chromatin is the complex of dna and histone proteins that packages eukaryotic dna into chromosomes .
the nucleosome is the repeating structural subunit of chromatin and consists of 147  bp of dna wrapped around a histone octamer core .
translational positioning of nucleosomes along the dna sequence influences the accessibility of regulatory sequences to the transcriptional machinery and can thereby regulate gene expression levels ( for review , see hughes and rando , 2014 , radman - livaja and rando , 2010 ) .
the average nucleosome - positioning profile over all yeast genes consists of a nucleosome - depleted region ( ndr ) of 150  bp , with well positioned 1 and  + 1 nucleosomes upstream and downstream of the ndr , respectively .
the transcription start site ( tss ) is located in the  + 1 nucleosome , there is a regularly spaced nucleosomal array over the first kb of the gene body , and nucleosome positions become fuzzier toward the middle and end of the coding sequence ( brogaard et  al . , 2012 ,
because ndrs are thought to facilitate transcriptional activation by enabling access of regulatory proteins to their binding sequences , ndr formation or loss can lead to gene activation or silencing , respectively .
the distribution of nucleosomes along the genome depends in part on the underlying dna sequence , with promoter regions enriched in poly a tracts mostly excluding nucleosomes ( kaplan et  al . , 2009 , yuan et  al . , 2005 ) .
in addition to poly a tracts that passively disfavor nucleosome assembly , ndrs can also be formed through active nucleosome removal from promoter regions by remodelers , such as rsc ( parnell et  al . , 2008 ) , or nucleosome displacement by general transcription factors ( tfs ) , such as abf1 and rap1 ( yarragudi et  al . , 2004 ,
whereas dna sequence composition contributes to nucleosome occupancy in yeast , it is the action of chromatin remodelers and the transcriptional machinery that establishes precise nucleosome positioning over genes ( gkikopoulos et  al . , 2011 , hughes et  al . , 2012 ,
, 2015 , pointner et  al . , 2012 , weiner et  al . , 2010 ) . indeed , in  vitro assembly of nucleosomes onto purified
yeast genomic dna results only in nucleosome depletion over poly a tracts but little evidence for nucleosome positioning , whereas addition of yeast extract to such reconstitutions yields a more - accurate recapitulation of nucleosome positioning patterns observed in  vivo ( zhang et  al . , 2011 ) .
purified atp - dependent remodelers , such as chd1 and swi / snf family  members , can generate ndrs at promoters and regularly spaced nucleosomal arrays over gene bodies similar to those seen in  vivo , even in the absence of transcription ( lieleg et  al . ,
however , such in  vitro nucleosome reconstitutions do not perfectly match nucleosome positions observed in  vivo ( hughes et  al . , 2012 ) .
in  vivo , the process of transcription plays a key role in nucleosome positioning , due both to the direct effects of rna polymerase on nucleosomes and to the effects of remodelers that are recruited to target genes during transcriptional activation or elongation ( bintu et  al . , 2011 , radman - livaja et  al .
, 2011 , studitsky et  al . , 1997 , weiner et  al . , 2010 ) .
contrary to the steady - state landscape of nucleosome positioning , chromatin structure dynamics over the cell cycle , during which chromosomes are subject to dramatic perturbations caused by replication and mitosis , are not well characterized .
dna replication initiates the disassembly of maternal nucleosomes ahead of the replication fork and their reassembly in its wake on one or the other daughter chromatid ( alabert and groth , 2012 ) .
as nucleosomes can influence transcription , depending on their precise locations , replication provides an opportunity for the cell either to re - establish the same nucleosome - positioning profiles or to rearrange the nucleosomal landscape and thereby maintain or change its gene expression program , respectively .
the process of nucleosome re - positioning after the disruption caused by replication is not well understood .
specifically , it is not known how and where nucleosomes re - position themselves on newly replicated dna and how long it takes them to reconstitute the canonical mid - log positioning pattern .
this is related to the questions of when transcription resumes after the disruption caused by dna replication , whether both new gene copies are transcribed , and whether transcription re - activation  is  a cause or a consequence of nucleosome positioning maturation . in order to address these questions ,
we have developed a method for genome - wide mapping of nucleosome positions on recently replicated dna in budding yeast : nascent chromatin avidin pull - down ( nchap ) . in this method , we isolate newly synthesized dna at varying times after a pulse of the nucleotide analog 5-ethynyl-2-deoxyuridine ( edu ) , which , along with micrococcal nuclease ( mnase ) digestion , allows us to follow genome - wide nucleosome - positioning dynamics after the passage of the replication fork on both leading and lagging dna copies .
we find that nucleosomes assume their mid - log positions with varying rates at different genomic loci .
because we find that highly transcribed genes exhibit a rapid return to their canonical chromatin architecture , we hypothesized that transcription participates in the regular phasing of nucleosomes in the gene body minutes after the passage of the fork .
consistent with this , treatment with the rna polymerase inhibitor thiolutin interfered with chromatin maturation over coding regions .
experiments in deletion mutants reveal a role for chd1 and isw1a in nucleosome phasing relative to the tss and a role for hir in determining the linker length between nucleosomes .
in contrast to transcription - dependent maturation of gene body chromatin , aspects of promoter packaging , such as the ndr midpoint and the position of the  + 1 nucleosome , appear to be determined earlier , possibly in the absence of transcription elongation .
together , our data illuminate the genomic landscape of chromatin maturation following replication , and our methodology enables future genetic interrogation of the mechanisms responsible for chromatin maturation .
in order to map the positions of nucleosomes on recently replicated dna , we developed nchap , which combines edu labeling of nascent dna ( sirbu et  al .
, 2011 , wirges et  al . , 2007 ) with mnase digestion of chromatin .
edu is a thymidine analog that is incorporated during the synthesis of new dna , and mnase is an endonuclease that preferentially cleaves dna in the linker regions between nucleosomes .
cells are arrested in g1 with  factor and released into s phase in the presence of edu .
aliquots are taken at regular intervals following release into s phase , and cells are fixed with formaldehyde .
fixed cells are then treated with mnase , and nucleosome - protected dna is isolated after cross - link reversal .
edu from purified 150-bp fragments is conjugated to biotin azide in a click reaction , and biotinylated dna fragments are isolated using streptavidin - coated magnetic beads .
illumina paired end sequencing libraries are prepared ( adapted from borodina et  al . , 2011 ) from the nascent dna attached to beads . in order to differentiate between the lagging and the leading strand copies ,
the edu - containing nascent strand is separated from its template using primer extension from one end of the adaptor - ligated fragment ( figure  1a ) .
upstream  of efficient replication origins will originate from the lagging strand copy , whereas the complementary crick reads will be from the leading copy .
several controls validate the ability of nchap to identify newly replicated dna in relatively unperturbed cells .
first , flow cytometry profiling of dna content shows that s phase progression is not impaired in the presence of edu ( figure  s1a ) .
second , we tested the ability of our protocol to specifically capture the single edu - bearing strand of dna , using in - vitro - generated fragments that have incorporated edu in only one strand ( figure  s2 ) .
this fragment was then subjected to the same procedure that we used to generate nchap libraries as outlined in figures 1a , s2a , and s2b .
following streptavidin pull - down of these test libraries , qpcr with strand - specific primers showed that the fraction of fragments with the edu - containing strand in the expected orientation was 70%85% . finally , we also generated dna fragments in which only one strand has incorporated edu in  vivo , taking advantage of the 53 resection and gap - filling steps that occur during the repair of a double - strand break at the matalpha1 locus ( reviewed in haber , 2012 ) , which we induced in the presence of edu ( figure  s2c ) .
here , we observe a 10-fold enrichment of  the edu - containing strand in the expected orientation .
overall , our tests show that our strand - specific library construction protocol can efficiently isolate the nascent dna strand and thus differentiate between leading and lagging strand copies of the genome .
we next applied nchap to cells released from g1 arrest into s  phase for varying lengths of time .
nchap data at early time points reveal strong peaks surrounding known origins of replication ( autonomously replicating sequence [ ars ] ; nieduszynski et  al . , 2007 ,
2002 ) , validating the ability of our protocol to specifically map nucleosomes assembled on newly synthesized dna .
the enrichment of nchap data ( blue peaks ) around origins is not due to mnase bias toward replicated regions , because read density distributions from mnase - digested input fractions ( before biotinylation and streptavidin pull - down ) exhibit the relatively even occupancy expected of the nucleosome landscape in yeast ( pink peaks ) .
average nucleosome profiles from the nascent chromatin fraction resemble classic average nucleosome profiles , indicating that edu incorporation does not interfere with mnase digestion or nucleosome assembly ( figure  s3a ) .
together , these data validate the ability of nchap to accurately identify nucleosome - protected regions of recently replicated dna in a strand - specific manner . during cell - cycle arrest and release , asynchrony among individual cells in the timing of g1 release and entry into s phase results in heterogeneity in the location of the replication fork in any given cell , meaning that , even for early firing regions of the genome , nchap will capture dna that has been replicated from 1 to as many as 20  min prior .
consequently , in order to characterize the dynamics of chromatin maturation following replication , we carried out nchap across a time course in which asynchronous yeast was subject to a brief pulse of edu followed by a thymidine chase for varying lengths of time .
an asynchronous population by definition contains cells at all stages of the cell cycle , including 15%40% of cells that are in s phase and which , as a population , will have replication forks located at every location along the genome . as a result
, a relatively short pulse of edu will label short stretches of replicating dna covering the entire genome over the whole cell population .
the thymidine chase stops further incorporation of edu , and subsequent fixation at regular time intervals provides snapshots of simultaneous nucleosome positioning changes shortly after replication in all replicating cells . in order to capture times as close as possible to the moment immediately after the passage of the replication fork
, we sought to identify the minimal duration of the edu pulse that provided appreciable incorporation into replicated dna .
we used flow cytometry profiling of cells labeled with fluorescein ( fam)-conjugated edu to monitor the kinetics of edu incorporation in asynchronous cells ( figure  s4 ) .
edu labeling is detectable within 15  min of its addition to the culture ( figure  s4a ) , and the majority of replicating cells have incorporated edu after 2530  min ( figure  s4 ) .
edu incorporation could be delayed and slowed by growing yeast at suboptimal temperatures to extend the length of the cell cycle ( figure  s4b ) . at 30c
, an initial lag phase of 15  min is followed by a gradual increase in the numbers of cells that have incorporated edu , as well as an increase in edu - fam fluorescence intensity per cell as genome replication progresses and more edu is incorporated in each s phase cell ( figure  s4c ) . a slower increase in the average cellular edu - fam intensity over time at 25c and 37c compared
to 30c is consistent with slower or stalled replication forks , delayed s phase entry , and/or slower edu uptake and processing ( figure  s4c ) . because the fraction of edu - positive cells in the asynchronous cell population increases gradually and cells
need to be incubated with edu for 25 , 40 , or 50  min ( if grown at 30c , 37c , or 25c , respectively ) before all cells that were in s phase at the moment of edu addition become edu positive ( figure  s4b ) , we conclude that the rates of edu import and processing can vary widely among different cells in the population , possibly due to variable expression of the edu transporter ( hent1 ) and thymidine kinase ( tk ) that were introduced into our yeast strain .
all subsequent experiments were performed at 30c . to assess whether we could effectively halt edu incorporation using an excess of cold thymidine , we pulsed asynchronous yeast with edu for 20 or 5  min and then assayed edu - fam at varying times after thymidine addition ( figure  s5 ) .
both the levels of edu - fam across the population and the fraction of edu - fam - positive cells stay constant up to 25  min after the thymidine chase ( figures s5b and s5c ) , and then as cells enter a new round of replication , the fraction of edu - positive cells decreases , as expected for a successful thymidine chase .
moreover , the average edu - fam intensity per cell remains low throughout the chase , consistent with edu being incorporated  into only a small fraction of the genome , as intended ( figure  s5e ) .
we carried out two separate pulse - chase time course experiments : one with a 20-min and another with a 5-min edu pulse ( figure  s5 ) .
we calculated the pearson correlation between the nucleosome - positioning profile for each gene in the yeast genome ( from 500  bp upstream of the tss to the stop codon ; xu et  al . , 2009 ) in our nchap data and the corresponding profile from a mid - log total chromatin standard ( weiner et  al . , 2010 ) for each time point ( figure  2a ; table s1 ) .
for each gene , we define its  maturation index  as the average correlation over the time course between the nucleosome profiles from nascent chromatin and the total chromatin standard , with individual genes exhibiting a wide range of maturation indices ( figures s3b and s3c ) .
importantly , data for total chromatin consistently exhibit higher correlations to the standard than do nascent chromatin data ( figure  2a ; plot below the heatmap ) , demonstrating that the wide range of maturation indices in nascent chromatin is not an artifact of variability in mnase digestion across the time course .
progressive changes in nucleosome positioning on nascent dna are also evident from whole - genome pairwise correlation analysis ( figure  s6a ; table s2 ) .
what distinguishes genes that rapidly adopt their mature chromatin state following replication ? sorting genes according to their maturation index from the 20-min edu pulse experiment , we find that genes that have the highest maturation index are also generally highly transcribed during active growth in rich medium ( kim et  al . , 2010 ) .
this trend is shown via a 50-gene running window average of rna pol2 occupancy and is even more evident when genes are divided into quintiles ( 1,245 genes each ) of maturation indices : the average rna pol2 occupancy in the highest quintile ( 5 ) is 10-fold higher than in the lowest quintile ( 1 ; figure  2a , top middle and right panels ) .
this suggests that the process of transcription plays a role in re - establishing nucleosome positions over genes after chromatin disruptions caused by dna replication .
box plot and pairwise t test analysis of correlations to the standard for quintiles 1 and 5 from two biological replicates of the 20-min edu pulse experiment show that the two quintiles are significantly different in all time points from both replicates ( p value of t test  0.05 ) and that most of the variability between replicates comes from early time points in the first quintile ( figures s6b and s6c ) .
although data from the 5-min edu time course differ quantitatively from data from the  20-min edu time course ( figure  2b ) , in both datasets , nascent chromatin increasingly matches the mid - log standard as the time course progresses ( figures 2a , bottom , and s7 ) , and this trend is also replicated in the second 20-min edu pulse experiment ( figure  s7 ) .
moreover , the correlation of maturation indices in each quintile ( defined above ) with average rna pol2 occupancy is also preserved in both time courses when focusing on robustly measured genes ( figure  2b ) . to examine the process of chromatin maturation more closely ,
we compared the average tss - aligned nucleosome profiles from nascent chromatin to the same profiles obtained from mid - log yeast , averaging data according to quintiles of genes grouped by maturation index . visual inspection of these averaged profiles reveals that nucleosomes become better defined over the first kb of the gene body over time ( figures 2c2e ) .
this improvement in nucleosome phasing can be quantitated using a measure of peak to trough for nucleosomes  a low peak - to - trough ratio can either be due to a low average nucleosome occupancy at that position across the cell population or fuzzy positioning ( nucleosomes are not placed at the same distance from the tss in all genes and in all cells ) .
the average peak - to - trough ratios for nucleosomes  + 2 to  + 7 on nascent chromatin after a 20-min edu pulse reach a plateau between the 4- and 8-min time points for rapidly maturing ( fifth quintile ) genes , whereas , in the first quintile , the ratios are stabilized only 10  min later ( figure  2d , top ) .
similar rates of average peak / trough increase were confirmed in a second biological replicate of the 20-min edu pulse experiment ( figure  s8 )
. a quadratic fit to the curves from figure  2d ( top ) reveals that genes in the first and fifth quintiles reach their half - maximal peak / trough 20 and 7  min after the chase , respectively .
consistent results were obtained using the data from the 5-min edu pulse ( figure  2e ) , albeit with somewhat more - rapid apparent rates of nucleosome phasing over gene bodies ( figures 2d , bottom , and 2e ) . as the genes that exhibit comparatively rapid phasing of nucleosomes over gene bodies ( the fifth quintile ) are relatively highly transcribed , we propose that the regular phasing of nucleosomes downstream of the tss is a consequence of transcription elongation . in contrast to the relatively slow chromatin maturation observed over gene bodies , the  + 1 nucleosome and the midpoint of the ndr are already in place at the beginning of our time course for the majority of genes ( figures 2c and 2e ) .
thus , the positions of the  + 1 nucleosome and the ndr are determined early after the passage of the replication fork .
indeed , promoter chromatin architecture is established so rapidly that it is impossible to pinpoint its exact kinetics using our assay , as even for the 5-min edu pulse time course , there is a 10-min window between edu addition and the first recorded time point , during which time edu is incorporated into dna at different moments in different cells ( figure  s4 ) . to directly test the involvement of transcription in nucleosome positioning maturation , we treated cells with the rna polymerase inhibitor thiolutin in a parallel experiment with the 5-min edu pulse time course shown in figure  2 ( figure  s9 ) .
chromatin maturation is greatly impaired upon treatment with the inhibitor ( figures 3 , s6 , and s7 ) , thus providing experimental support to our hypothesis that transcription elongation is involved in the reordering of nucleosomes on nascent dna .
importantly , thiolutin specifically affects nascent chromatin maturation , as total chromatin fractions from thiolutin - treated and untreated cells are nearly indistinguishable ( figures 3 , s6 , and s7 ) .
note that thiolutin has been added after the edu pulse to avoid negative effects of the inhibitor on replication and edu incorporation .
as expected , the average peak / trough ratios in thiolutin - treated cells are lower than in non - treated cells .
we can still detect slow nucleosome phasing maturation , with peak / trough ratios increasing at a similar rate in the first and fifth quintiles , even in the absence of transcription ( figure  3c ) , suggesting that there is also a transcription - independent mechanism responsible for nucleosome reorganization .
still , we can not exclude the possibility that some residual transcription occurs in thiolutin - treated cells and accounts for the observed slow chromatin maturation . in any case
, we conclude that transcription plays a central role in establishing chromatin architecture over gene bodies . in order to better understand the mechanisms involved in nucleosome positioning maturation , we repeated the 5-min edu pulse - chase experiment in mutants with deletions of hir1 , chd1 , or ioc3
chd1 and isw1a are both atp - dependent chromatin remodelers that associate with the gene body during transcription and are involved in nucleosome array organization over coding sequences ( gkikopoulos et  al . , 2011 ,
radman - livaja et  al . , 2012 , smolle et  al . ,
hir1 is a subunit of the hir nucleosome assembly complex , which participates in histone turnover and replication - independent nucleosome assembly ( green et  al . , 2005 , lopes da rosa et  al . , 2011 , ray - gallet et  al . , 2002 ) .
nucleosome phasing is globally reduced in all three mutants ( although to a somewhat lesser extent in hir1 ) compared to wild - type ( wt ) profiles ( figure  4b , left ) .
the level of disorganization of nucleosomal arrays in gene bodies is comparable between nascent and total chromatin profiles , suggesting that perturbations caused by dna replication persist after s phase in the absence of these chromatin remodelers .
more striking , however , is the difference in linker lengths between nascent and total chromatin profiles ( figure  4b , right ) .
nucleosomes appear to be less densely packed shortly after replication , with an average linker length of 20  bp compared to 13  bp in total chromatin in wt , chd1 , and ioc3 cells alike . in hir1 cells ,
total and nascent chromatin have the same 20-bp linker length , suggesting that hir activity tightens the  spacing between nucleosomes after replication , consistent with a recent report on hir1 effects on nucleosome positioning in nascent chromatin ( fennessy and owen - hughes , 2016 ) .
as detailed above , the strand separation step in our library - generation  protocol enables us to distinguish between nucleosome - positioning profiles on the leading and lagging daughter chromatids . the leading or lagging copy annotation
is assigned according to the position of a gene relative to its closest replication origin and whether its reads map to the watson or the crick strand 
watson reads upstream and downstream of an origin will be lagging and leading copies , respectively .
however , due to varying efficiencies of yeast origins ( every origin is not activated in every s phase cell ; yang et  al . , 2010 ) , leading and lagging annotations can only be unambiguously assigned to genes located near efficient origins of replication , leaving us with a set of 1,064 genes ( figures s10a and s10b ; experimental procedures ) .
nucleosome profiles of lagging and leading copies of each gene in this set and from every time point were compared to the corresponding profiles from the final ( 22  min ) time point in the 20-min edu pulse - chase experiment ( figure  2 ) .
the resulting correlation profiles were ordered according to the average difference ( for all three experiments ) in maturation indices between the lagging and the leading strand copies of each gene ( figure  5a ; table s3 ) .
we could detect comparable differences in nucleosome positioning maturation on leading and lagging copies in the three datasets at 433 genes out 1,064 , with 200 genes showing significant differences between leading and lagging copies ( figures 5b and s11 ) , suggesting that nucleosome re - positioning can occur independently on the two daughter chromatids .
there is no global effect of leading or lagging strand replication on nucleosome re - positioning because maturation indices are higher on the leading or the lagging copy in equal proportions .
lagging and leading profiles from total chromatin also show asymmetry in their correlations , albeit to a lesser extent than the nascent profiles ( figures 5a and 5e ) .
this is not due to mnase sequence bias toward the watson or the crick strands of individual genes , as all genes were compared to the corresponding watson or crick profiles from the 22-min time point standard .
it is more likely that the asymmetry we observe in total chromatin profiles comes from the substantial fraction of s phase cells , which in this experiment represents 40% of the population ( figure  s9b ) .
the relatively slower - maturing copies in the top and bottom quartile of the heatmap in figure  5a are enriched for genes in which the newly synthesized strand also serves as the template for transcription ( figure  5c ) .
this observation is consistent with at least two hypotheses : ( 1 ) edu incorporation on the template strand potentially interferes with rna pol2 initiation or elongation , thus delaying transcription - coupled chromatin maturation ( figures s10c and s10d ) or ( 2 ) asymmetric recruitment of chromatin remodeling enzymes and/or tfs to one copy , resulting in the preferential transcription of that gene copy .
for example , asymmetric transcription following replication could result from transcription preferentially occurring on the leading strand copy when the newly synthesized copy of a gene s promoter and the replication fork are oriented in the same direction , potentially as a result of the underassembly of chromatin on the lagging strand immediately behind the fork .
several observations suggest that asymmetric chromatin maturation results from differential expression of the two gene copies after replication .
first , differences between maturation indices of the leading and lagging copies are substantially reduced in the presence of thiolutin , suggesting that differences in chromatin maturation dynamics on the two copies of a gene may be due to differences in transcription rates of the two copies , i.e. , when neither gene copy is transcribed , chromatin maturation is equally slow on either copy ( figures 5d and s11 ) .
second , to test whether edu interferes with transcription when it is incorporated in the template strand , we compared steady - state mrna levels in mid and late s phase of edu - treated and untreated synchronized cells using gene expression microarrays in two independent biological replicates ( figure  6 ) .
consistent with recent studies ( voichek et  al . , 2016 ) , we find that rna levels of 95% of cell - cycle - independent genes do not change from mid to late s phase , which could be a consequence of either a 2-fold decrease in transcription rates on both copies or the expression of only one gene copy . for this group of genes ,
edu also had no effect on rna levels ( figures 6a and 6b ) , arguing against the hypothesis that asymmetric chromatin maturation is an artifact of edu effects on transcription . moreover , although a small group of 343 genes ( whose expression was not buffered after s phase ) exhibited edu - dependent inhibition of transcription ( figures 6b and 6c ) , these genes are not enriched for genes that show differences in chromatin maturation between the leading and the lagging gene copies .
the non - buffered gene set is enriched for ribosomal genes and genes involved in translation ( figure  6d ) .
we propose that , for the majority of genes , rna production is buffered after genome replication by the suppression of transcription in one of the two copies ( probably the one with the nascent strand as the transcription template ) , which we detect as a difference in transcription - dependent chromatin maturation between the two gene copies ( figure  5 ) .
future studies will focus on the mechanisms that regulate gene expression levels in the genome after replication , which should clarify whether the two gene copies are differentially transcribed following replication .
chromatin features change throughout the cell cycle , with the biggest perturbations occurring during dna replication and mitosis .
histone proteins on the maternal genome are removed from the dna ahead of the replication fork and are recycled on one or the other daughter chromatid , with newly synthesized histones restoring a full complement of nucleosomes to both daughter genomes . here
, we describe a method for following nucleosome positioning dynamics on newly replicated dna , which we call nchap .
nchap allows us to isolate nascent chromatin and follow in parallel changes in nucleosome positioning on the leading and lagging strand chromatids shortly after the passage of the replication fork . whereas other studies have concentrated on proteomic analysis of bulk nascent chromatin ( alabert et  al . , 2014 ,
2011 ) , we provide a genome - wide locus - specific timeline of nucleosome footprint changes after replication . an earlier study , which mapped okazaki fragments , reported an enrichment of okazaki fragment ends at the positions of nucleosome dyad axes ( smith and whitehouse , 2012 ) . however , it is not clear whether nucleosome assembly on the lagging strand precedes okazaki fragment ligation , as proposed . given that we find that nucleosome maturation is not significantly faster on leading or lagging strand genomes ( figure  5 ) , we conclude that any lagging strand - specific nucleosome deposition processes must occur rapidly relative to the 10-min time resolution achieved here and that the nucleosome positioning maturation process described here takes place after okazaki fragment maturation .
we propose that gene expression programs are maintained from one cell generation to the next by the formation of ndrs on daughter chromatids very early after the passage of the replication fork ( figure  7a ) . due to the heterogeneous rates of edu import in the population
, we can only conclude that promoter maturation happens within 10  min after replication fork passage , and this process most likely occurs independently of transcriptional elongation , because transcription - dependent nucleosome phasing is detected later on in our time course .
it may involve a dna sequence that  repels  nucleosomes ( such as poly a tracts ) and consequently favors the ( re)binding of tfs , chromatin remodelers , and other components of the transcription machinery that were probably present at the locus before replication . or an initially bound nucleosome may be evicted through the action of a chromatin remodeler recruited to the site by a sequence - specific tf , or the bound tf itself may prevent nucleosome binding .
these tfs / remodelers presumably then help establish the positions of the  + 1 and 1 nucleosomes .
a similar model was recently proposed for promoter architecture re - establishment after replication in drosophila cells ( ramachandran and henikoff , 2016 ) . in regions without nucleosome - repelling sequences or without sequences for available tfs ,
nucleosomes are deposited at regular intervals shortly after the passage of the replication fork . at this stage , nucleosomes are slightly delocalized ( i.e. , not exactly in the same position ) , both on the two nascent copies within the same cell and in different cells in the population , resulting in average nucleosome profiles with low peak - to - trough ratios .
remodelers that accompany the elongating rna pol2 ( chd1 or isw1a ) reorder nucleosomes in its wake and position them at more - regular intervals .
consequently , nucleosomes on genes with higher rna pol2 occupancy will be better phased over the whole population .
concomitantly , hir reduces the spacing between nucleosomes from on average 20  bp ( found in nascent chromatin ) to 13  bp ( measured in total chromatin ) .
we nevertheless found a significant number of genes with low rna pol2 occupancy and high maturation indices , which , as suggested by our results with thiolutin - treated cells , is probably due to a transcription - independent chromatin maturation process . this could be a consequence of the action of transcription - independent chromatin remodelers , although it is not clear why these remodelers only act on a subset of poorly transcribed genes .
it is also possible that , in the absence of transcription , nucleosomes are reorganized passively through statistical positioning within discrete domains located between boundaries akin to chromosomal interaction domains ( cids ) recently observed in yeast ( hsieh et  al . , 2015 ) .
alternatively , a transient burst of transcription right after replication could also be responsible for high maturation indices on these genes .
a detailed analysis of rna pol2 occupancy kinetics on nascent dna will be necessary to distinguish between these possibilities . because the nascent strand in the slower - maturing copy tends to be the transcription template strand , observed differences in maturation rates between leading and lagging gene copies could be a consequence of gene expression buffering . whereas rna pol2 progression may be impaired when it encounters edu in the template strand , transcription of both copies of the gene after replication in the absence of edu is only seen at 343
mostly highly transcribed genes ( note that these are not the same genes that exhibit copy - specific maturation behaviors ) .
the reason there is no detectable edu effect on steady - state rna levels in most genes could be a consequence of recently reported buffering effects of h3k56 acetylation by rtt109 ( voichek et  al .
, 2016 ) that potentially operate via the preferential expression of only one gene copy after replication .
we propose that the suppressed copy would generally be the one with the nascent strand as the transcription template strand .
consequently , differences in maturation rates between the two gene copies might be caused by asymmetric h3k56 acetylation and recruitment of chromatin - remodeling enzymes and/or tfs to one of the copies , which would cause differences in transcription rates and nucleosome positioning maturation rates .
interestingly , when the lagging nascent strand is the transcription template , the replication fork and rna polymerase advance in the same direction .
conversely , replication and transcription travel in opposite directions when the transcription template strand is the leading nascent strand .
it is therefore possible that tfs bound to promoters ahead of the fork rebind preferentially to the leading or lagging copy after replication as a consequence of the differential rates of okazaki fragment ligation and fork speed . as illustrated in figure  7b , it is less likely that rna pol2 ( on the yet un - replicated promoter ) and the replication fork will collide when replication and transcription travel in the same direction . as the fork travels unhindered , okazaki fragment ligation may lag behind the fork and tfs bound to the promoter ahead of the fork are more likely to rebind to the leading copy , thus favoring transcription and consequently chromatin maturation of the leading copy as observed . on the other hand , when the two travel in opposite directions , a head - on collision of the fork and rna pol2 that is ahead of the fork is more likely .
the fork then possibly stalls and slows down , and okazaki fragment ligation now happens almost simultaneously with synthesis , which allows tfs to bind to the leading or lagging copy of the promoter .
it is , however , difficult to imagine why there would be a bias toward the lagging copy as our results predict .
future studies will test whether only one gene copy is transcribed after replication and consequently find out which copy is suppressed . taken together ,
future studies should further illuminate the mechanisms responsible for rapid establishment of nucleosome positions and could potentially identify subtle consequences of slow maturation on genome function .
detailed protocols are available in the supplemental experimental procedures . all experiments ( except those in figure  s2c )
were done with the strain pv1 ( mata ade2 - 1 trp1 - 1 can1 - 1000 leu2 - 3,112 his3 - 11,15 gal psi+ rad5 + ura3::ura3/gpd - tk(7x ) aur1c::adh - hent1 bar1::kanr ) .
the experiment in figure  s2c was done with the cvy61ho strain ( mata ade2 - 1 his3 - 11,15 trp1 - 1 leu2 - 3,112 can1 - 100 bar1::hisg trp1::brdu  inc [ brdu  = hsv - tk  + hent1 ] pjh132 [ gal::ho ura3 ] ) .
mutant strains from figure  4 are as follows : hir1 : strain ac5 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 hir1::nat bar1::kanr);chd1 : strain rz12 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 chd1::leu2 ) ; andioc3 : strain rz15 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 ioc3::kanr ) .
hir1 : strain ac5 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 hir1::nat bar1::kanr ) ; chd1 : strain rz12 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 chd1::leu2 ) ; and ioc3 : strain rz15 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 ioc3::kanr ) . for the synchronization experiment in figure  1 ,
cells were arrested in g1 with  factor and then transferred into preheated media containing 10  m edu and pronase .
aliquots were taken and fixed with 1% formaldehyde right before release and then at regular intervals after release starting at 25  min from media change . in the edu - thymidine pulse - chase experiments , exponentially growing cells were transferred to media with 10  m edu preheated at 30c .
thymidine was added after 5 or 20  min and incubated for another 5 or 10  min .
cells were then pelleted and transferred into fresh media with thymidine ( and thiolutin when indicated ) , and aliquots were taken at indicated time points and fixed as above .
cells from frozen pellets were spheroplasted by bead beating in the bullet blender ( next advance ) .
spheroplasts were treated with mnase , which was adjusted to the cell density in each tube in order to obtain 80%90% mononucleosomal - sized fragments after 20  min at 37c .
after cross - link reversal , dna was extracted with phenol- chloroform - iso amyl alcohol ( pci ) , and 150-bp mono - nucleosomal sized fragments were subsequently purified from 2% agarose gels .
purified 150-bp fragments were mixed with biotin azide in a cubr solution . after a 2-hr incubation at 37c ,
biotinylated dna was incubated with streptavidin - coated magnetic beads ( blocked with salmon sperm dna ) .
following ligation with illumina genome sequencing adaptors with in - line barcodes , dna was subjected to primer ( illumina pe primer 2.0 ) extension with 2deoxyuridine 5-triphosphate ( dutp ) to separate the nascent strand from its complement .
after degradation of the dutp - containing strand with user enzyme , the nascent dna strand was pcr amplified .
paired - end sequencing was done on a hiseq 2000 ( illumina ; cnag ) or on a next seq sequencer ( illumina ) in o.j.r.s laboratory .
measurements were made with facscalibur ( bd biosciences ; fl-1 filter ; forward scattered light [ fcs ] size cutoff : 70 ) .
genomic dna was isolated from g1-arrested flash - frozen cell pellets and sonicated with the bioruptor pico cup sonicator .
cells were released into s phase in media with or without 10  m edu , as above .
fifty - milliliter aliquots were flash frozen in liquid nitrogen 32 and 40  min after release for rna isolation .
total rna was isolated from frozen pv1 cell pellets with trizol and treated with dnase i. rna was reverse transcribed using oligodt as primers .
the resulting cdna was dye coupled with cy5 or cy3 n - hydroxysuccinimide ( nhs ) esters and purified as described previously ( liu et  al . , 2005 ) .
the cy5- or cy3-labeled cdna was mixed with cy3- or cy5-labeled genomic dna , respectively , and hybridized to agilent 8x15k yeast gene expression arrays .
images were scanned with the innoscan 710 microarray scanner ( innopsys ) and processed with the mapix software .
data were normalized by dividing the cy5/cy3 ratio for each probe with the average cy5/cy3 ratio for the whole array .
sequences were aligned to the s.  cerevisiae genome using blast - like alignment tool ( blat ) .
we kept reads that had at least one uniquely aligned 100% match in the paired - end pair .
read count distribution was normalized to one by dividing each base pair count with the genome - wide average base - pair count .
genes that are replicated from efficient origins were filtered as follows : ( 1 ) efficient origins were defined as origins whose read density peak heights were  0.6 at the 25-min time point in the experiment from figure  1 .
( 2 ) genes that were within the boundaries of the read density area around efficient origins at the 25-min time point were considered as being replicated from that particular origin in most cells .
analysis was done using in - house perl and r scripts ( available upon request ) .
the analysis in figures s6 , s7 , and s11 was performed using perl ( statistics::ttest ) and r scripts as detailed in the supplemental experimental procedures and figures s6 , s7 , and s11 .
m.r .- l . developed nchap , designed the experiments , performed the experiments in figure  s2 , developed analysis tools , analyzed the data , and wrote the manuscript .
o.j.r . initiated the development of nchap , advised on experimental design and data analysis , and wrote the manuscript .
 summarychromatin is thought to carry epigenetic information from one generation to the next , although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication . here 
 , we measure a key aspect of chromatin structure dynamics during replication  how rapidly nucleosome positions are established on the newly replicated daughter genomes . by isolating newly synthesized dna marked with 5-ethynyl-2-deoxyuridine ( edu ) 
 , we characterize nucleosome positions on both daughter genomes of s.  cerevisiae during chromatin maturation . 
 we find that nucleosomes rapidly adopt their mid - log positions at highly transcribed genes , which is consistent with a role for transcription in positioning nucleosomes in  vivo . 
 additionally , experiments in hir1 mutants reveal a role for hir in nucleosome spacing . 
 we also characterized nucleosome positions on the leading and lagging strands , uncovering differences in chromatin maturation dynamics at hundreds of genes . 
 our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions