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 H3K9methyltransferase 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.Metazoan centromeres are mostly composed of repetitive DNA upon which the kinetochore assembles to mediate chromosome segregation. Epigenetic factors contribute to the establishment and maintenance of kinetochores at particular sites, which are composed of CENP-A chromatin (1-4). However, the primary signals specifying the site of CENP-A chromatin, and thus kinetochore assembly, are unknown.In Drosophila, human, and fission yeast, kinetochores are embedded in heterochromatin (5-7). Fission yeast centromeres have two distinct domains: outer repeats (otr) that flank the central kinetochore domain composed of innermost repeats (imr) and central core (cnt/cc) DNA (3, 8) (Fig. 1A).Heterochromatin, containing Swi6 (heterochromatin protein 1 or HP1) bound to histone H3 dimethylated on lysine 9 (H3K9me2), forms over the otr, whereas CENP-A Cnp1 replaces histone H3 in the central domain (5, 7, 9-11). Centromeric heterochromatin contributes to centromere function by recruiting cohesin and mediating cohesion between sister centromeres (12, 13). Here, we test the idea that heterochromatin marks sites for CENP-A chromatin assembly (14) Fission yeast minichromosomes must contain at least part of an otr and a large portion of central domain (imr-cc) DNA to allow kinetochore assembly, segregation function, and minichromosome retention (3,(14)(15)(16)(17). Small interfering RNAs (siRNAs) originating from noncoding otr transcripts direct H3K9 methylation and heterochromatin formation on these repeats (8, 18). Defects in various components cause loss or reduction in H3K9 methylation, Swi6, and centromeric cohesin (5, 9, 13). Endogenous chromosomes segregate without centromeric heterochromatin, because sister chromatids remain tethered by arm cohesin. However, mitotic stability of small circular minichromosomes is obliterated (19). Minichromosomes containing otr and cc DNA have centromere activity, resulting in a low lo...

Human centromere genomics: now it's personal

“…In addition to direct sequence-based interactions with inner kinetochore proteins, centromeric sequences also accommodate a hierarchy of constitutive and dynamic protein-based interactions that bridge the inner kinetochore and the outer kinetochore in a protein-mediated transfer of tension from spindle forces to those proteins that directly bind DNA (Hori et al 2008;Screpanti et al 2011). Moreover, proper assembly of pericentromeric heterochromatin, typically correlated with functional kinetochores, may be influenced by sequence-based factors, such as transcriptional regulation and specialized chromatin compaction (Folco et al 2008). Our understanding of the genomic definition of human centromeres will require a much broader understanding of the sequence-based interactions that define the centromere-kinetochore interface.…”

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

“…Of central importance to centromeres are nucleosomes with the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A) (1). Centromeres are intimately associated with heterochromatin, which is frequently formed adjacent to an array of CenH3 nucleosomes, upon which the kinetochore is assembled, and heterochromatin is required to promote CenH3 assembly at centromeres (2). In both mammals and plants, constitutively heterochromatic megabase-sized centromeric regions are rich in highly repetitive tandem arrays of satellite DNA and moderately repetitive sequences, such as transposons, and show a low level of transcription (3).…”

Epigenetics: heterochromatin meets RNAi

“…The levels of eight proteins have been shown to be controlled by Dcr1 in S. pombe [84]. A novel function of RNAi has recently been discovered by its requirement for de novo establishment of CENP-A chromatin at the centromere [85]. CENP-A is a specialized Histone H3 variant, found at active centromeres and functioning as a platform for kinetochore assembly, and is highly conserved in eukaryotes [86].…”