Previous evidence has demonstrated that the HIRA histone chaperone is required for transcriptional silencing at heterochromatic loci (16
). Here, we demonstrate that its function is not limited to heterochromatin, because HIRA represses transcripts from numerous promoters distributed throughout the genome, including subtelomeric genes, LTR retrotransposons, and their remnants, and it also limits the levels of cryptic antisense transcripts. Furthermore, loss of HIRA leads to increased access of genotoxic agents to the genome, indicating that HIRA is required for maintenance of the protective functions of chromatin.
Our data revealed a functional overlap between the HIRA complex and the class I HDAC Clr6 with respect to both promoter silencing and prevention of cryptic transcription. The simplest explanation of this finding is that HIRA is required to reassemble or repair nucleosomes that are subsequently modified by Clr6-Sin3 complexes. Interestingly, class I HDACs have been linked to HIRA function in vertebrate cells, as HDAC-2 stably interacts with chicken HIRA through an LXXLL motif located in the HIRA C-terminal region (1
). However, neither Hip1 nor Slm9 has LXXLL motifs, and there is no evidence of a stable interaction between the fission yeast HIRA complex and Clr6. Indeed, large-scale affinity purification of Clr6-interacting subunits did not identify any of the components of the HIRA complex (30
). Furthermore, a large-scale purification of Prw1, a homologue of RbAp48 that is present in all fission yeast Clr6-Sin3 complexes, also did not identify any subunits of the HIRA complex (unpublished data).
Analysis of the expression of individual Tf2 elements indicated that the high levels of Tf2 mRNA present in hip1Δ and slm9Δ cells result from increased expression levels of all 13 copies of this element. It is possible that Tf2 elements are located within regions of the genome that are associated with HIRA-dependent forms of chromatin. Arguing against this, HIRA-dependent repression of a Tf2 reporter was also maintained when it was moved to a novel site in the genome, suggesting that these elements contain sequences that limit their own expression.
Packaging of retrotransposons into repressive chromatin structures is known to occur in many cells types and is thought to limit the potentially harmful spread of these elements (25
). The chromatin structures that suppress the expression of Tf2 retrotransposons appear to be dependent upon the HIRA complex. It is possible that the interaction of HIRA with these elements is transient because, as yet, we have been unable to detect binding of HIRA to Tf2 LTRs by chromatin immunoprecipitation. Global silencing of Tf2 elements is also dependent upon HDACs (Clr6, Clr3, and Hst4) and Cenp-B homologues (7
). While the influence of these factors on the expression of individual elements has not been formally investigated, chromatin immunoprecipitation analysis has indicated that Clr3 and Cenp-B proteins are located at multiple Tf2 elements (7
), implying that they are required for silencing of all Tf2 elements.
The involvement of HIRA proteins in the regulation of retrotransposons is not limited to fission yeast, because Hir1 and Hir2 have been implicated in the regulation of Ty1/Copia elements in S. cerevisiae
. Indeed, hir
mutants were found to suppress the deleterious effects of the insertion of a Ty LTR (δ element) into the HIS4
). Furthermore, mutation of HIR
genes in combination with CAC
genes also increases Ty1 transposition frequency, although this increased transposition was not associated with increased levels of Ty1 mRNA (37
). It is important to note that the Tf2 elements of S. pombe
are members of the Gypsy group of LTR retrotransposons and as such are only distantly related to the Ty1/Copia elements of S. cerevisiae
). The finding that HIRA complexes regulate such distinct elements suggests that this chaperone regulates other classes of LTR retrotransposons in other eukaryotic organisms.
The substantial population of solo LTR elements present in the S. pombe
genome have been generated by recombination between two LTRs resulting in the removal of the internal retrotransposon coding sequences. Thus, solo LTRs are the remnants of retrotransposons and mark the positions of previous insertion events. Our data, along with those of others (7
), are consistent with these elements being assembled into silent chromatin in order to limit the production of spurious noncoding transcripts. While in many cases expression from solo LTRs would not be advantageous, there are an increasing number of examples of cells exploiting retrotransposable elements to regulate gene expression (14
). Indeed, in S. pombe
a set of solo LTRs that are closely related to the LTRs associated with full-length Tf2 elements are known to function as oxygen-responsive promoters (39
). Some of these confer oxygen-dependent expression to neighboring genes. Our data indicate that HIRA-dependent repression is not restricted to elements that are induced in response to low oxygen levels, as all but one of the solo LTR elements examined in this work lack a consensus SRE element and we also identified Tf1-type LTR elements that were repressed by Hip1 and Slm9.
The finding that HIRA mutants have high levels of spurious antisense transcription is consistent with this histone chaperone being required to maintain the integrity of chromatin in transcribed regions. Furthermore, mutation of hip1
being synthetic lethal with rrp6
Δ is consistent with HIRA having a major role in prevention of such antisense transcription in fission yeast. This result would not necessarily have been predicted from studies of S. cerevisiae
where mutation of HIR
genes did not result in high levels of internal initiation at the synthetic FLO8-HIS3
reporter gene, although it did increase the level of spurious sense transcripts observed in a spt2
Δ background (31
). Nonetheless, our analysis is the first report that HIRA proteins are required to prevent cryptic antisense transcripts at naturally occurring genes.
Passage of RNA polymerase II through chromatin has been proposed to result in partial or complete disassembly of nucleosomes (48
). That antisense transcription (and by implication access to cryptic promoters) at the thi4+
gene is dependent upon high levels of sense transcription is entirely consistent with this notion. Notably, loss of the HIRA complex apparently leads to continued access to these promoters even in the absence of high levels of sense transcription. We propose that the HIRA complex restores chromatin structure in the wake of RNA polymerase II passage. Consistent with this idea, Hir1 and Hir2 have been shown to localize to transcribed regions in S. cerevisiae
). In higher cells, HIRA is linked to deposition of variant histone H3.3 at actively transcribed genes (3
), and so it will be interesting to determine whether this contributes to the prevention of spurious transcription initiation.
We also show that the HIRA complex plays an important role in protecting cells against double-strand breaks. The loss of HIRA led to an increased susceptibility to bleomycin-induced chromosomal breaks, suggesting that HIRA is required to maintain the protective functions of chromatin. This may not be the only role for HIRA in the response to DNA damage, as other histone chaperones, such as S. cerevisiae
Asf1, have been shown to be required for restoration of chromatin structures following DNA repair (8
). Thus, it will be important to determine whether or not HIRA is also required for removal or replacement of nucleosomes during repair of double-strand breaks.