cellular genomes are constantly exposed to various sources of dna damage ( ciccia and elledge , 2010 , hanawalt , 2015 , hoeijmakers , 2009 , jackson and bartek , 2009 ) , threatening not only genome stability , but also the integrity of chromatin organization ( adam et  al . , 2015 , lukas et  al . , 2011 ,
the basic unit of chromatin is the nucleosome core particle , in which dna is wrapped around a histone protein octamer comprising an ( h3-h4)2 tetramer flanked by two h2a - h2b dimers ( kornberg , 1974 , oudet et  al . , 1975 , luger et  al . , 1997 ) .
variations at the level of this repetitive unit , through histone variants and post - translational modifications ( bannister and kouzarides , 2011 , maze et  al . ,
2014 , talbert and henikoff , 2010 ) , as well as further chromatin compaction , constitute a major source of information that regulates gene expression and cell identity ( filipescu et  al . , 2014 , probst et  al . , 2009 ) .
how chromatin is reorganized in response to dna damage and to which extent the information that it carries can be preserved is thus of fundamental importance .
our current view of chromatin dynamics following dna damage in human cells is based on the access - repair - restore ( arr ) model ( polo and almouzni , 2015 , smerdon , 1991 ) .
this model postulates that chromatin is first disorganized in response to dna damage , which facilitates access to repair factors , followed by restoration of chromatin structure .
yet , it is still unclear whether , when , and by which mechanisms the pre - damage chromatin state is restored ( dabin et  al . , 2016 ) .
notably , chromatin restoration after damage involves the deposition of newly synthesized histones ( adam et  al .
, 2013 , luijsterburg et  al . , 2016 , polo et  al . , 2006 ) , which could potentially replace damaged histones and leave a mark of the damage experience .
thus , assuming that nucleosome density remains at a steady state , a subset of parental histones should be evicted from chromatin during the access step , which would limit the capacity to recover the original epigenetic information at damaged sites ( figure  1a ) .
consistent with this idea , recent reports provide evidence for nucleosome destabilization and histone eviction during dna double - strand break repair ( goldstein et  al . , 2013 ,
li and tyler , 2016 , xu et  al . , 2010 ) and in response to uvc irradiation ( lan et  al . , 2012 ,
uvc damage sites also show reduced histone density , promoted by the uv damage sensor ddb2 ( dna damage - binding protein 2 ) ( luijsterburg et  al . , 2012 ) . however , a  massive loss of parental histones from damaged chromatin would certainly threaten epigenome maintenance . to fully understand how chromatin integrity is preserved or altered in response to genotoxic stress , it is critical ( 1 ) to examine the fate of parental histones present in chromatin before damage and which carry the original epigenetic information and ( 2 ) to track them long enough after damage to examine their contribution to repaired chromatin together with newly deposited histones .
we developed two complementary approaches for specific tracking of parental histones following uvc damage in human cells . challenging our current view of damaged chromatin rearrangements ,
we show that rather than being massively evicted and lost from damaged chromatin , parental histones redistribute in a conservative manner and subsequently recover .
our mechanistic studies demonstrate that both the redistribution and recovery of parental histones are tightly coordinated with repair progression through the uvc damage sensor ddb2 .
we thus propose a conservative model by which parental histone dynamics coupled to dna damage repair contribute to the maintenance of epigenome integrity during the response to uvc damage .
we developed two complementary approaches combining uvc laser micro - irradiation with specific tracking of parental histones in living cells .
we first took advantage of the snap - tag technology , which we exploited previously to follow new histone deposition at uvc damage sites ( adam et  al . , 2013 ) , to fluorescently
we used u2os cells that stably express h3.3-snap histones ( dunleavy et  al . , 2011 ) and a gfp - tagged version of the repair factor xpc ( xeroderma pigmentosum c ) for visualizing damage sites in live cells ( figure  s1a ) .
real - time imaging of parental h3.3 dynamics after local uvc irradiation revealed a rapid reduction of the red fluorescence associated with parental h3.3 , which was restricted to the damaged chromatin area marked by gfp - xpc and detectable at least for 1  hr after irradiation ( figure  1b ) .
we observed a similar decrease of parental h3.3 signal in cells that do not express gfp - xpc ( figure  1c ; movie s1 ) , thus showing that exogenous expression of the repair factor xpc does not alter the histone response to uvc .
uvc damage led to a progressive reduction of the red fluorescence associated with parental histones , with a maximum of 40% loss 10  min after dna damage infliction ( figure  1c ) .
we verified that the region of low histone density observed after damage did not correspond to a nucleolus ( figure  s1b ) .
we also ruled out the possibility that the decrease in parental h3.3 signal could result from photo - bleaching of the red fluorescence by the uvc laser , as irradiating paraformaldehyde - fixed cells with uvc did not reduce the red signal intensity ( figure  1c ) .
thus , the observed decrease in old h3.3 signal in uvc - irradiated chromatin regions actually reflects enhanced dynamics of parental h3.3 histones in response to genotoxic stress . considering that snap - tag - based labeling may interfere with parental h3 dynamics
, we also developed a complementary method to track parental histones based on photo - activation of pa - gfp ( photoactivatable gfp ) in u2os cells engineered to stably express h3.3-pa - gfp and rfp - xpc ( figures s1a and s1c ) .
consistent with our previous findings , we observed a marked reduction of parental h3.3 signal in uvc - damaged chromatin regions within minutes after laser micro - irradiation in live cells and not in paraformaldehyde - fixed cells ( figures s1d and s1e ) .
thus , using two distinct methods to monitor old histone dynamics , our data reveal a local loss of parental h3.3 histone signal in damaged chromatin regions .
furthermore , we observed an altered distribution of parental histones upon uvc damage in all irradiated cells throughout interphase ( figure  s2a ) , and this was not restricted to the h3.3 variant since parental h3.1 signal was also reduced at uvc damage sites ( figures s2a and s2b ) .
we recapitulated these results in mcf7 cancer cells and in bj primary fibroblasts and observed similar dynamics for parental histone h4 ( figures s2c  s2f ) . collectively , these results reveal a rapid reduction of parental h3 and h4 histone density in uvc - damaged chromatin .
we next sought to determine whether the local reduction in parental h3 staining upon uvc irradiation actually reflects parental histone loss from damaged chromatin .
for this , we measured changes in old h3.3 fluorescence within the entire cell nucleus after local uvc damage . to maximize sensitivity in this assay , we reduced the size of the area of interest by photo - bleaching h3.3-snap - associated fluorescence in the whole nucleus apart from a small patch ( figure  2a ) .
next , photo - bleaching 40% of the fluorescence inside this patch led to a 20% fluorescence reduction in the entire nucleus ( figure  2b ) .
in contrast , targeting uvc irradiation to the fluorescent patch , while leading to a comparable 40% loss of signal in the irradiated area , did not result in a detectable loss of fluorescence from the entire nucleus ( figure  2b ) . from these results , we conclude that parental h3 histones mobilized early after genotoxic stress remain in the damaged nucleus and do not undergo massive degradation , as also supported by biochemical analyses of parental h3.3 levels after uvc damage ( figure  s2 g ) .
we then refined our analysis by measuring the spatial distribution of parental h3.3 histones around the damaged area ( figure  2c ) .
notably , we observed that the reduction of parental histone signal in the damaged region was counterbalanced by an increase of signal in the surrounding area ( figures 2d and 2e ) .
importantly , this conservative redistribution of parental histones did not occur upon local photo - bleaching of parental h3.3 fluorescence ( figures 2c and 2d ) , and we obtained similar results by photo - activation - based tracking of parental histones ( figures s3a  s3d ) .
furthermore , the area showing reduced histone density gradually increased over time post - irradiation ( figure  3a ) , which reveals that parental histone dynamics proceed radially outward . such redistribution of parental histones argues against their eviction from damaged chromatin , because , if solubilized in the nucleoplasm , they would not be retained in a defined space in the periphery of the irradiated region .
supporting the fact that mobilized histones remain chromatin associated , detergent extraction of live cells after uvc irradiation did not alter  the redistribution pattern of parental histones ( figure  s3e ) .
altogether , these data demonstrate that parental h3 histones redistribute to the periphery of damaged chromatin .
such redistribution can involve parental nucleosomes sliding away from dna lesions and/or an expansion of chromatin in the irradiated region , pushing away surrounding undamaged chromatin fibers . to test these hypotheses and gain further insights into the mechanisms underlying parental histone dynamics in uvc - damaged regions
thus , we found that parental h3.3 redistribution was  accompanied by a decrease in dna density within uvc - damaged regions ( figures 3b and s3f ) , indicative of chromatin opening .
the area of uvc - damaged dna increased by a factor of 1.26 within 15  min post - damage ( figure  3c ) , consistent with the 20% dna density loss measured in the same experimental conditions ( figure  3b ) .
this strongly supports the idea that the reduced dna density in the damaged region results from chromatin opening .
interestingly , while histone and dna signal loss both increased with the exposure time to uvc laser , histone signal loss exceeded dna signal loss in all conditions examined ( figure  3d ) .
this observation can not be accounted for by chromatin opening only , which would lead to an equal reduction in histone and dna densities in the damaged area .
since we already ruled out the possibility of histone dissociation from chromatin and extensive histone degradation ( figures 2 and s3 ) , a possible explanation is that the extra loss in histone density reflects histone mobilization on chromatin away from damage sites ( figure  3e ) .
the biphasic shape of the curves for histone and dna signal loss as a function of uvc damage ( figure  3d ) also points to a possible dual mechanism driving parental histone redistribution , each plateau corresponding to the saturation of a distinct process ( figure  3f ) .
our findings suggest that chromatin opening along with histone mobilization on damaged chromatin drive parental histone redistribution to the periphery of uvc - damaged regions . to search for molecular determinants of parental h3 redistribution upon uvc damage
, we examined the connection with uvc damage repair by knocking down factors involved at different steps in the ner ( nucleotide excision repair ) pathway ( figure  4a and s4a ; reviewed in alekseev and coin , 2015 , marteijn et  al . , 2014 ) . decreasing the expression of the late repair factor xpg ( xeroderma pigmentosum g ) , required for excision of
the damaged oligonucleotide before repair synthesis , only moderately reduced parental h3.3 redistribution upon uvc irradiation ( figure  s4b ) .
similarly , knocking down the early repair factor ercc6 ( excision repair cross - complementing 6 ) involved in damage recognition within transcribed genes did not markedly impair parental h3.3 dynamics ( figure  s4b ) .
consistent with this , cells treated with a transcription inhibitor prior to uvc irradiation did not show major defects in old h3.3 redistribution ( data not shown ) .
these results indicate a relatively minor contribution of transcription - coupled repair and late repair steps to the reduced parental h3 histone density at damaged sites .
in contrast , when knocking down the uvc damage sensor ddb2 involved in global genome repair , we observed a striking impairment in parental h3.3 , h3.1 , and h4 density loss at uvc damage sites ( figures 4a and s4c  s4e ) .
we did not observe comparable defects in parental h3.3 dynamics upon downregulation of xpc , another early repair factor involved in global genome repair ( figure  s3f ) , or upon knockdown of the ddb2 partners ddb1 and cul4a ( cullin 4a ) ( figure  4a ) , which are part of an e3-ubiquitin ligase complex that modifies various substrates at sites of uvc damage ( nouspikel , 2011 ) .
consistent with a minor contribution of ddb1 and cul4a to parental h3.3 density loss , we found that preventing de novo ubiquitylation reactions by treating cells with a proteasome inhibitor or with a neddylation inhibitor did not markedly alter the redistribution of old h3.3 in damaged chromatin ( figures s4f and s4 g ) .
these data indicate that the ubiquitylation activity of the ddb1-ddb2-cul4a - containing complex does not play a major role in parental histone dynamics following uvc damage .
parental histone redistribution to the periphery of damaged chromatin regions is thus coupled to the earliest steps of global genome ner with a prominent role for ddb2 . to further characterize the contribution of ddb2 to parental histone redistribution upon uvc damage , we tested the effect of ddb2 overexpression . expressing exogenous
ddb2 significantly increased the area of parental histone signal loss after local uvc irradiation : this area was 50% larger in cells overexpressing ddb2 than in cells overexpressing another early ner factor , xpc ( figure  4b ) .
these results thus indicate that ddb2 levels are limiting for parental histone redistribution in uvc - irradiated  chromatin regions .
furthermore , artificial tethering of ddb2 to a laco ( lactose operator ) array in the absence of dna damage led to a marked reduction of parental h3.3 histone density at the laco array ( figure  4c ) .
ddb2 tethering also triggered an expansion of the laco array , as previously reported ( luijsterburg et  al .
these results reveal that ddb2 binding to chromatin is sufficient for parental histone redistribution even in the absence of dna damage .
to gain mechanistic insights into ddb2 effect on parental histone dynamics , we tested the potential contribution of chromatin remodelers with reported connections to the ddb2 complex , namely alc1 ( amplified in liver cancer 1 ) and ino80 ( inositol - requiring 80 ) ( jiang et  al . , 2010 , pines et  al . , 2012 ) .
knocking down these remodelers did not recapitulate the effect of ddb2 downregulation ( figures s5a and s5b ) . similarly , interfering with poly(adp - ribosyl)ation , which has been associated with ddb2 and uv - damaged chromatin decompaction ( luijsterburg et  al . , 2012 ,
pines et  al . , 2012 ) , did not impact parental histone redistribution at uvc damage sites ( figures s5c and s5d ) .
however , our analyses of dna and histone signal loss at uvc sites upon ddb2 knockdown ( figure  s5e ) indicate a major contribution of ddb2 to chromatin opening only , consistent with previous observations ( luijsterburg et  al . , 2012 ) , suggesting that additional factors come into play to promote histone mobilization on chromatin . collectively , our data put forward the early repair factor ddb2 as a master regulator of parental histone redistribution by chromatin opening at uvc sites . as we previously demonstrated that ddb2 controls the recruitment of the histone chaperone hira ( histone regulator a ) , which promotes the deposition of newly synthesized histones h3.3 at uvc damage sites ( adam et  al . , 2013 ) , we investigated the potential coupling between parental and new h3.3 dynamics in response to uvc irradiation . for this , we labeled parental and new histones in different colors within the same sample and compared the kinetics of parental histone density loss and new histone deposition upon local uvc damage ( figure  5a ; movies s2 and s3 ) . while parental h3.3 histones are redistributed within minutes after damage induction , new histone h3.3 accumulation at damage sites becomes detectable only 30  min after local uvc irradiation ( figure  5a ) .
we obtained similar results when we swapped the snap reagents , labeling old h3.3 in green and new h3.3 in red ( data not shown ) .
thus , our assay reveals that parental histone density loss precedes new histone deposition at uvc damage sites .
given that the histone chaperone hira promotes the deposition of newly synthesized h3.3 at uvc damage sites ( adam et  al .
, 2013 ) , we tested whether the same chaperone was responsible for parental h3.3 dynamics in uvc - damaged regions .
interestingly , hira downregulation did not impair old h3.3 signal loss at uvc sites ( figure  5b ) , showing that this histone chaperone does not participate in any significant manner in parental h3.3 dynamics after uvc damage .
consistent with this , preventing the synthesis of new h3.3 by sirna ( small interfering rna ) ( figure  5c , green ) did not interfere with parental h3.3 displacement from uvc - damaged regions ( figure  5c , red ; note that this treatment did not affect parental h3.3 levels ) . altogether , these data demonstrate that parental histone h3.3 redistribution in uvc - damaged regions is functionally independent of new h3.3 deposition .
since parental histones are still detected in the periphery of uvc - damaged regions early after dna damage , we investigated whether and to which extent they contribute to chromatin restoration after damage . for this purpose
, we examined both parental and new h3.3 dynamics in parallel with repair progression in u2os cells stably expressing h3.3-snap and cfp - xpc ( figure  s6a ) .
this analysis revealed that parental histones recovered in chromatin undergoing uvc damage repair , reaching almost complete recovery ( 90% of the initial signal ) 9  hr after uvc irradiation , which mirrors the slow kinetics of uvc damage repair ( figure  6a ) . a similar extent of parental histone recovery at uvc damage sites
was measured in u2os cells stably expressing h3.3-pa - gfp ( figure  s6b ) and in mfc7 cells transiently expressing h3.3-snap ( figure  s6c ) .
noteworthy , parental histone recovery was delayed compared to new histone incorporation ( figure  6a ) , suggesting that they involve distinct mechanisms . to gain insight into how parental histones recover
, we compared their dynamics to basal histone mobility assessed by frap ( fluorescence recovery after photo - bleaching ) . 
while parental histone recovery in uvc - damaged regions reached 80% within 12  hr after irradiation , it did not exceed 20% in an undamaged chromatin region of the same nucleus within the same time frame ( figure  s6d ) , consistent with a previous report ( kimura and cook , 2001 ) .
this demonstrates that parental h3.3 recovery in chromatin undergoing repair does not result from basal histone turnover but actually reflects the relocation of parental histones that were mobilized away from uvc - damaged regions .
notably , when measuring the area of parental histone density loss over time after uvc damage , we observed that parental histone recovery proceeded radially inward ( figure  6b ) , suggesting that displaced histones were moving back by an opposing mechanism to their initial redistribution .
the area of new histone accumulation by contrast did not shrink ( figure  s6e ) , arguing that chromatin restoration does not proceed solely via re - compaction . as a result , recovered parental histones mix with newly synthesized histones in repairing chromatin ( figure  s6f ) .
we next sought to determine whether parental h3.3 recovery in damaged chromatin was coupled to repair progression .
for this , we depleted the late repair factor xpg , which interferes with repair progression with no major effect on the early redistribution of parental histones in damaged chromatin ( figure  s3b ) .
parental h3.3 recovery , however , was markedly impaired in xpg - depleted cells ( figure  6c ; movies s4 and s5 ) down to a rate similar to basal histone turnover ( figures s6d and s6 g ) .
these results indicate that parental histone recovery in damaged chromatin is dependent on repair progression .
given that xpg depletion also significantly delayed ddb2 release from chromatin , we assessed more directly the role of ddb2 in parental histone recovery . for this , we triggered lacr - ddb2 release from the laco array by iptg addition ( figure  6d ) .
this resulted in rapid recovery of old h3.3 at the laco array , which highlights the key role of ddb2 in controlling parental histone dynamics in uvc - damaged chromatin .
collectively , our results underline the major contribution of parental histones to chromatin restoration coupled to repair and establish that parental histone recovery is coordinated with repair progression through ddb2 release from damaged chromatin .
by exploiting real - time tracking of parental h3 and h4 histones after local uvc damage in human cells , we provide novel insights into epigenome maintenance in response to dna damage .
our study indeed identifies a conservative process , tightly coordinated with repair progression , whereby parental histones rapidly redistribute away from uvc - damaged chromatin regions and subsequently recover ( figure  7 ) .
we propose that parental histones are kept in the periphery of the damaged areas to help restore chromatin organization after dna repair , which may contribute to preserving a memory of chromatin identity in response to dna damage .
chromatin rearrangements coupled to the early stages of the  ddr , although considered to be critical for efficient dna repair , still remained poorly characterized . here , we provide evidence for a redistribution of parental histones to the periphery of uvc - damaged chromatin regions , which prompt us to re - evaluate  our views on the access - repair - restore model . even though histone solubilization has been reported in response to genotoxic stress ( goldstein et  al . , 2013 , wang et  al .
, 2006 , xu et  al . , 2010 ) , our data support a model where parental h3 histones do not massively dissociate from chromatin but are redistributed away from uvc - damaged sites , possibly via nucleosome sliding and chromatin opening .
this is consistent with a recent study suggesting that h2b histones are not solubilized following uv irradiation in human cells ( morisaki and mcnally , 2014 ) .
the extent of chromatin rearrangements in response to local dna damage is a matter of debate . while one study indicates that chromatin destabilization affects the whole nucleus upon local uvc irradiation ( rubbi and milner , 2003 ) , several lines of evidence rather support the idea that chromatin is locally disorganized upon genotoxic stress ( dinant et  al . , 2013 , goldstein et  al . , 2013 , hinde et  al . , 2014 ,
2013 ) . here , we reconcile these data by demonstrating that a local loss of parental histone density in uvc - damaged areas has a long - range impact on chromatin , potentially affecting the whole nucleus , because parental histone redistribution actually spreads over long distances away from the damaged regions ( figure  2e ) .
whether and how histone redistribution facilitates access to damaged dna and repair progression are not entirely clear .
the recruitment of the damage sensor ddb2 to uvc lesions precedes and orchestrates parental histone dynamics .
it is tempting to speculate that this may also contribute to protect parental histones from modifications by histone - modifying enzymes recruited to regions of ongoing repair , thereby promoting the maintenance of the original epigenetic information .
mechanistically , both histone mobilization on chromatin and chromatin opening potentially contribute to chromatin disorganization in response to uvc damage .
whether they use similar regulatory factors as those promoting chromatin mobility in response to dna breaks ( dion and gasser , 2013 ) is an attractive possibility . in terms of molecular players ,
we identify the damage sensor ddb2 as a master regulator of parental histone dynamics at sites of uvc lesions , mostly via its ability to promote chromatin opening .
interestingly , while the ubiquitylation activity of ddb2-containing complex is required for new histone deposition in uvc - damaged chromatin ( adam et  al . , 2013 ) , it is largely dispensable for parental histone dynamics .
consistent with our findings , ubiquitylation - deficient mutants of ddb2 induce chromatin expansion like wild - type ddb2 when artificially tethered to chromatin ( luijsterburg et  al . , 2012 ) .
thus , ddb2-mediated chromatin expansion is largely independent of the other members of the uvc damage recognition complex .
whether ddb2 acts alone or in association with other factors to fulfill this activity will be important to investigate in future studies .
remarkably , ddb2 has very strong affinity for uvc - damaged dna ( kulaksiz et  al .
2005 ) , and ddb2 binds damaged dna on nucleosomes ( osakabe et  al . , 2015 ) .
we can speculate that ddb2 binding to damaged chromatin may on its own push away surrounding nucleofilaments leading to chromatin opening .
given that ddb2 does not display atpase / helicase or histone - binding domains , we rather favor a model where it works in concert with other factors directly involved in chromatin dynamics  such as histone chaperones and/or chromatin remodeling complexes that remain to be identified  to promote chromatin disorganization in damaged regions .
our study identifies a pathway that may contribute to preserving the integrity of chromatin architecture in response to dna damage .
we unraveled that damaged chromatin reorganization is a two - step process with new histone incorporation preceding parental histone recovery .
the biphasic nature of chromatin restoration is consistent with an early model based on the accessibility to nucleases of chromatin undergoing ner ( reviewed in smerdon , 1991 ) . while we can not formally exclude that a limited amount of parental histones are ultimately degraded after uvc damage as reported for hyperacetylated histones in response to ionizing radiation ( qian et  al . , 2013 ) , the conservative nature of parental histone redistribution around uvc damage sites and the almost complete recovery of parental histones during repair progression argue against massive histone degradation .
furthermore , the retention of parental histones proximal to the site of damage offers a possible redirection to the original site to promote a conservative restoration of chromatin architecture
. it will be important to develop higher - resolution approaches to determine whether parental histones retrieve their original positions on the dna sequence upon recovery and whether the topological organization of parental chromatin is fully re - established .
the major contribution of parental histones to the composition of repaired chromatin is crucial to envision mechanisms for epigenome maintenance after dna damage .
indeed , it opens up the possibility that parental marks can be preserved and transferred to the new histones , which initially carry their own set of post - translational modifications ( ptms ) ( loyola et  al . , 2006 ) . in this respect
, a parallel can be drawn between the restoration of chromatin after dna damage and dna replication ( alabert and groth , 2012 , groth et  al .
the maintenance of chromatin identity is achieved by old histone recycling with their ptms and by subsequent modifications of new histones to mirror the parental ones ( alabert et  al . , 2015 ) .
noteworthy , while cells have to cope with 50% histone renewal during replication , most parental histones recover in damaged chromatin regions , which could facilitate the re - establishment of the original chromatin landscape . it is tempting to speculate that the presence of parental and new histones in neighboring nucleosomes may allow old ptm transmission to newly deposited histones after repair
. finally , our data open up new avenues for understanding the  etiology of several human diseases , including h3 mutant - associated cancers ( reviewed in kallappagoudar et  al . , 2015 , yuen and knoepfler , 2013 ) and ner disorders ( reviewed in digiovanna and kraemer , 2012 , marteijn et  al . , 2014
considering that ddb2 dynamics strongly impact the fate of parental histones in response to uvc damage , the phenotype of xpe patients harboring ddb2 mutations that prevent its binding to damaged dna may not only reflect a dna repair defect but also altered chromatin plasticity in response to genotoxic stress .
similarly , h3 mutations could contribute to the development of human cancers by affecting the resetting of the epigenome following dna damage . in conclusion ,
our work sheds new light to our current view of dna damage - induced chromatin rearrangements and suggests that parental histone dynamics are critical to the maintenance of epigenome integrity in response to genotoxic stress
. our findings also pave the way for the identification of new factors that  contribute to restoring damaged chromatin identity and for understanding how this protects cells against pathological conditions .
snap labeling of histone proteins was done as described ( bodor et  al . , 2012 ) .
photo - activation experiments were performed in u2os cells stably expressing h3.3-pa - gfp on a zeiss lsm710 confocal microscope using the 30 mw 405  nm laser focused through an ld lci plan - apochromat 25/0.8 oil objective .
uvc laser micro - irradiation was done as described ( adam et  al . , 2013 , dinant et  al . , 2007 ) .
details for the frap procedure and for image acquisition and analysis are in supplemental experimental procedures .
sirnas ( supplemental experimental procedures ) were transfected into cells using lipofectamine rnaimax ( invitrogen ) .
cells were transiently transfected with plasmid dna ( 1  g / ml final , supplemental experimental procedures ) using lipofectamine 2000 ( invitrogen ) 48  hr before subsequent cell treatment .
these procedures , including lists of antibodies and primer pairs , are described in supplemental experimental procedures .
p values for mean comparisons between two groups were calculated with a student s t test , including welch s correction when necessary , using r software .
multiple comparisons were performed by one - way anova with bonferroni post - test using graphpad prism .
( equal contribution ) , and s.e.p . designed and performed experiments , analyzed the data , and wrote the manuscript .
g.a . provided inputs for experimental design using the uvc laser technology and snap tagging and for data analysis and writing .
 summarychromatin integrity is critical for cell function and identity but is challenged by dna damage . to understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress , we established a system for real - time tracking of parental histones , which characterize the pre - damage chromatin state . focusing on histone h3 dynamics after local uvc irradiation in human cells , we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin . 
 importantly , parental histones almost entirely recover and mix with new histones in repairing chromatin . 
 our data further define a close coordination of parental histone dynamics with dna repair progression through the damage sensor ddb2 ( dna damage - binding protein 2 ) . 
 we speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to dna damage . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions