MOLECULAR BIOLOGY MCM2 promotes symmetric inheritance of modified histones during DNA replication Nataliya Petryk 1,2 *, Maria Dalby 3 *, Alice Wenger 1,2 , Caroline B. Stromme 1 †, Anne Strandsby 1 ‡, Robin Andersson 3 §, Anja Groth 1,2 § During genome replication, parental histones are recycled to newly replicated DNA with their posttranslational modifications (PTMs). Whether sister chromatids inherit modified histones evenly remains unknown. We measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading-strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry.This work reveals how histones are inherited to sister chromatids and identifies a mechanism by which the replication machinery ensures symmetric cell division. H istone posttranslational modifications (PTMs) contribute to the establishment and maintenance of epigenetic chromatin states that regulate transcriptional pro- grams during development (1, 2), but the mechanisms that ensure transmission of his- tone PTM patterns to daughter cells remain un- clear. Chromatin is disrupted upon replication fork passage, and nucleosomes are rapidly re- assembled on newly synthesized DNA through recycling of evicted parental histones and de novo deposition of new histones (3, 4). The re- cycling of modified parental histones is a critical step in histone PTM transmission (5), and early studies suggested that parental histones segre- gate randomly to both daughter DNA strands (6, 7). However, whether histone PTM inheri- tance is truly symmetric and how parental his- tones are segregated to the leading and lagging strands of the replication fork remain open ques- tions. Multiple replication origins are used to replicate large metazoan chromosomes, and rep- lication fork directionality (RFD) and leading- and lagging-strand replication therefore alternate along chromosomes (8, 9). Potential biases in seg- regation of modified parental histones during replication will thus result in a specific pattern of sister chromatid asymmetry. We investigated the distribution of parental and new histones on sister chromatids and linked this distribution to RFD to understand histone segregation. We developed SCAR-seq (sister chromatids after replication by DNA sequencing) to track his- tone recycling and de novo deposition genome- wide (Fig. 1A and methods). We differentiated old and new histones H4 by dimethylation at lysine 20 (H4K20me2) (fig. S1A), present exclusively on >80% of old H4 in nascent chromatin (5, 10), and acetylation at lysine 5 (H4K5ac), present on >95% of new H4 (3, 11). Mouse embryonic stem cells were labeled with 5-ethynyl-2′-deoxyuridine (EdU), and nascent mononucleosomes carrying H4K20me2 or H4K5ac were purified sequentially by chromatin immunoprecipitation (ChIP) and streptavidin capture of biotinylated EdU-labeled DNA. The new and parental DNA strands were separated (fig. S1B) and sequenced in a strand- specific manner to score genome-wide sister chro- matid histone partition (Fig. 1A and fig. S1C). To determine locally which sister chromatid was replicated preferentially by the leading strand, we measured RFD by Okazaki fragment sequencing (OK-seq) (methods) (8). Replication initiation zones (n = 2,844) (fig. S2A) were com- parable to those in humans ( 8) and Caenorrhabditis elegans (12), ranging in size and efficiency (fig. S2, B and C), and were mostly intergenic (fig. S2D), enriched in enhancer-associated features [H3 acetylation at lysine 27 (H3K27ac), H3 monometh- ylation at lysine 4 (H3K4me1), p300 occupancy, and deoxyribonuclease I–hypersensitive sites], and flanked by active genes [marked by H3 trimethyl- ation at lysine 36 (H3K36me3) and lysine 4 (H3K4me3)] (fig. S2E). Around initiation zones, the partition of old and new H4 showed a weak reciprocal shift, with H4K20me2 and H4K5ac skewed toward leading- and lagging-strand rep- lication, respectively (Fig. 1, B to D, and fig. S3, A to C). The partition amplitude was considerably lower than RFD, suggesting that old histones segregate to both strands but not entirely sym- metrically. Analysis of the parental DNA strands showed the complementary partition shift (fig. S3, D and E), excluding an effect of EdU on partition measurements. The partition skew was most pro- nounced around highly efficient initiation zones (fig. S3F), indicating that DNA replication drives the observed sister chromatid asymmetries. His- tone partition skew also tracked with RFD at higher genomic scales (fig. S4)—for example, across replication units with early-replicating borders and late-replicating centers, termed U-domains (8, 9, 13). Together, these results dem- onstrate that parental histones segregate to both arms of the replication fork with a slight pref- erence for the leading strand, whereas de novo deposition has a comparable bias toward the lagging strand. Replication timing is related to chromosome organization in topologically associating domains (TADs) (9, 14, 15). TAD borders (16) are enriched in initiation zones (fig. S5A) (8, 13) and showed a reciprocal histone partition skew (Fig. 2, A and B, and fig. S5B). B-compartment TADs (transcrip- tionally inactive) displayed stronger RFD and partition shifts than transcriptionally active A- compartment TADs (17) (Fig. 2A), possibly be- cause of increased internal initiation within active TADs (Fisher’s exact test, odds ratio 2.2, P < 2.2 × 10 -16 ) or an effect of transcription. To investigate partition asymmetries over genes, we tracked H3K36me3 (fig. S5C) present on pa- rental histones in gene bodies ( 5, 18, 19). H3K36me3 partition skewed moderately toward leading- strand replication, consistent with H4K20me2 partition (Fig. 2A and fig. S5, C to E), and was stronger over active genes and codirectional with transcription (Fig. 2C) (8). Further, the correla- tion of chromatin interaction directionality with histone partition was weaker than the correlation between chromatin interaction directionality and RFD (Fig. 2B), suggesting that although histone partitioning is driven by RFD it can be affected by transcription. Active enhancers often coincided with initiation zone centers, and promoters tended to be flanking (20) (fig. S5F), suggesting that en- hancer activity affects partitioning in neighbor- ing regions. Consistently, both RFD and histone partition asymmetries were greater around active enhancers (21) and super-enhancers that control cell type–specific genes (22) (Fig. 2D) (Mann- Whitney U test, P < 1.1 × 10 -16 ). MCM2, part of the replicative helicase, is proposed to recycle parental histone H3-H4 via its N-terminal histone-binding domain (HBD) ( 11, 23–25). Using genome editing, we mutated two critical residues in the HBD [Tyr 81 →Ala (Y81A) and Tyr 90 →Ala (Y90A), yielding MCM2-2A] (24, 25) (fig. S6A) that disrupt histone binding (fig. S6B) (25) without affecting cell cycle progres- sion (fig. S6C). Notably, in MCM2-2A mutants, partition of old and new histones was strongly skewed toward leading and lagging strands, re- spectively, generating partition ratios similar to RFD in amplitude and pattern (Fig. 3, A to C, and figs. S6D and S7). Moreover, partition of new and RESEARCH Petryk et al., Science 361, 1389–1392 (2018) 28 September 2018 1 of 3 1 Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. 2 Novo Nordisk Foundation Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. 3 The Bioinformatics Centre, Department of Biology, Faculty of Science, University of Copenhagen, 2200 Copenhagen, Denmark. *These authors contributed equally to this work. †Present address: Novo Nordisk, 2860 Soborg, Denmark. ‡Present address: Virus and Microbiological Special Diagnostics, Statens Serum Institut, 2300 Copenhagen S, Denmark. §Corresponding author. Email: [email protected] (R.A.); [email protected] (A.G.) on November 3, 2020 http://science.sciencemag.org/ Downloaded from
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MOLECULAR BIOLOGY
MCM2 promotes symmetricinheritance of modified histonesduring DNA replicationNataliya Petryk1,2*, Maria Dalby3*, Alice Wenger1,2, Caroline B. Stromme1†,Anne Strandsby1‡, Robin Andersson3§, Anja Groth1,2§
During genome replication, parental histones are recycled to newly replicated DNA withtheir posttranslational modifications (PTMs). Whether sister chromatids inherit modifiedhistones evenly remains unknown. We measured histone PTM partition to sisterchromatids in embryonic stem cells. We found that parental histones H3-H4 segregateto both daughter DNA strands with a weak leading-strand bias, skewing partition attopologically associating domain (TAD) borders and enhancers proximal to replicationinitiation zones. Segregation of parental histones to the leading strand increased markedlyin cells with histone-binding mutations in MCM2, part of the replicative helicase,exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones areinherited to sister chromatids and identifies a mechanism by which the replicationmachinery ensures symmetric cell division.
Histone posttranslational modifications(PTMs) contribute to the establishmentand maintenance of epigenetic chromatinstates that regulate transcriptional pro-grams during development (1, 2), but the
mechanisms that ensure transmission of his-tone PTM patterns to daughter cells remain un-clear. Chromatin is disrupted upon replicationfork passage, and nucleosomes are rapidly re-assembled on newly synthesized DNA throughrecycling of evicted parental histones and denovo deposition of new histones (3, 4). The re-cycling of modified parental histones is a criticalstep in histone PTM transmission (5), and earlystudies suggested that parental histones segre-gate randomly to both daughter DNA strands(6, 7). However, whether histone PTM inheri-tance is truly symmetric and how parental his-tones are segregated to the leading and laggingstrands of the replication fork remain open ques-tions. Multiple replication origins are used toreplicate large metazoan chromosomes, and rep-lication fork directionality (RFD) and leading-and lagging-strand replication therefore alternatealong chromosomes (8, 9). Potential biases in seg-regation of modified parental histones duringreplication will thus result in a specific patternof sister chromatid asymmetry. We investigated
the distribution of parental and new histones onsister chromatids and linked this distribution toRFD to understand histone segregation.We developed SCAR-seq (sister chromatids
after replication by DNA sequencing) to track his-tone recycling and de novo deposition genome-wide (Fig. 1A andmethods).We differentiated oldand new histones H4 by dimethylation at lysine20 (H4K20me2) (fig. S1A), present exclusively on>80% of old H4 in nascent chromatin (5, 10), andacetylation at lysine 5 (H4K5ac), present on >95%of new H4 (3, 11). Mouse embryonic stem cellswere labeled with 5-ethynyl-2′-deoxyuridine(EdU), and nascent mononucleosomes carryingH4K20me2 orH4K5acwere purified sequentiallyby chromatin immunoprecipitation (ChIP) andstreptavidin capture of biotinylated EdU-labeledDNA. The new and parental DNA strands wereseparated (fig. S1B) and sequenced in a strand-specificmanner to score genome-wide sister chro-matid histone partition (Fig. 1A and fig. S1C).To determine locally which sister chromatid
was replicated preferentially by the leadingstrand, we measured RFD by Okazaki fragmentsequencing (OK-seq) (methods) (8). Replicationinitiation zones (n = 2,844) (fig. S2A) were com-parable to those inhumans (8) andCaenorrhabditiselegans (12), ranging in size and efficiency (fig. S2,B and C), and were mostly intergenic (fig. S2D),enriched in enhancer-associated features [H3acetylation at lysine 27 (H3K27ac),H3monometh-ylation at lysine 4 (H3K4me1), p300 occupancy,anddeoxyribonuclease I–hypersensitive sites], andflanked by active genes [marked by H3 trimethyl-ation at lysine 36 (H3K36me3) and lysine 4(H3K4me3)] (fig. S2E). Around initiation zones,the partition of old and newH4 showed a weakreciprocal shift, with H4K20me2 and H4K5acskewed toward leading- and lagging-strand rep-lication, respectively (Fig. 1, B to D, and fig. S3, Ato C). The partition amplitude was considerably
lower than RFD, suggesting that old histonessegregate to both strands but not entirely sym-metrically. Analysis of the parental DNA strandsshowed the complementary partition shift (fig. S3,D and E), excluding an effect of EdU on partitionmeasurements. The partition skew was most pro-nounced around highly efficient initiation zones(fig. S3F), indicating that DNA replication drivesthe observed sister chromatid asymmetries. His-tone partition skew also tracked with RFD athigher genomic scales (fig. S4)—for example,across replication units with early-replicatingborders and late-replicating centers, termedU-domains (8, 9, 13). Together, these results dem-onstrate that parental histones segregate to botharms of the replication fork with a slight pref-erence for the leading strand, whereas de novodeposition has a comparable bias toward thelagging strand.Replication timing is related to chromosome
organization in topologically associating domains(TADs) (9, 14, 15). TAD borders (16) are enrichedin initiation zones (fig. S5A) (8, 13) and showed areciprocal histone partition skew (Fig. 2, A and B,and fig. S5B). B-compartment TADs (transcrip-tionally inactive) displayed stronger RFD andpartition shifts than transcriptionally active A-compartment TADs (17) (Fig. 2A), possibly be-cause of increased internal initiation withinactive TADs (Fisher’s exact test, odds ratio 2.2,P < 2.2 × 10−16) or an effect of transcription. Toinvestigate partition asymmetries over genes,we tracked H3K36me3 (fig. S5C) present on pa-rental histones in genebodies (5, 18, 19).H3K36me3partition skewed moderately toward leading-strand replication, consistent with H4K20me2partition (Fig. 2A and fig. S5, C to E), and wasstronger over active genes and codirectional withtranscription (Fig. 2C) (8). Further, the correla-tion of chromatin interaction directionality withhistone partitionwasweaker than the correlationbetween chromatin interaction directionality andRFD (Fig. 2B), suggesting that although histonepartitioning is driven byRFD it can be affected bytranscription. Active enhancers often coincidedwith initiation zone centers, andpromoters tendedto be flanking (20) (fig. S5F), suggesting that en-hancer activity affects partitioning in neighbor-ing regions. Consistently, both RFD and histonepartition asymmetries were greater around activeenhancers (21) and super-enhancers that controlcell type–specific genes (22) (Fig. 2D) (Mann-Whitney U test, P < 1.1 × 10−16).MCM2, part of the replicative helicase, is
proposed to recycle parental histone H3-H4 viaits N-terminal histone-binding domain (HBD)(11, 23–25). Using genome editing,wemutated twocritical residues in the HBD [Tyr81→Ala (Y81A)and Tyr90→Ala (Y90A), yielding MCM2-2A](24, 25) (fig. S6A) that disrupt histone binding(fig. S6B) (25) without affecting cell cycle progres-sion (fig. S6C). Notably, in MCM2-2A mutants,partition of old and new histones was stronglyskewed toward leading and lagging strands, re-spectively, generating partition ratios similar toRFD in amplitude and pattern (Fig. 3, A to C, andfigs. S6D and S7). Moreover, partition of new and
RESEARCH
Petryk et al., Science 361, 1389–1392 (2018) 28 September 2018 1 of 3
1Biotech Research and Innovation Centre (BRIC), Faculty ofHealth and Medical Sciences, University of Copenhagen,2200 Copenhagen, Denmark. 2Novo Nordisk FoundationCenter for Protein Research (CPR), Faculty of Health andMedical Sciences, University of Copenhagen, 2200Copenhagen, Denmark. 3The Bioinformatics Centre,Department of Biology, Faculty of Science, University ofCopenhagen, 2200 Copenhagen, Denmark.*These authors contributed equally to this work. †Present address:Novo Nordisk, 2860 Soborg, Denmark. ‡Present address: Virus andMicrobiological Special Diagnostics, Statens Serum Institut, 2300Copenhagen S, Denmark.§Corresponding author. Email: [email protected] (R.A.);[email protected] (A.G.)
old histones showed strong anticorrelation inMCM2-2A (Fig. 3D and fig. S7D), indicating seg-regation to opposite strands. H3K36me3 occu-pancywas not altered inMCM2-2A cells (fig. S8,A and B), indicating that histone partitioningrather than recycling was perturbed. The associ-ation betweenH3K36me3partition and transcrip-tional directionality was reduced in MCM2-2Acells (fig. S8, C to E), further indicating increasedreplication-driven sister chromatid asymmetryin parental and new histones. Sister chromatidasymmetry was also strongly increased at TADborders, around enhancers (fig. S8, F and G),
and across important developmental loci (e.g., theHox clusters) (fig. S9) in MCM2-2A cells, whichthus provides a model to address histone PTMinheritance in development. The high correla-tion betweenH4K20me2 partition inMCM2-2Acells and RFD prompted us to map H4K20me2partition break points. They showed strong co-localizationwith initiation zonesmapped by OK-seq (Fig. 3E andmethods), suggesting H4K20me2SCAR-seq in MCM2-2A as a method to mapreplication dynamics.In summary, SCAR-seq revealed that parental
histone segregation is almost symmetrical, with
a weak inherent preference for the leading strand(Fig. 3F, left), creating modest sister chromatidasymmetries that might be mitotically trans-mitted as new histones acquire PTMs with slowkinetics (5). Importantly, MCM2 histone chap-erone activity promotes balanced segregationof old histones to leading and lagging strands,thereby ensuring inheritance of histone-basedinformation to both sister chromatids. This isconsistent with chaperoning of old histones byMCM2 (11, 23) and cryo–electron microscropydata placing the MCM2 HBD in front of thefork (26, 27). We envisage that MCM2 recycles
Petryk et al., Science 361, 1389–1392 (2018) 28 September 2018 2 of 3
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Fig. 2. TAD borders and genes flanking active enhancers show skewed histone PTM inheritance to sisterchromatids. (A) Average RFD and partition of H4K5ac, H4K20me2, and H3K36me3 across scaled TADs split bycompartment class (16, 17). (B) Spearman correlation of Hi-C directionality index (16) with RFD, histone PTMpartition [colors as in (A)], and transcriptional directionality measured by precision nuclear run-on (PRO-seq)(29). (C) Density distribution of H3K36me3 partition and RFD in active and inactive forward (red)- and reverse(blue)-strand genes and intergenic regions. (D) Average RFD and histone PTM partition centered at enhancers(21) and super-enhancers (22).
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Fig. 1. Parental histones segregate to both sister chromatids with aweak bias toward the leading strand. (A) SCAR-seq technique. Partitionof old and new histones is calculated as the proportion of forward (F) (red)and reverse (R) (blue) counts in genomic windows [the range is between−1 (100% reverse strand) and 1 (100% forward strand)]. (B) RFD andpartition of H4K20me2 and H4K5ac at a genomic region. Initiation zonecenters (lines), active gene orientation (arrowheads), and active enhancers
(bars) are shown. Chr3, chromosome 3. RFD is calculated as the proportionbetween right- and left-moving replication forks [the range is between −1(100% left moving) and 1 (100% right moving)]. (C) Average RFD (blue) andpartition of old (H4K20me2) and new (H4K5ac) histones around initiationzones. (D) Partition at downstream (leading strand) and upstream (laggingstrand) edges of initiation zones, with significant partition difference in eachreplicate (rep) (paired Wilcoxon signed-rank test, P < 5.3 × 10−14).
parental histones to the lagging strand (Fig. 3F,right), whereas a separate pathway depositsparental histones on the leading strand. In thisvein, it is conceivable that histone segrega-tion can be regulated during development todrive asymmetric cell fates (28).
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We thank P. Lansdorp for initial discussions on SCAR-seq;K. Stewart-Morgan and C. Hammond for comments on themanuscript; A. Fossum for help with fluorescence-activated cellsorting; and the Groth, Andersson, and Brakebusch laboratories fordiscussions. Funding: Research in the Groth lab was supportedby the Independent Research Fund Denmark (4092-00404), theEuropean Research Council (CoG no. 724436), the Novo NordiskFoundation (NNF14OC0012839), the Lundbeck Foundation(R198-2015-269), and the Danish Cancer Society. Research inthe Andersson lab was supported by the Independent ResearchFund Denmark (6108-00038B) and the European Research Council(StG no. 638173). Author contributions: N.P. and A.G. conceivedof the project and designed experiments. N.P. developed andperformed SCAR-seq. M.D. and R.A. developed computationalmethods. M.D. performed computational analyses with supportfrom N.P. and R.A. C.B.S. and A.S. performed genome editing.A.W. characterized MCM2-2A cells and assisted with SCAR-seq.A.G. and R.A. supervised the project. N.P. and M.D. wrote themanuscript with input from A.G., R.A., and A.W. Competinginterests: The authors declare no competing interests. Data andmaterials availability: Data have been deposited to NCBI GEOunder accession number GSE117274.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/361/6409/1389/suppl/DC1Materials and MethodsFigs. S1 to S9Tables S1 to S3References (30–42)Data S1
30 April 2018; accepted 6 August 2018Published online 16 August 201810.1126/science.aau0294
Petryk et al., Science 361, 1389–1392 (2018) 28 September 2018 3 of 3
Fig. 3. MCM2 histone binding is required for parental histone recycling to the lagging strand.(A) Average RFD and partition of old (H4K20me2 and H3K36me3) and new (H4K5ac) histones inthe wild type (WT) (solid lines) and MCM2-2A (dashed lines) around initiation zones. (B) RFD andhistone PTM partition at a genomic region in the WT and MCM2-2A. Initiation zone centers (lines),active gene orientation (arrowheads), and active enhancers (bars) are shown. (C) Scatterplots ofRFD and histone PTM partition in the WT and MCM2-2A. Spearman’s rank correlation coefficientis shown in the top left corner of each plot. (D) Scatterplot of H4K20me2 versus H4K5ac partitionin MCM2-2A. Spearman’s rank correlation coefficient is shown in the top right corner. (E) Fraction ofinitiation zones with the nearest distance to predicted H4K20me2 partition break points or randomH4K20me2 bins in the WTand MCM2-2A. Horizontal dotted lines represent random mean fractions.(F) Model for segregation of parental histones H3-H4 in WT and MCM2-2A cells.
MCM2 promotes symmetric inheritance of modified histones during DNA replicationNataliya Petryk, Maria Dalby, Alice Wenger, Caroline B. Stromme, Anne Strandsby, Robin Andersson and Anja Groth
originally published online August 16, 2018DOI: 10.1126/science.aau0294 (6409), 1389-1392.361Science
, this issue p. 1386, p. 1389; see also p. 1311Sciencein mouse cells, the replicative helicase MCM2 counters the leading-strand bias.were different. Yeasts use subunits of DNA polymerase to prevent the lagging-strand bias of parental histones, whereas
mousePerspective by Ahmad and Henikoff). However, the mechanisms ensuring this symmetric inheritance in yeast and therespectively, that modified histones are segregated to both DNA daughter strands in a largely symmetric manner (see
found in mouse and yeast,et al. and Petryk et al.do the two sister chromatids inherit modified histones equally? Yu Parental histones with modifications are recycled to newly replicated DNA strands during genome replication, but
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