Abstract
SummaryUnlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle [1] and is incorporated into chromatin independent of replication [2]. Recently, H3.3 has been implicated in the cellular response to ultraviolet (UV) light [3]. Here, we show that chicken DT40 cells completely lacking H3.3 are hypersensitive to UV light, a defect that epistasis analysis suggests may result from less-effective nucleotide excision repair. Unexpectedly, H3.3-deficient cells also exhibit a substantial defect in maintaining replication fork progression on UV-damaged DNA, which is independent of nucleotide excision repair, demonstrating a clear requirement for H3.3 during S phase. Both the UV hypersensitivity and replication fork slowing are reversed by expression of H3.3 and require the specific residues in the α2 helix that are responsible for H3.3 binding its dedicated chaperones. However, expression of an H3.3 mutant in which serine 31 is replaced with alanine, the equivalent residue in H3.2, restores normal fork progression but not UV resistance, suggesting that H3.3[S31A] may be incorporated at UV-damaged forks but is unable to help cells tolerate UV lesions. Similar behavior was observed with expression of H3.3 carrying mutations at K27 and G34, which have been reported in pediatric brain cancers. We speculate that incorporation of H3.3 during replication may mark sites of lesion bypass and, possibly through an as-yet-unidentified function of the N-terminal tail, facilitate subsequent processing of the damage.
Highlights
H3.3-Deficient DT40 Cells Are Viable but Exhibit Alterations in Gene Expression H3.3 is incorporated throughout the cell cycle [2, 4], in regions of the genome in which histones need to be displaced, such as transcribed genes or regulatory elements [5, 6]. Incorporation in these contexts depends on the histone chaperone HIRA [7] and helps maintain chromatin structure by filling gaps left by loss of H3.1/H4 [5, 8]
H3.3 is incorporated in some repressed loci and at telomeres and pericentric heterochromatin, where deposition depends on
Because H3.3 has been implicated in processes related to nucleotide excision repair (NER) [3], we examined its genetic relationship to NER by performing epistasis analysis of H3.3 with XPA, a key component of the NER pathway. xpa DT40 cells are highly sensitive to UV light, considerably more so than h3.3 (Figure 2B)
Summary
Resistance to DNA Damage Requires the H3.3-Specific Chaperone-Binding Patch and S31 H3.3 differs at two sites from H3.2, the single canonical H3 in chickens (Figure 2C). We created h3.3 clones stably expressing H3.3GFP carrying either a substitution of the AIG patch with the did not complement the UV sensitivity of h3.3 cells (H3.3[S31D]; Figure 2E).
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