Heterochromatin and RNAi are required to establish CENP-A chromatin at centromeres

Abstract

Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A(Cnp1) and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A(Cnp1) chromatin on naïve templates. Once assembled, CENP-A(Cnp1) chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified.

Fig. 1

Testing roles for Clr4 in establishment of CENP-A^Cnp1 chromatin at centromeres. (A) Fission yeast centromere DNA and minichromosomes used (20). Association of CENP-A^Cnp1 and H3K9me2 with pH-cc2 (B), pHH-cc2 (C), or pcc2 (D) introduced into wild-type (wt) or clr4 cells by crossing (X) or transformation (T) is shown (see also fig. S1). Polymerase chain reaction (PCR) product positions on minichromosomes are indicated (A). fbp1 is the control noncentromeric locus. In CENP-A^Cnp1 ChIP, enrichment of pcc2 product was compared with input DNA (T) relative to fbp1. Enrichment of endogenous cc1/3 relative to fbp1 was also assessed. In H3K9me2 ChIP, enrichment at plasmid–outer repeats J2 (B) or K″K″ (C) products was compared with the PCR of input DNA relative to the fbp1 product. Enrichment of endogenous otr sequences was also assessed (B and C). Quantitative PCR confirms these results [lower panels (B) and (C) and right (D) and fig. S3] (20). Histograms show percent enrichment on plasmid relative to endogenous cc1/3.

Fig 2

Clr4 is required to establish, but not to maintain CENP-A^Cnp1 and kinetochore proteins on the large cen3 minichromosome. Association of CENP-A^Cnp1 and H3K9me2 in (A), and CENP-C^Cnp3 and Sim4 in (B) were determined by ChIP in wild-type and clr4 strains containing pH-icc3i-H introduced by crossing (X) or transformation (T). fbp1 or act1 are control noncentromeric loci. In CENP-A^Cnp1, CENP-C^Cnp3, and Sim4 ChIPs enrichment of plasmid cc3 product was compared with input DNA (T) relative to the act1 product. Positive control: Enrichment of endogenous centromeric cc1/3 was assessed. H3K9me2 ChIP: Enrichment of plasmid–outer repeat J3 product was compared with PCR of input DNA (T) relative to act1. Positive control: Enrichment of endogenous otr centromeric repeats was assessed.

Fig 3

clr4⁺ reintroduction triggers CENP-A^Cnp1 recruitment to minichromosome and minichromosome stability. (A) Association of H3K9me2 and CENP-A^Cnp1 with pH-icc3i-H in clr4 before and after clr4⁺ reintroduction (clr4⁺clr4). Primers and conditions as in Fig. 2. (B) Wild-type (wt) and clr4⁺clr4 cells containing pH-icc3i-H, are indistinguishable. Serial dilution assay on 1/10th adenine assesses minichromosome stability [red colonies (lost); white colonies (retained)] or full adenine ± 10 g/μl TBZ.

Fig 4

Swi6 and RNAi components are required for CENP-A establishment. CENP-A^Cnp1 and H3K9me2 ChIP on pHH-cc2 after direct transformation with plasmid DNA and selection in dcr1, chp1, swi6, and wild-type (wt) strains. Primers and conditions as in Fig 1C. Quantitative PCR confirms these results (lower panel and fig. S3) (20).