Silencing Mediated by the Schizosaccharomyces pombe HIRA Complex Is Dependent upon the Hpc2-Like Protein, Hip4

Abstract

BackgroundHIRA (or Hir) proteins are conserved histone chaperones that function in multi-subunit complexes to mediate replication-independent nucleosome assembly. We have previously demonstrated that the Schizosaccharomyces pombe HIRA proteins, Hip1 and Slm9, form a complex with a TPR repeat protein called Hip3. Here we have identified a new subunit of this complex.Methodology/Principal FindingsTo identify proteins that interact with the HIRA complex, rapid affinity purifications of Slm9 were performed. Multiple components of the chaperonin containing TCP-1 complex (CCT) and the 19S subunit of the proteasome reproducibly co-purified with Slm9, suggesting that HIRA interacts with these complexes. Slm9 was also found to interact with a previously uncharacterised protein (SPBC947.08c), that we called Hip4. Hip4 contains a HRD domain which is a characteristic of the budding yeast and human HIRA/Hir-binding proteins, Hpc2 and UBN1. Co-precipitation experiments revealed that Hip4 is stably associated with all of the other components of the HIRA complex and deletion of hip4+ resulted in the characteristic phenotypes of cells lacking HIRA function, such as temperature sensitivity, an elongated cell morphology and hypersensitivity to the spindle poison, thiabendazole. Moreover, loss of Hip4 function alleviated the heterochromatic silencing of reporter genes located in the mating type locus and centromeres and was associated with increased levels of non-coding transcripts derived from centromeric repeat sequences. Hip4 was also found to be required for the distinct form of silencing that controls the expression of Tf2 LTR retrotransposons.Conclusions/SignificanceOverall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci.

The double face of the histone variant H3.3

“…The pattern of yeast H3 DSI deposition thus resembles the one mediated by the HIRA complex for H3.3 incorporation in "higher" eukaryotes. Of note, in both S. cerevisiae and S. pombe, a homolog of the HIRA complex was identified with orthologs characterized for each component of this complex [82][83][84]. In S. cerevisiae, the Hir complex is one of the factors required for nucleosome reassembly after the passage of the RNA polymerase II, a mechanism that is proposed to play a critical role in transcription-coupled H3 deposition [85].…”

HIRA, a Conserved Histone Chaperone, Plays an Essential Role in Low-dose Stress Response via Transcriptional Stimulation in Fission Yeast

“…Mass spectrometric analysis of the fission yeast Slm9 has led to the identification of the 19S ATPase complex as the interacting proteins (27). The 19S ATPase complex is recruited to chromatin in a histone H2B ubiquitylation-dependent manner and plays a nonproteolytic role in Pol II transcriptional elongation in the budding yeast (58 -60).…”

“…2A). As Slm9 and Hip1 form a complex with two other proteins, Hip3 and Hip4 (26,27), we also examined these HIRA complex subunit disruptants. We found that both hip3⌬ and hip4⌬ cells showed the cross-tolerance-defective phenotype, similar to slm9⌬ and hip1⌬ cells (Fig.…”

“…Whereas higher eukaryotes have a single HIRA protein (19 -22), the fission yeast possesses two HIRA proteins (Slm9 and Hip1) (23,24), same as the budding yeast Saccharomyces cerevisiae (Hir1 and Hir2) (25). Fission yeast HIRA proteins stably associate with two other proteins, Hip3 and Hip4, and form a tetrameric complex (HIRA complex) (26,27). Recently, Cabin1 and UBN1 were identified as the human counterparts of Hip3 and Hip4, respectively (28 -30), suggesting that the HIRA complex is evolutionarily conserved.…”

“…In human cells, the formation of senescence-associated heterochromatin foci depends on HIRA (39). Loss of the fission yeast HIRA complex components results in silencing defects at the centromere and mating type loci (27). A recent study has also demonstrated that a complex formed by the histone chaperone Asf1 and HIRA spreads across silenced regions via its association with the chromodomain protein Swi6 to facilitate histone deacetylation and heterochromatin spreading in the fission yeast (40).…”