dna in all eukaryotic organisms is bound by nucleosomes , forming a physiological chromatin context in which all molecular processes involving dna operate .
the integrity of the chromatin template is constantly compromised by fundamental biological processes , such as dna replication , repair , and transcription , following which the normal chromatin structure is restored with the help of histone chaperone proteins ( gurard - levin et  al . , 2014 , ransom et  al . , 2010 ) .
advances in recent years have shed light on some of the molecular players involved in chromatin assembly and maintenance , separating ( on the molecular level ) dna replication - dependent and -independent pathways ( burgess and zhang , 2013 ) .
histone proteins themselves are central to these processes : the expression and incorporation of canonical histones is tightly coupled to dna replication ; in contrast , histone variants can be incorporated into chromatin independent of the cell cycle ( maze et  al . , 2014 ) . while it has become increasingly clear that the activity of histone chaperone proteins is of critical importance during dna replication and repair ( adam et  al . , 2013 , hoek and stillman , 2003 , polo et  al . , 2006 , ransom et  al . , 2010 ) ,
studies that have attempted to dissect the importance of histone replacement in the interphase nucleus have revealed only a limited contribution of histone chaperones and/or variants to transcriptional regulation ( banaszynski et  al . , 2013 ,
goldberg et  al . , 2010 , hdl and basler , 2009 , sakai et  al . , 2009 ) .
these studies , however , have been complicated by the use of proliferative cell systems that could ( at least partially ) restore chromatin integrity through replication - coupled chromatin assembly ( banaszynski et  al .
, 2013 , hdl and basler , 2009 , sakai et  al . , 2009 , wyrick et  al . , 1999 ) .
thus , the extent to which basic physiological processes , such as transcription , are dependent on or regulated by histone replacement remains unclear . to overcome the limitations of these previous studies , we took advantage of the unique system presented by mammalian oogenesis . over an extended time span and in the absence of dna replication , postnatal mammalian oocytes execute the oogenesis - specific developmental program , involving widespread transcriptional changes and de novo dna methylation , ultimately acquiring the competencies required for fertilization and embryogenesis ( de la fuente , 2006 , li and albertini , 2013 , tomizawa et  al . , 2012 ) . to address the importance of histone turnover during this process
, we have generated a mouse oocyte - specific knockout of the histone chaperone hira . as opposed to caf1 , which is implicated in the replication - coupled deposition of canonical histones h3.1 and h3.2 ,
hira has been shown to deposit the h3.3 variant during replication - independent chromatin assembly ( ray - gallet et  al . , 2002 ,
our findings show that depletion of hira in primordial oocytes causes a severe developmental defect associated with extensive oocyte death . on the molecular level
this causes significantly increased dna accessibility , accumulation of dna damage , and chromosome segregation defects .
the lack of normal chromatin structure has a striking impact on transcriptional regulation : lack of normal h3.3/h4 replacement prevents the oocytes from maintaining full dynamic range of gene expression , and it leads to compromised transcriptional transitions normally associated with the oocyte development .
furthermore , we show that histone replacement is necessary for silencing of spurious transcription and , in the context of the oocyte , also for the efficient deposition of de novo dna methylation .
our results thus uniquely demonstrate the critical relationship between continuous histone replacement , the structural integrity of the chromatin template , the normal regulation of transcription , and the establishment of dna methylation , in the context of an in  vivo developmental system .
in the mouse , female germ cells arrest in prophase of the first meiotic division at embryonic day ( e)13.5 . the female gonocytes subsequently resume growth in postnatal ovaries where they undergo oogenesis and progress through meiosis ( de la fuente , 2006 ) .
this process involves the following : ( 1 ) pronounced transcriptional changes as the developing oocytes progress from primordial to large antral follicle stages ; ( 2 ) the deposition of de  novo dna methylation starting in the secondary follicle stage ;  ( 3 ) the silencing of transcription in the germinal vesicle ( gv ) stage ; and , finally , ( 4 ) the condensation of chromosomes in the mii stage , resulting in an oocyte that is competent to undergo fertilization and support early embryonic development ( figure  1a ) .
we first addressed the capacity of developing oocytes to incorporate histones by microinjection of mrna for tagged versions of histones . in agreement with the absence of replication during oocyte maturation
, growing oocytes did not show any incorporation of canonical histones h3.1 and h3.2 ( figure  1b ; akiyama et  al . , 2011 ) .
in contrast , microinjected flag - tagged h3.3 and h2a.x , previously shown to be enriched in the developing oocyte ( akiyama et  al . , 2011 ,
2010 ) , were readily incorporated into the chromatin of growing oocytes ( figure  1b ) .
we thus conclude that h3.3 is the only h3 subtype incorporated during postnatal oocyte development . in further support of this observation , oocytes isolated from our h3.3b - egfp knockin mice ( figure  s1
g ) confirmed that both growing and gv oocytes contain high levels of nuclear h3.3 ( figure  1e ) .
however , we also detected a high level of chromatin incorporation for flag - tagged h2a.x , h4 , and h3.3 upon injection of tagged histone mrna into transcriptionally inactive gv - stage oocytes ( figure  1c ) , demonstrating that the chromatin in the mature gv oocyte is still dynamic even after global transcription has ceased .
previous studies have shown that the majority of h3.3 incorporation is critically dependent on the presence of histone chaperone hira ( goldberg et  al . , 2010 , pchelintsev et  al . , 2013 ) .
in agreement with the observed h3.3 incorporation , hira is expressed and shows nuclear localization during postnatal oocyte development ( figure  1d ) . to investigate whether hira is specifically required for the observed h3.3 incorporation during oogenesis
, we took advantage of gdf9- and zp3-driven expression of cre recombinase and deleted hira in the primordial and primary stages of follicle development , respectively ( figures 1a and s1a ) .
we confirmed that both gdf9-cre- and zp3-cre - deleted oocytes undergo genetic recombination ( figure  s1b ; data not shown ) and are depleted of hira mrna , protein and complex ( figures 1d and s1c  s1f ) .
the absence of hira severely decreased h3.3 content and completely abolished the incorporation of microinjected tagged h3.3 in both growing and gv - stage oocytes ( figures 1b , 1c , and 1e ) .
the observed impairment of h3.3 incorporation occurred despite the presence of an alternative atrx / daxx chaperone complex that has been well characterized to incorporate h3.3 ( burgess and zhang , 2013 , maze et  al . ,
, the presence and the localization of this complex in the gv oocyte were not affected by hira deletion ( figure  s2a ) , and we could observe h3.3b - egfp loci overlapping with atrx staining in hira 
 gdf9-cre , h3.3b - egfp gv oocytes at higher laser detection intensity ( figure  s2c ) .
the severe reduction of h3.3 incorporation in hira 
 gdf9-cre oocytes thus demonstrates that the atrx / daxx chaperone complex can not compensate for the lack of hira , as was also observed in mouse embryonic stem cells ( escs ) ( goldberg et  al . , 2010 ) .
the lack of hira and h3.3 incorporation also was not compensated for by incorporation of the canonical histones h3.1 or h3.2 ( figure  1b ) , and it coincided with the lack of detectable incorporation of histone h4 ( figure  1c ) . taken together ,
these findings demonstrate that the histone  variant h3.3 , rather than h3.1 or h3.2 , is actively incorporated into chromatin during oocyte development , and confirm that hira is responsible for the incorporation of the majority of h3.3 in conjunction with histone h4 ( figures 1b1e and s2b ) . of note , although hira - deficient oocytes failed to incorporate tagged h3 and h4 histones , the incorporation of tagged h2a.x was readily detectable ( figures 1b and 1c ) , confirming that distinct molecular pathways are implicated in the deposition of h3/h4 and h2a / h2b histones ( burgess and zhang , 2013 ) .
assessment of females with oocyte - specific hira deletion revealed that loss of hira led to a severe ovarian phenotype .
significantly lower numbers of mii oocytes could be recovered following superovulation ( figure  2a ) ; zp3-cre - driven deletion of hira led to a more than 50% reduction in the number of ovulated mii oocytes , and this effect was even more dramatic upon earlier gdf9-cre - driven deletion ( figure  2a ) . furthermore , gv oocytes ( figure  1a ) isolated from both hira 
 zp3-cre and hira 
 gdf9-cre ovaries showed dramatically reduced competency to complete the gv - to - mii transition during in  vitro maturation ( figure  s2e ) .
hira 
 gdf9-cre ovaries were significantly smaller ( figures 2b and s2f ) ; and , although we could recover some gv oocytes from 3-week - old females , the ovaries of older mice contained only very few oocytes , which were predominantly at early developmental stages ( figures 2c and s2 g ) .
further analysis of the superovulated hira 
 gdf9-cre mii oocytes revealed frequent defects in chromosome alignment and segregation and instances of aberrant polar body extrusion ( figure  2d ) . in line with a severe ovarian phenotype and in agreement with recently published reports ( inoue and zhang , 2014 , lin et  al .
2005 ) , the surviving hira - depleted oocytes were unable to either reprogram the paternal pronucleus or support early embryonic development after fertilization . in agreement with the observed enrichment of h3.3 and hira in the paternal pronucleus of control zygotes ( figures s3a  s3c ) , fertilized hira 
 gdf9-cre oocytes could not fully de - condense the male pronucleus ( figure  s3d ) , incorporate h3.3 ( figure  s3e ) , or support normal zygotic development ( figure  s3f ) .
failure to incorporate h3.3 occurred despite the efficient removal of protamines ( figure  s4a ) , indicating that protamine removal and chromatinization of the paternal genome are mechanistically distinct ( inoue and zhang , 2014 , lin et  al . , 2014 ) .
although the histone load of the paternal pronucleus was severely reduced upon maternal hira deletion , we could detect a residual amount of histones on the paternal dna by immunofluourescent staining ( figure  s4b ) .
however , it remains to be addressed whether the residual histones were inherited from sperm or whether they were incorporated in a hira - independent manner . in agreement with the severe chromatinization defect ,
the partially decondensed paternal pronucleus of zygotes in which hira had been maternally deleted failed to undergo dna demethylation ( figure  s4c ) and could not provide a normal template for zygotic replication or transcription ( figures s4d and s4e ; lin et  al .
, 2014 ) . apart from the failure to support normal reprogramming of the paternal pronucleus following fertilization , hira - depleted oocytes showed severe maternal defects .
the atp content was significantly reduced in hira - depleted mii oocytes ( figure  s2h ) , and , upon parthenogenetic activation , only a fraction of these oocytes reached the two - cell stage and none progressed further , contrary to the control oocytes that efficiently supported development of parthenogenetic blastocysts ( figure  2e ) . considering the pronounced oocyte phenotype , we set out to understand the molecular mechanisms by which hira regulates oocyte development .
we hypothesized that , in the absence of h3/h4 incorporation , the chromatin structure in hira - depleted oocytes would be severely affected . in agreement with this ,
the histone content of hira - depleted gv - stage oocytes was severely reduced ( figures 3a and s2b ) , and the resulting chromatin displayed altered structure and increased dnase i sensitivity ( figures 3b and 3c ) .
moreover , in accordance with the predicted role of histones in protecting genetic material from environmental stress and mutagenesis ( ljungman and hanawalt , 1992 ) , hira - depleted oocytes showed clear signs of dna damage both by increased levels of -h2ax ( figure  3d ) and upregulation of dna damage - responsive genes ( figure  3e ) .
the importance of hira and continuous h3.3 replacement has been previously assessed in the context of proliferating mouse pluripotent escs ( banaszynski et  al .
these studies surprisingly revealed that hira deletion led to only minor transcriptional changes ( banaszynski et  al .
the severity of the phenotype observed following the deletion of hira in oocytes , we next asked whether the reduced h3/h4 incorporation and increased dna accessibility ( figures 1b1e , 3a , and 3c ) affected the transcriptional program of the hira - depleted oocytes . surprisingly , although the lack of maternal hira severely affected the amount of transcription emanating from the paternal pronucleus following fertilization ( figure  s4e ; lin et  al . , 2014 )
, hira - depleted and control growing oocytes showed similar global levels of transcription , as judged by eu incorporation ( figure  4a ) . to address the role of hira in transcription during oocyte development in depth , we carried out single - cell rna sequencing ( scrna - seq ) on four hira 
 gdf9-cre and four hira control oocytes .
we chose to analyze ovulated mii oocytes that had successfully completed their first meiotic division to avoid inconsistencies due to differences in staging or developmental progression .
of note , consistent with the identical patterns of eu incorporation in the hira 
 gdf9-cre and control growing oocytes ( figure  4a ) , normalization to the ercc rna spike - ins included in our scrna - seq library preparations revealed no significant differences in the total quantity of poly - adenylated rna between hira 
 gdf9-cre and control mii oocytes ( figure  4b ) . in agreement with the observed severe developmental phenotype , the initial data analysis revealed pronounced transcriptional differences upon deletion of hira ( figures 4c and s6a ) .
expression analysis identified 2,101 differentially expressed genes ( false discovery rate [ fdr ]  < 0.1 ) between hira 
 gdf9-cre and control oocytes ( 1,274 upregulated in hira 
 gdf9-cre oocytes relative to controls ; 827 downregulated ) , representing over 15% of all annotated transcripts detected ( table s1 ) .
gene ontology ( go ) analysis of differentially upregulated genes showed enrichment for cell membrane , biological adhesion molecules , and cell junctions ( figure  4d ) .
in contrast , go analysis on differentially downregulated genes confirmed enrichment for processes important in late - stage oocytes , including chromosome , nucleolus , mitochondria , and pronucleus ( figure  4e ) .
surprisingly , we detected significantly more annotated transcripts ( 7.7% increase , p value  < 0.01 ) in hira 
 gdf9-cre oocytes relative to controls ( figure  4f ) . the increased number of transcripts was not evenly distributed across all expression levels ( figure  4 g ) ; rather , deletion of hira resulted in relatively fewer transcripts with very low ( first decile ) or very high ( ninth and tenth deciles ) expression ( figure  4 g ) . furthermore , differentially down- and upregulated genes showed higher and lower than expected expression , respectively ( figure  4h ) ; and , while the genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure  4i ; gene set enrichment analysis [ gsea ] , fdr  < 0.001 ; data not shown ) , genes with very high expression in normal oocytes ( top 20% )
were specifically downregulated in hira 
 gdf9-cre oocytes ( figure  4i ; gsea , fdr  <
this also was evident by the greatly reduced variance in expression levels of annotated transcripts within the hira - depleted oocytes ( figure  4j ; p value  < 0.001 ) , suggesting that hira is necessary to maintain the full dynamic range of gene expression .
thus , contrary to previous findings in proliferating pluripotent escs , hira appears to be critical for normal transcriptional regulation during oocyte development .
the major transcriptional transition during oogenesis occurs between primordial stage oocytes ( when gdf9-driven cre is first expressed ) and primary stage oocytes ( when zp3-driven cre is first expressed ) , during which the major wave of gene activation is observed ( pan et  al . ,
2005 ) . to test whether the observed transcriptional defects are due to hira 
 gdf9-cre oocytes failing to execute this transcriptional activation or to other dominant secondary effects
, we additionally carried out scrna - seq on four hira 
 zp3-cre and three hira control oocytes ( figure  s6 ; table s3 ) .
remarkably , a nearly identical transcriptional phenotype was observed in hira 
 zp3-cre and hira 
 gdf9-cre oocytes : differentially down- and upregulated genes showed higher and lower than average expression in the control oocytes , respectively ( figure  s6e ) , and genes normally not expressed or expressed at very low levels ( bottom 20% ) were upregulated upon hira deletion ( figure  s6f ) , while genes with very high expression in normal oocytes ( top 20% ) were specifically downregulated in hira 
 gdf9-cre oocytes ( figure  s6f ) .
similar to the effect observed in hira 
 gdf9-cre oocytes , the variance in expression levels of annotated transcripts within the hira  zp3-cre oocytes was greatly reduced ( figure  s6 g ; p  value  < 0.001 ) .
the developing oocyte is a unique experimental system , as it allows us to assess the importance of continuing histone h3.3/h4 replacement not only for steady - state transcription , but also for affecting transcriptional transitions associated with development , such as activation or repression of dynamically regulated genes .
to address this , we used k - means clustering of published expression data ( pan et  al . , 2005 ;
geo : gse3351 ) to identify eight clusters of genes that show distinct expression patterns over the course of oocyte development ( figures s5a and s5b ) , and we compared these to the expression profiles in hira 
 gdf9-cre and control oocytes .
remarkably , genes downregulated upon hira depletion ( hira 
 gdf9-cre ) were specifically enriched for genes whose transcription is activated either between the earliest stages of oocyte development ( figure  5a , cluster 4 , primordial and primary follicle stages ; gsea , fdr  < 0.001 ) or between early and late oogenesis ( figure  5a , cluster 5 ; gsea , fdr  < 0.001 ) .
in contrast , genes upregulated upon hira deletion were most significantly enriched for clusters of genes that all uniquely show downregulation of expression between early and late oogenesis ( figure  5a , cluster 2 ; fdr  = 0.017 ) or between primordial and primary follicle stages ( figure  s5b , clusters 6 and 8) . of note ,
similar phenotypes were observed upon zp3-cre - driven deletion of hira ( figure  s6h ) . to confirm that these observations were not an artifact of the reduced dynamic range observed in the hira - depleted mii oocytes
, we carried out a similar gsea analysis on only median expressed genes ( i.e. , genes in the second , third , or fourth expression quintile ) .
cluster 2 ( genes progressively downregulated during oogenesis ) remained enriched among upregulated genes in hira 
 gdf9-cre oocytes ( gsea , fdr  = 0.08 ; data not shown ) ; similarly , cluster 4 ( genes upregulated in early oogenesis ) still showed enrichment ( although this was less pronounced ) among genes downregulated in the absence of hira ( gsea , fdr  = 0.26 ; data not shown ) . to further test the direct link between normal transcriptional activation and hira , we carried out differential expression analysis between hira 
 gdf9-cre and hira 
 zp3-cre mii oocytes .
given the same observed reduction of the dynamic range of transcription in both systems ( figures 4 g , 4j , and s6 g ) , we hypothesized that , if normal transcriptional activation was dependent on hira , genes that are specifically upregulated between primordial ( when gdf9-driven cre is first expressed ) and primary ( when zp3-driven cre is first expressed ) stage oocytes should show higher expression in hira 
 zp3-cre mii oocytes .
indeed , this hypothesis was confirmed by the gsea analysis ( figure  s6i ; gsea , fdr  < 0.001 ) . taken together ,
our data bring to light the critical role of hira ( and h3.3/h4 turnover ) for affecting changes to the transcriptional state .
histone h3.3/h4 replacement is required for normal transcriptional activation of genes associated with developmental progression of oocytes , and , surprisingly , it is also necessary for genes to become or to remain transcriptionally silent .
nucleosomes have been hypothesized to constitute a barrier for  transcription ( teves et  al . , 2014 ) .
considering the failure of hira - deleted oocytes to silence genes , we asked whether the increased dna accessibility in the absence of continuous h3.3/h4 replacement may lead to aberrant transcription from regions not normally transcribed within the genome . to address this possibility , we used a previously described approach that allows for the identification of unannotated transcripts originating entirely from intronic or intergenic regions ( trapnell et  al . , 2010 ) .
consistent with our previous mapping , we observed an increase ( 7.3% , p value  <
0.05 ) in the number of annotated transcripts detected in hira 
 gdf9-cre oocytes ( figure  5b ) .
remarkably , a much larger increase was observed in the total number of transcribed fragments ( 37.7% , p value  < 0.05 ; figure  5b ) , with the largest increases observed in unannotated transcripts originating entirely from intronic ( 112.6% increase , p value  < 0.05 ) and intergenic ( 92.0% increase , p value  < 0.05 ) regions ( figure  5c ) of the hira - deleted oocytes . of note , while expression of unannotated intergenic transcripts was clearly detectable ( 90% transcripts with > 1 fragments per kilobase of transcript per million mapped reads [ fpkm ] ) and often at high level ( 8% transcripts with > 10 fpkm ) in individual knockout samples , expression of a given unannotated intergenic transcript was only ever observed in one biological replicate , reflecting stochastic events consistent with spurious transcription ( table s2 ) . also of note , a nearly identical phenotype was observed upon zp3-cre - driven deletion of hira ( figures s6d , s6j , and s6k ; table s4 ) .
progression through oocyte development is accompanied by the  accumulation of dna methylation . during this process
, the largely hypomethylated genome of primordial oocytes acquires dna methylation through the activity of the de novo dna methyltransferase dnmt3a and its co - factor dnmt3l ( kaneda et  al . , 2004 , smallwood and kelsey , 2012 , smallwood et  al . , 2011 ) .
acquisition of dna methylation in oocytes is known to be positively correlated with transcription , which could relate to the recruitment of dnmt3a/3l to gene bodies through binding of its pwwp domain to h3k36me3 , a histone modification associated with transcriptional elongation ( chotalia et  al . , 2009 , dhayalan et  al .
although h3k36me3-driven dna methylation in gene bodies has recently been attributed to the enzymatic activity of dnmt3b in mouse escs ( baubec et  al . , 2015 , yang et  al . , 2014 )
, dnmt3a is responsible for the accumulation of oocyte dna methylation as dnmt3b is absent in growing oocytes ( kaneda et  al . , 2004 , shirane et  al . , 2013 ) .
given the widespread transcriptional changes observed in the hira knockout oocytes , we set out to examine the potential impact of hira deletion on de novo dna methylation in the oocyte .
although the total amount of transcription was not altered upon hira deletion ( figures 4a and 4b ) , the observed reduced histone load was connected with lower levels of global h3k36me3 in hira - depleted gv oocytes ( figures 3a and s7a ) .
interestingly , initial immunofluorescence ( if ) detection of 5mc indicated that hira 
 gdf9-cre gv oocytes also contain lower levels of dna methylation ( figure  s7b ) . further quantitative assessment by liquid chromatography - mass spectrometry ( lc - ms ) revealed a  dramatic reduction of 5mc in hira - deleted oocytes , with a more pronounced effect following earlier gdf9-cre - driven deletion ( figure  6a ) .
importantly , the reduction of dna methylation occurred despite the transcriptional upregulation of dnmt3a and without statistically significant changes in the expression of other dna methyltransferases ( figures s5c , s7c , and s7d ) . to further understand the relationship among hira deletion , transcriptional changes , and global changes in dna methylation ,  we profiled dna methylation genome - wide by bisulphite sequencing . because of the very limited availability of hira - deleted oocytes , we performed single - cell bisulphite sequencing ( scbs - seq ) on 14 individual hira 
 gdf9-cre mii oocytes and 14 individual hira control mii oocytes , followed by in  silico merging of the individual datasets , as previously described ( smallwood et  al . , 2014 ) .
we first confirmed that our scbs - seq dataset represented an accurate depiction of oocyte methylation , as we observed a high correlation ( r  = 0.96 ) between our merged scbs - seq control oocytes and an independent whole - genome bisulphite sequencing ( wgbs ) dataset of pooled oocytes ( shirane et  al .
, 2013 ; dna data bank of japan : dra000570 ; figures 6c and s7e ) .
in agreement with the lc - ms measurements and if data , our wgbs analysis confirmed the dramatic reduction in dna methylation in both cpg ( 42.7% decrease ) and chh ( 73.2% decrease ) contexts ( figures 6b and s7f ) .
further analysis revealed that only 3.8% of the 3-kb genomic windows classed as methylated in wild - type oocytes ( > 80% methylation ) remained methylated to the same extent in hira - depleted oocytes , with the majority of methylated ( > 80% ) regions showing less than 50% dna methylation in hira - depleted oocytes ( figure  6d ) . at the whole - genome level
, 40.2% of 3-kb genomic windows displayed a statistically significant loss of dna methylation in hira 
 gdf9-cre oocytes ( figure  6e ; chi - square test , p  <  0.001 ) .
this extensive reduction of dna methylation was observed across all types of genomic regions , including genic regions , cpg islands , maternally methylated imprint control regions , and repetitive elements ( figures 6f , 6 g , s7 g , and s7h ) . in agreement with previously published observations ( kobayashi et  al . , 2012 ,
2015 ) , we observed a positive correlation between transcription and the level of dna methylation in control oocytes ( figure  6h ) .
although the methylome of hira 
 gdf9-cre oocytes was severely reduced in comparison to control oocytes , they too retained a positive correlation between transcription and dna methylation across most of the transcriptional range ( figure  6h ) ; however , this correlation was abolished for highly expressed genes ( figure  6h ) .
consequently , the effect on dna methylation in hira - deleted oocytes was more pronounced at highly expressed genes compared to median expressed genes ( figure  s7i ) .
surprisingly , beyond the observed relationship between transcription level and dna methylation described above , on average , dna methylation was reduced both at genes whose expression was unaffected by hira deletion and also at genes differentially expressed between control and hira 
 gdf9-cre oocytes , regardless of whether they were up- or downregulated ( figure  6i ) .
cumulatively , these observations suggest that , although de novo methylation is still targeted to transcribed regions in hira - depleted oocytes , this process appears to be inefficient , clearly indicating that transcription is not sufficient to ensure normal levels of de novo dna methylation in this system . considering the high levels of dnmt3a and dnmt3l in hira 
 gdf9-cre oocytes ( figures s7c and s7d ) , we reasoned that the observed effect was likely caused either by inefficient recruitment or reduced enzymatic activity on chromatin of the dnmt3a/3l complex .
we first asked whether chromatin association of dnmt3a is grossly altered following hira depletion in hira 
 gdf9-cre oocytes . in agreement with our rna - seq and qpcr data ,
hira knockout oocytes contained higher levels of dnmt3a ( figures s7c , s7d , and s5c ) .
importantly , dnmt3a remained associated with chromatin following triton pre - extraction of soluble proteins , confirming that altered chromatin structure in hira - deleted oocytes does not completely abolish recruitment of this enzyme ( figure  s7j ) . in this context , the pwwp domain of dnmt3a has previously been shown to interact in  vitro with h3k36me3 ( dhayalan et  al . , 2010 ) .
although the recognition of this histone modification by dnmt3b ( and not by dnmt3a ) has recently been linked to gene body dna methylation in mouse escs ( baubec et  al . , 2015 ) , it is possible that reduced h3k36me3 levels observed in hira - depleted gv oocytes contribute to the observed loss of dna methylation in this system .
in addition , using in  vitro biochemical assays , it has recently been shown that the enzymatic activity of dnmt3a is specifically enhanced by binding to the n - terminal tail of histone h3 lacking methylation at lysine 4 ( h3k4me0 ) ( guo et  al . , 2015 ) . despite the lack of h3.3/h4 replacement , growing hira 
 gdf9-cre oocytes remained highly transcriptionally active ( figure  4a ) .
it is , thus , conceivable that passage of the transcriptional machinery would result in histone h3 depletion and increased dna accessibility , both of which we observed ( figures 3a and 3c ) . although our developmental system precludes direct analysis of locus - specific histone turnover due to severely limited material , a very strong correlation had been observed previously between transcription levels and the degree of histone ( and specifically h3.3 ) replacement , a relationship that is conserved among various model organisms including mouse ( deal et  al . , 2010 , kraushaar et  al . , 2013 ,
dna methylation was most affected at genes with the highest level of transcription ( figures 6h and s7i ) .
we also note that , while the combination of both compromised dnmt3a/3l recruitment and reduced enzymatic activity would explain the reduction of dna methylation in transcribed regions , reduced histone load is likely to underpin the methylation defect observed in other non - transcribed parts of the genome ( figure  6f ; guo et  al . , 2015 ) .
to address the biological significance of continuous histone replacement in a physiological context , we have generated a genetic deletion of the histone chaperone hira in the early stages of mouse oogenesis .
developing mouse oocytes represent a unique experimental system as postnatal oocytes undergo developmental transitions , including major transcriptional changes and widespread de novo dna methylation , in the absence of dna replication .
our results show that the chromatin of developing oocytes is highly dynamic , with histone turnover being observed also in the transcriptionally inert gv - stage oocytes ( figures 1c and 4a ) .
we demonstrate that constant histone replacement is necessary for the maintenance of normal chromatin homeostasis in  vivo .
depletion of hira during early oocyte development leads to a severe reduction of histone load , compromised developmental progression , and progressive oocyte loss ( figures 2 , 3a , and s2 g ) .
we note that , albeit pronounced , our observed phenotype is milder in comparison to the recently reported oocyte death in h3.3 knockout mice ( tang et  al . , 2015 ) .
we attribute this difference to the presence of an alternative atrx / daxx h3.3 chaperone complex , which , although present in our system , can not functionally replace hira - driven h3.3 deposition ( figures s2a  s2c ) .
our experiments document that , while lacking the normal ability to incorporate h3.3 and h4 , hira - deleted growing oocytes remain transcriptionally active , which results in chromatin with severely reduced histone content , increased dnase i sensitivity , and signs of dna damage ( figures 3 and s2b ) .
reminiscent of the phenotype associated with hira depletion in yeast ( blackwell et  al .
, 2004 , greenall et  al . , 2006 ) and h3.3 depletion in mice ( bush et  al .
, 2013 , lin et  al . , 2013 ) and drosophila ( sakai et  al . , 2009 ) , the compromised chromatin structure leads to chromosome segregation defects and aberrant first polar body extrusion ( figure  2d ) .
however , the observed chromosome segregation defects in the hira - depleted oocytes are not linked with aberrant cenpa incorporation , as previously suggested in somatic cells ( figure  s2c ; bush et  al . , 2013 ) .
non - replicative hira - depleted oocytes uniquely reveal the importance of histone replacement on transcriptional regulation in the absence of replication - coupled chromatin assembly .
although transcription can continue from histone - depleted chromatin , our study shows that the lack of histone replacement has a major impact on the dynamic range of gene expression . in the context of histone - depleted chromatin
additionally , the lack of normal histone occupancy leads to increased spurious transcription from otherwise not - transcribed regions of the genome , suggesting an evolutionarily conserved role for the hira histone chaperone complex ( anderson et  al . , 2009 ) .
our results additionally reveal an unexpected connection among continuous h3.3 replacement , transcription , and de novo dna methylation in developing oocytes .
following hira depletion , more accessible chromatin with reduced histone load leads to significantly reduced dna methylation ( figures 6a6 g ) .
we note that , although in other systems dna methylation changes result in pronounced transcriptional outcomes ( yang et  al . , 2014 ) ,
reduced dna methylation is not likely to contribute to the observed transcriptional changes in hira - depleted oocytes , as dnmt3l knockout oocytes lacking dna methylation do not display any transcriptional phenotype ( kobayashi et  al . , 2012 ) .
our data suggest that the observed methylation phenotype can not be attributed to downregulation of the dnmt3a/3l complex , and , although we can not exclude locus - specific effects on dnmt3a/3l recruitment , the observed reduced dna methylation is likely due to altered enzymatic activity of dnmt3a in the absence of normal levels of chromatin - bound h3 ( figures 1c , 1e , and s2b ) . in this context , the n - terminal part of h3 has been shown in  vitro to be required for allosteric activation of dnmt3a catalytic activity ( guo et  al . , 2015 ) .
our study thus provides the first support for this effect in an in  vivo setting .
the observed effect is more pronounced in highly expressed genes ( figure  s7i ) , in agreement with the expected role of transcription in inducing nucleosome depletion in hira - depleted oocytes .
furthermore , cpg island methylation is greatly reduced in hira - depleted oocytes ( figure  6 g ) and methylation loss is more pronounced at regions with high cpg density ( figure  s7k ) . as the dnmt3a enzyme has been shown to operate in a non - processive manner ( dhayalan et  al . ,
2010 ) , stimulatory effect of the unmodified ( h3k4me0 ) h3 tail might be necessary to ensure a high level of methylation at these regions .
we also considered that loss of hira early during oocyte development ( gdf9-cre - driven deletion ) might lead to a variety of secondary effects that could contribute to the observed phenotypes . in this context
it is important to note that deletion of hira at  a later time point during oogenesis ( zp3-cre system ) fully recapitulated the transcriptional and dna methylation defects observed in hira 
 gdf9-cre oocytes ( figures 6a and s6 ) , providing further support for our findings and indicating that the observed effects are not solely attributable to compromised development of the oocytes .
although we can not exclude that some of the effects could be secondary to alterations in gene expression , the most parsimonious view is that the abnormal chromatin resulting from impaired h3.3/h4 deposition is responsible for the majority of the phenotypes we observed . in addition , while it is important to consider that the observed transcriptional and methylation effects could be connected with the specific absence of the h3.3 variant upon hira depletion in oocytes ( santenard et  al . , 2010 ) , it is likely that the lack of normal chromatin structure due to the inability to continuously replace h3/h4 is the main cause underlying the described phenotype .
consistent with this view , the pronounced transcriptional phenotype observed in hira - depleted oocytes is in stark contrast to the only limited transcriptional changes previously described in hira knockout escs ( banaszynski et  al . , 2013 , goldberg et  al . , 2010 ) .
in that case , replication - dependent deposition of canonical h3 by the caf1 complex was likely to compensate in the absence of hira - mediated h3.3 deposition in rapidly dividing escs ( banaszynski et  al .
indeed , h3.3 depletion does not lead to pronounced changes in nucleosome occupancy in proliferating escs ( banaszynski et  al . ,
in contrast , our findings document that post - replicative cells in  vivo require continuous histone replacement in order to maintain an intact chromatin structure , which in turn is required for normal regulation of transcription and for de novo dna methylation in the context of developing oocytes ( figure  7 ) .
interestingly , the critical role of continuous histone replacement also has been demonstrated recently in post - mitotic neurons , where the lack of h3.3 incorporation had an impact on both transcription and physiological function of neurons ( maze et  al
collectively , our studies thus provide new insights into the histone dynamics in  vivo and highlight the importance of studying chromatin in a physiological context and in non - proliferating post - mitotic cells .
oocyte - specific hira depletion was achieved by crossing hira mice with gdf9-icre or with zp3-cre mice , respectively .
the h3.3b - egfp knockin strain was generated in the mrc csc transgenic facility and protamine 1-egfp strain was generated by dr .
all animal experiments were carried out under a uk home office project license in a home office - designated facility .
detailed information is available in the supplemental experimental procedures . in  vitro fertilization ( ivf ) , in  vitro maturation ( ivm ) , parthenogenesis , mrna microinjection , tunel assay , if , and h&e staining were carried out as described previously ( hajkova et  al . , 2010 ,
the scrna - seq was performed on hira , hira 
 gdf9-cre , and hira 
 zp3-cre mii oocytes using the smarter ultra low input rna kit ( clontech laboratories ) with ercc rna spike - in mix 1 ( life technologies ) .
detailed information is available in the supplemental experimental procedures . an scbs - seq described previously ( smallwood et  al . ,
2014 ) was used to profile the dna methylation landscape of hira and hira 
 gdf9-cre oocytes .
ultra - sensitive lc - ms was used to determine overall 5-methylcytosine level . for detailed information ,
 summarythe integrity of chromatin , which provides a dynamic template for all dna - related processes in eukaryotes , is maintained through replication - dependent and -independent assembly pathways . to address the role of histone deposition in the absence of dna replication 
 , we deleted the h3.3 chaperone hira in developing mouse oocytes . we show that chromatin of non - replicative developing oocytes is dynamic and that lack of continuous h3.3/h4 deposition alters chromatin structure , resulting in increased dnase i sensitivity , the accumulation of dna damage , and a severe fertility phenotype . on the molecular level , abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression , the appearance of spurious transcripts , and inefficient de novo dna methylation . 
 our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non - replicative system in  vivo . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions