Hip3 Interacts with the HIRA Proteins Hip1 and Slm9 and Is Required for Transcriptional Silencing and Accurate Chromosome Segregation

Abstract

The fission yeast HIRA proteins Hip1 and Slm9 are members of an evolutionarily conserved family of histone chaperones that are implicated in nucleosome assembly. Here we have used single-step affinity purification and mass spectrometry to identify factors that interact with both Hip1 and Slm9. This analysis identified Hip3, a previously uncharacterized 187-kDa protein, with similarity to S. cerevisiae Hir3. Consistent with this, cells disrupted for hip3 ؉ exhibit a range of growth defects that are similar to those associated with loss of Hip1 and Slm9. These include temperature sensitivity, a cell cycle delay, and synthetic lethality with cdc25-22. Furthermore, genetic analysis also indicates that disruption of hip3 ؉ is epistatic with mutation of hip1 ؉ and slm9 ؉ . Mutation of hip3 ؉ alleviates transcriptional silencing at several heterochromatic loci, including in the outer (otr) centromeric repeats, indicating that Hip3 is required for the integrity of pericentric heterochromatin. As a result, loss of Hip3 function leads to high levels of minichromosome loss and an increased frequency of lagging chromosomes during mitosis. Importantly, the function of Hip1, Slm9, and Hip3 is not restricted to constitutive heterochromatic loci, since these proteins also repress the expression of a number of genes, including the Tf2 retrotransposons.Centromeres play a critical role in the precise segregation of chromosomes, and as a result, defects in centromere function lead to aneuploidy (1). The fission yeast Schizosaccharomyces pombe provides an excellent system for the study of centromeres (2). In contrast to the budding yeast Saccharomyces cerevisiae, which has simple "point" centromeres (3), S. pombe has large complex centromeres that occupy between 35 and 110 kb and are arranged as a central core (cnt) flanked by arrays of repeated (imr and otr) elements (2). In this respect, S. pombe centromeres are reminiscent of the complex regional centromeres of metazoans. Furthermore, ultrastructural studies have revealed that the overall architectural organization of fission yeast centromeres is conserved with their human counterparts (4). Fission yeast centromeres are organized into distinct chromatin domains (2, 5). An inner domain is assembled into specialized chromatin in which core histone H3 is replaced by Cnp1, the fission yeast homologue of CENP-A (6), whereas the outer regions are associated with chromatin that resembles the pericentric heterochromatin of higher cells (2). Marker genes inserted into these outer regions are subject to heritable inactivation (7,8), which is dependent upon the RNA interference machinery, the methylation of histone H3 on lysine 9, and the association of a number of proteins, including Swi6, a homologue of mammalian HP1 (heterochromatin protein 1) (2, 5, 9 -12). In addition, the integrity of pericentric heterochromatin in fission yeast is dependent upon the two HIRA proteins Hip1 and Slm9 (13,14), since loss of either protein alleviates silencing in the otr centromeric repeats and results in...

Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging

“…However, the apparent inconsistency in this idea is merely an extension of an existing paradox. Specifically, HIRA and its orthologs in other species are typically involved in gene silencing and formation of heterochromatin (13,50,64,67,97,116,117,119,123), whereas HIRA's favored deposition substrate, histone H3.3, is linked to transcriptional activation (3,77,80,112,133). However, to our knowledge, histone H3.3 per se has not been shown to directly cause or contribute to transcription activation, and a proportion of histone H3.3 does carry posttranslational marks characteristic of transcriptionally silent chromatin (55,74,77).…”

“…For example, pRB recruits the histone methylase Suv39h1 to chromatin and, as described above, this enzyme is required for oncogene-induced formation of heterochromatin in murine T-cells (16). HIRA and ASF1a are two histone chaperones whose physical interaction is required to form transcriptionally silent heterochromatin from yeast to humans (13,50,64,67,97,116,145). One simple model to describe the cooperation of the pRB and HIRA/ASF1a pathways is that pRB initiates heterochromatin formation at the promoters of E2F target genes, and this heterochromatin acts as a nucleation site for HIRA/ASF1a-mediated large scale chromosome condensation.…”

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

“…To see whether Tf2 derepression is always associated with Rad52 binding to Tf2 cDNA, we carried out SPI-seq analysis on ams2D, as well as two other mutants defective in Tf2 silencing, the HIRA histone chaperone mutant slm9D and the CENP-B mutant cbp1D (Greenall et al 2006;Cam et al 2008). In both ams2D and slm9D, we observed Rad52 enrichment on Tf2, with patterns similar to those in D1D3 (Fig.…”

Host genome surveillance for retrotransposons by transposon-derived proteins

“…We next determined whether Abp1-mediated silencing of retrotransposons intersects with that of HIRA/Hip1 histone chaperone, implicated in Tf2 silencing 25 . The levels of Tf2 expression were higher in abp1D hip1D cells compared with single mutant abp1D or hip1D cells (Fig.…”