Silencing mediated by the Schizosaccharomyces pombe HIRA complex is dependent upon the Hpc2-like protein, Hip4.
BACKGROUND
HIRA (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.


RESULTS
To 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
Overall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci.
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, United Kingdom.
Holly E
HE
Anderson
Alexander
A
Kagansky
Josephine
J
Wardle
Juri
J
Rappsilber
Robin C
RC
Allshire
Simon K
SK
Whitehall
D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D020868:Gene Silencing; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000502:physiology*
D017931:DNA Primers; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C543473:hip1 protein, S pombe