The fission yeast HIRA complex contains Hip1, Slm9 and a structurally unrelated protein called Hip3 
. In order to identify additional components of the HIRA complex, and to identify other factors that interact with this complex, we employed a rapid affinity purification protocol that facilitates the isolation of large protein assemblies 
. Extracts from cells expressing a Slm9-FLAG fusion, were affinity purified and the resulting proteins were identified by liquid chromatography-tandem mass spectrometry. Numerous proteins were found to reproducibly co-purify with Slm9-FLAG (). Multiple components of the chaperonin containing TCP-1 complex (CCT, also called TRiC) 
were present in Slm9-FLAG purifications. CCT is responsible for the folding of numerous proteins 
and this suggests that it is required for the biogenesis of the HIRA complex. This analysis also suggests that HIRA interacts with the 19S regulatory particle of the proteasome 
because multiple ATPase subunits (Rpt1, Rpt2, Rpt3, Rpt4 and Rpt5) from this complex co-purified with Slm9. Analysis of the list of Slm9-interacting proteins also revealed SPBC947.08c, a previously uncharacterised protein that we named Hip4 for H
. Hip4 has limited homology to S. cerevisiae
Hpc2 and human UBN1; Hpc2 is a component of the S. cerevisiae
HIR complex 
and during the course of this study Banumathy and co-workers reported that UBN1 functions in conjunction with human HIRA 
. Although the overall level of sequence identity of SPBC947.08c to both Hpc2 and UBN1 is low, it does contain a short sequence motif, termed an Hpc2-related domain (HRD), which is a characteristic of these proteins (). In order to determine whether Hip4 also interacts with the other components of the HIRA complex its chromosomal locus was tagged with the TAP epitope (hip4-TAP
). Whole cell extracts from hip4-TAP
cells were partially purified using IgG magnetic beads and analysed by western blotting. This confirmed that Slm9 co-purifies with Hip4 () and furthermore, similar experiments revealed that both Hip1 and Hip3 co-precipitate with Hip4 (). Therefore, Hip4 is stably associated with all the components of the S. pombe
An Hpc2/UBN1 related protein in fission yeast.
Hip4 is stably associated with the subunits of the HIRA complex.
In order to investigate the function of this protein we constructed a hip4Δ
strain. Cells lacking Hip4 were found to be viable but exhibited a number of phenotypes that are reminiscent of cells lacking the function of the HIRA complex (). Like other HIRA mutants, the hip4Δ
strain was temperature sensitive showing a limited ability to proliferate at 37°C (). Furthermore, microscopic examination of hip4Δ
cells revealed that they had an elongated phenotype similar to hip1Δ
cells (), which are known to have a G2 cell cycle delay 
. The introduction of a plasmid ectopically expressing hip4
(pRep41Pk-Hip4) rescued the elongated cell phenotype of hip4
Δ cells () confirming that this defect is due to lack of Hip4 function.
Phenotypes associated with deletion of hip4+.
The HIRA complex is required for accurate chromosome segregation and consistent with this hip1Δ
cells exhibit hypersensitivity to thiabendazole (TBZ), a drug which depolymerises microtubules and thus impairs the function of the mitotic spindle 
. Deletion of hip4+
also resulted in a marked increase in TBZ sensitivity, suggesting that Hip4 is also required for accurate chromosome segregation (). Increased sensitivity to TBZ is often correlated with dysfunctional centromeric heterochromatin. In fission yeast, centromeric heterochromatin is assembled onto arrays of dg
repeats that flank a central core domain 
. Marker genes inserted into dg
repeats are subjected to strong transcriptional silencing and so cells carrying the ade6+
gene in this region form dark red colonies when adenine is limiting 
(). Deletion of hip1+
results in reduced trans
-gene silencing and the formation of pink colonies 
. Similarly, introduction of the hip4Δ
allele into this background resulted in the formation of pink colonies indicating that Hip4 is also required for reporter gene silencing at dg-dh
Loss of Hip4 impairs transcriptional silencing.
Paradoxically, the proper assembly of ‘silent’ centromeric heterochromatin requires transcription of the dg-dh
repeats from both strands 
. These non-coding transcripts are processed by the RNAi machinery to form siRNAs which play an important role in the assembly of heterochromatin at the centromere. Transcription the dg-dh
repeats is differentially regulated: the reverse strand (cenREV) is transcribed in wild type cells at low levels and rapidly processed by the RNAi machinery whereas transcription in the forward direction (cenFOR) is limited by heterochromatin 
. Accordingly, mutations that impair heterochromatin function often result in increased levels of cenFOR transcripts. Quantitative RT-PCR analysis revealed that transcripts from dg-dh
repeats were increased (1.59 fold [±0.1]) in a slm9
Δ background. We therefore used strand-specific RT-PCR to further analyse dg-dh
transcripts and found that increased levels of cenFOR transcripts were detectable in hip1Δ
cells (). This is consistent with a role for the HIRA complex in the function of pericentric heterochromatin.
Heterochromatin is also found at the mat
locus, although at this region the assembly of heterochromatin is only partially dependent upon the RNAi machinery 
. Marker genes that are inserted into the mat
region are subjected to strong transcriptional silencing that is dependent upon the HIRA complex 
(see also ). Deletion of hip4+
also abolished the silencing of an ade6+
reporter located at mat3-M
(). This indicates that the function of the HIRA complex at heterochromatin requires the Hip4 subunit.
The genome of the sequenced S. pombe
strain (972), harbours 13 full-length copies of the Tf2
LTR retrotransposon 
. These elements are silenced by a mechanism that is distinct from the mat
locus and centromeres being independent of H3K9 methylation 
. Instead the transcriptional silencing of these elements requires CENP-B homologues 
and multiple histone deacetylases (Clr6, Clr3 and Hst4) 
. It is also dependent upon the function of the HIRA complex as global Tf2
mRNA levels are dramatically increased in hip1
Δ and hip3
Δ backgrounds 
. Furthermore, northern blotting and quantitative RT-PCR analyses revealed that Tf2
mRNA levels were similarly increased in hip4Δ
cells (), indicating that LTR retrotransposon silencing requires Hip4. Taken together our data indicate that Hip4 is an integral component of the HIRA complex that is required for silencing at multiple loci.
Hip4 represses the expression of Tf2 retrotransposons.