recruitment of modifying enzs, direct H3K9Me A patch of fully intact centromeric heterochromatin is required to establish centromere

8. Key findings • RNAi-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A cnp-1 and kinetochore assembly over the central domain. Figure S6. Model. An intact domain of heterochromatin with all components (RNAi, Swi6, Clr4 H3K9me2) is required for the initial establishment of CENP-ACnp1 chromatin and consequently kinetochore proteins on central domain DNA. However, once established, this specialised CENP-ACnp1 structure can be propagated independently of heterochromatin.

9. Questions raised • Fig 3B, clr4Δ-X is also sensitive to TBZ. This indicates that although CENP-ACnp1 is formed (IP positive), but the centromere function can still be impaired. • Fig 4, in the absence of chp1, dcr1, or swi6, CENP-ACnp1 did not associate with cc2 on minichromosome even though itis associated with endogenous centromere. This indicates either RNAi is required on naked DNA or it might be other components are defected in the artificial minichromosome (ex.cohesin). Crossing to mutants may address the difference is due to the fact that DNA is naked or preassembled.