h3.3 is incorporated throughout the cell cycle [ 2 , 4 ] , particularly 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   and helps maintain chromatin structure by filling gaps left by loss of h3.1/h4 [ 5 , 8 ] . h3.3 deposition at transcriptionally active loci has also been proposed to help maintain active expression , possibly by creating a more accessible chromatin structure [ 2 , 9 ] . however , h3.3 is also incorporated in some repressed loci and at telomeres and pericentric heterochromatin , where deposition depends on the atrx - daxx chaperone complex [ 1012 ] . although h3.3 is not essential for transcription in drosophila , its loss results in significantly decreased fertility and reduced viability during embryogenesis . mouse embryonic stem cells with no h3.3b and depleted of h3.3a exhibit altered regulation of polycomb - dependent gene expression that interferes with their ability to differentiate . mice lacking h3.3b exhibit a semilethal phenotype with reduced growth , anaphase bridging , and karyotypic abnormalities . recently , h3.3 has also been implicated in the response to ultraviolet ( uv ) irradiation , because its chaperone hira is required to promote transcription restart after uv damage . in order to examine the effect of complete loss of h3.3 in a differentiated vertebrate cell line , we created an h3.3 null variant of the chicken bursal lymphoma dt40 . in chicken , as in mammals , h3.3 is encoded by two loci , h3.3a on chromosome 18 and h3.3b on chromosome 3 . despite considerable divergence of the cdna sequence of h3.3a and h3.3b , they encode identical proteins , which also have the same sequence as human h3.3 . rna deep - sequencing analysis ( rna - seq ) of dt40 revealed that h3.3b contributes over 90% of the total pool of h3.3 transcript in chicken dt40 b cells ( figure  1a ) . to create h3.3 null dt40 cells , we first disrupted both alleles of h3.3b by homologous recombination using a targeting strategy that removed the majority of the coding sequence ( figure  1b ; supplemental experimental procedures available online ) . this resulted in a substantial reduction of total h3.3 protein levels ( figure  1c ) , as predicted by the rna - seq data ( figure  1a ) . we then disrupted both alleles of h3.3a by removing the whole h3.3a coding sequence ( figure  1b ) . h3.3 cells proliferate more slowly than wild - type ( c. 15 versus c. 11  hr ; figure  1d ) . their unperturbed cell - cycle profile suggests that this is at least in part explained by an increase in spontaneous apoptosis ( figure  1e ) . we next examined the extent of transcriptional dysregulation in cells lacking h3.3 by rna - seq . this analysis revealed that 557 of 16,396 gene transcripts ( 3.4% ) exhibited a > 2-fold and significant ( p  < interestingly , the number of genes exhibiting a significant decrease in expression ( 235 ) is actually slightly exceeded by those increasing in expression ( 324 ) , supporting recent evidence that h3.3 , or its modifications , is not just important for actively expressed loci [ 12 , 14 ] . we observed no underlying pattern to the chromosomal locations of affected genes ( figure  s1 ) . thus , loss of h3.3 is linked to significant changes in gene expression , but affects a relatively small fraction of loci in dt40 cells . in addition to being incorporated during transcription , recent experiments have shown that h3.3 is deposited at sites of uv - induced dna damage by the histone chaperone hira , where it facilitates the recovery of transcription after repair of the damage . h3.3 cells were modestly , but consistently , hypersensitive to uv irradiation ( figure  2a ) . this is unlikely to be a secondary effect , because no known dna damage response genes exhibited significantly dysregulated expression in h3.3 cells ( table s1 ) . further , the sensitivity of h3.3 cells to uv light was reversed by stable expression of h3.3 c - terminally tagged with gfp ( figure  2a ; figure  s2 ) . in fact , ectopic expression of h3.2 appeared to cause further sensitization to uv , as previously observed in yeast . because h3.3 has been implicated in processes related to nucleotide excision repair ( ner ) , 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 ) . a double h3.3/xpa mutant was no more sensitive than xpa alone , suggesting that xpa may be epistatic to h3.3 and that h3.3 acts  to facilitate excision repair of a subset of uv lesions . however , although the uv colony survival assay has the dynamic range to detect additional sensitivity over and above that of the xpa mutant ( figure  2b ) , the very large difference in the sensitivities of the h3.3 and xpa mutants means that epistasis in this instance must be interpreted with some caution . h3.3 differs at two sites from h3.2 , the single canonical h3 in chickens ( figure  2c ) . s31 , in the n - terminal tail region and an alanine in h3.2 , has been reported to be phosphorylated during mitosis , although the function of this modification is not yet understood . h3.3 also has three residues at the base of  helix 2 that differ from h3.2 . these are a87/i89/g90 , which are s87/v89/m90 in h3.2 ( hereafter referred to as thus , the aig patch is required for binding of h3.3 to daxx   and is required for replication - independent chromatin deposition [ 2 , 4 ] , that is dependent on hira . we created h3.3 clones stably expressing h3.3-gfp carrying either a substitution of the aig patch with the svm patch of h3.3 or an s31a substitution and ensured matched expression levels by monitoring gfp by flow cytometry ( figure  s2 ) . neither the aig patch nor s31a h3.3 mutants complemented the uv hypersensitivity of h3.3 cells ( figure  2d ) , suggesting that the chaperone binding specificity of h3.3 and a serine at position 31 are required . a potentially phosphomimetic substitution of s31 with aspartic acid also did not complement the uv sensitivity of h3.3 cells ( h3.3[s31d ] ; figure  2e ) . recently , mutations in the n - terminal tail of h3.3 , in the vicinity of s31 , have been linked to a number of pediatric cancers , including glioblastoma , chondroblastoma , and giant cell tumors of bone [ 1921 ] . understanding the mechanistic basis for the clinical effects of these apparently driver mutations has focused on their effects on posttranslational modifications of h3 . thus , mutations at g34 affect the global distribution of h3k36me3 and changes in gene expression . likewise , mutation of h3.3k27 , a residue whose trimethylation is associated with polycomb complex - mediated transcriptional repression , results in reduced global h3k27me3 and derepression of multiple  transcripts . because s31 lies close to these residues , we wondered whether the cancer - associated mutations k27 m , g34r , and g34v [ 19 , 20 ] might also confer sensitivity to dna damage . interestingly , all three h3.3 mutants exhibit uv sensitivity similar to the h3.3 knockout , suggesting that these residues are also required for the  role played by h3.3 in facilitating excision repair ( figure  2e ) . this somewhat  surprising result suggests the possibility that h3.3 cancer - associated mutations could impact on dna repair as well as on transcriptional regulation , a point that merits further exploration . although the deposition of h3.3 is primarily replication independent , we asked whether the absence of h3.3 affected replication by monitoring fork progression in stretched dna fibers . we pulse labeled cells sequentially with two different halogenated nucleosides ( iododeoxyuridine and chlorodeoxyuridine ; 20  min each ) , stretched the extracted dna on glass slides , and revealed the replicons with antibodies specific for the halogenated nucleotides ( figure  3a ) . we observed a small , but not significant , decrease in median fork velocity in h3.3 cells but no change in replication origin density ( figures s3a  s3c ) . however , after uv irradiation , applied at the same time as the second label , replication fork progression in the second 20  min was dramatically reduced in h3.3 cells in comparison to wild - type ( figures 3b and 3c ) . it is likely that at least some of these forks remain persistently blocked , because a greater fraction of h3.3 cells accumulate in late s phase 24  hr after uv exposure , suggesting a delay in completion of replication ( figure  s3d ) . the delayed fork progression following uv exposure in h3.3 cells was reduced to wild - type levels by expression of h3.3-gfp but not h3.2-gfp ( figure  3c ) . although this defect is reminiscent of cells lacking the translesion polymerase rev1 [ 25 , 26 ] , we could observe robust translesion synthesis of uv ( 6 - 4 ) photoproducts in xpa / h3.3 cells using a replicating plasmid assay   and , further , the frame infidelity characteristic of photoproduct bypass in rev1-deficient cells   was not evident ( figures s3e thus , delayed replication fork progression after uv damage in h3.3 cells does not appear to result from a significant defect in rev1-dependent translesion dna synthesis . we then asked whether the role of h3.3 at the replication fork was also dependent on both the aig patch and s31 , as for uv sensitivity . whereas the aig - to - svm patch mutant failed to complement the defective fork progression after uv ( figure  3c ) , the h3.3[s31a ] mutant restored wild - type behavior ( figure  3c ) , as did the cancer - associated mutants g34v , g34r , and k27 m ( figure  3d ) . in view of the apparent epistasis of h3.3 and xpa , we considered whether the delayed fork progression in h3.3 cells reflected defective excision repair . however , h3.3 cells exhibit a much more prominent defect in fork progression after uv than xpa cells , the response of which is similar to wild - type ( figure  3e ) . this is not consistent with the fork progression defect seen after uv in h3.3 cells resulting from defective ner at the fork , an event that in any case would likely be deleterious to cell survival due to strand incision at the lesion causing replication fork collapse . finally , we asked whether the role of h3.3 in the response to uv was also seen with other forms of dna damage . in addition to uv , h3.3 cells exhibit mild hypersensitivity to the interstrand crosslinking agent cisplatin and the alkylating agent methyl methanesulfonate ( mms ) but not to x - rays ( figures 4a4c ) . in the case of cisplatin , both the aig patch and n - terminal tail mutants discussed above exhibit hypersensitivity ( figures 4d and 4e ) , as observed with uv . however , for neither cisplatin nor mms is there any exacerbation of the delay in fork progression induced by these agents ( figures 4f and 4 g ) , a point we consider further below . our observations provide the first clear evidence of the involvement of a variant histone in replication fork progression , and suggest that forks require a supply of h3.3 when they encounter uv damage to maintain processive replication . although our experiments are not able to show directly that h3.3 is incorporated by the replication fork during replication of uv dna damage , by analogy with the effect of histone supply on bulk dna replication , we suggest that the defective fork progression in h3.3 cells is a result of failure of a process that would normally see h3.3 incorporated . we speculate that h3.3 incorporation during the replication of uv lesions at the fork , and possibly during postreplicative lesion bypass , may facilitate subsequent access and repair ( figure  s4 ) . hira would seem to be a strong candidate given its documented role in h3.3 incorporation at sites of uv damage , although the same study reported that hela cells depleted for hira are not uv sensitive . whether atrx plays any role in replicating uv - damaged dna is unknown , but it has been implicated in the replication of g quadruplex dna   and , recently , atrx - deficient cells have been shown to exhibit replication defects , suggesting that it contributes to limiting fork stalling during s phase . although cells lacking h3.3 are sensitive to uv , mms , and cisplatin , a fork progression defect , as assessed in labeled dna fibers , is only observed after uv exposure . this suggests a broad requirement for h3.3 in facilitating dna repair , but that incorporation of this histone variant may not only take place  on the fly  at the replication fork when it encounters dna damage but also , for instance , during lesion bypass in postreplicative gaps . loss of this latter role would not be detectable as  a defect in the dna fiber assay . the basis for this specificity remains unclear , but we speculate that it may be related to the mechanisms and complexes cells bring into play at different lesions , which in turn may affect the timing of lesion bypass . indeed , such damage - dependent specificity is now well documented in translesion synthesis   and , recently , damage caused by uv and by mms has been shown to induce quite distinct bypass responses in human cells . however , much further work is needed to understand the contexts in which h3.3 is required for processive replication of damaged dna . finally , how might h3.3 incorporation facilitate subsequent dna repair and survival ? because it has been proposed that h3.3-containing chromatin has a more open and accessible structure , its incorporation may be particularly important for promoting ner in highly condensed regions of the genome . additionally , the damage sensitivity of the h3.3 cells may also be related to its ability to promote transcriptional recovery after repair , possibly through its ability to interact  specifically with components of the fact chromatin remodeling complex , which has itself been implicated in transcriptional recovery after ner and in resistance to uv damage . a.f . performed the experiments , analyzed the data , and wrote the paper . t.l . with a.f . performed the uv dna fiber analysis . performed the cell - cycle analysis and global analysis of replication by dna combing and analyzed the rna - seq data .
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 2 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 .