www.sciencemag.org/cgi/content/full/science.

aau0294/DC1

Supplementary Materials for

MCM2 promotes symmetric inheritance of modified histones during
DNA replication
Nataliya Petryk*, Maria Dalby*, Alice Wenger, Caroline B. Stromme, Anne Strandsby,
Robin Andersson†, Anja Groth†
*These authors contributed equally to this work.
†Corresponding author. Email: robin@binf.ku.dk (R.A.); anja.groth@bric.ku.dk (A.G.)
Published 16 August 2018 on Science First Release
DOI: 10.1126/science.aau0294

This PDF file includes:

Materials and Methods

Figs. S1 to S9
Tables S1 to S3
Caption for Data S1
References

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.aau0294/DC1)

Data S1
 Submitted Manuscript: Confidential

Materials and Methods

Cell culture

5 Mouse embryonic stem cells E14TG2a (kind gift from K. Helin lab) were grown on gelatin-

coated dishes in 2i/LIF media (30) at 37 °C with 5 % CO2. Custom-made 2i media (2i SILAC,

Gibco), containing a 1:1 mix of DMEM/F-12 and Neurobasal media, 2 mM Glutamax and 1 mM

sodium pyruvate but depleted for L-arginine and L-lysine, was supplemented with 50 µM beta-

mercaptoethanol, 1x Penicillin-Streptomycin (Gibco), N-2 and B-27 supplements (Gibco), 3

10 µM GSK3 inhibitor (CHIR99021), 1 µM MEK inhibitor (PD0325901), Leukemia Inhibitory

Factor (LIF; homemade by K. Helin lab), 548 µM L-arginine hydrochloride (Sigma) and 648

µM L-lysine hydrochloride (Sigma). Cells were passaged using Trypsin-EDTA (Gibco) and

Soybean Trypsin inhibitor (Gibco) in PBS (final concentration 0.25 mg/mL). Cells were

routinely tested for mycoplasma contamination.

15

SCAR-seq

Step-by-step scheme of the protocol is shown in Fig. 1A. Cells were grown to 70-80%

confluency and EdU (Jena Bioscience) was added with a final concentration of 10 µM for 15 or

20 30 minutes as indicated (data S1). Cells were washed with ice-cold Phosphate-Buffered Saline

(PBS), harvested by scraping and collected by centrifugation at 300 g for 10 minutes at 4°C.

Native ChIP was performed essentially as described (31) with following modifications. MNase

(Warthington) was added at 30°C for 20 minutes at final concentration of 2 U per 106 cells. For

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each IP, 50 µg of chromatin (measured as DNA concentration) and 16 µg of antibody was used

(table S1). H4K20me2 antibody (Diagenode, C15200205) specificity was validated by dot blot

(Fig. S1A) as well as by the manufacturer (dot blot on peptide array). H4K5ac antibody (Abcam

ab51997) specificity was validated by the manufacturer (dot-blot, peptide array and ELISA).

5 H3K36me3 antibody (Abcam ab9050) specificity was validated of a peptide array by the

manufacturer and correlation of K36me3 SCAR-seq enrichment with ENCODE H3K36me3.

Immunoprecipitated chromatin fragments were washed 3 times with 500 µL of low salt washing

buffer (TRIS-HCl pH 8.0 20 mM, EDTA 2 mM, NaCl 150 mM, Triton X100 1 %, SDS 0,1 %), 3

times with 500 µL of high salt washing buffer (TRIS-HCl pH 8.0 20 mM, EDTA 2 mM, NaCl

10 500 mM, Triton X100 1 %, SDS 0,1 %) and subsequently eluted in 200 µl ChIP elution buffer

(TRIS-HCl pH 8.0 20 mM, EDTA 10 mM, SDS 1 %) for 30 minutes at 37°C, purified with

Minelute Reaction Clean-up column (Qiagen) and eluted into 50 µl of EB (10mM Tris-HCl pH

8,5). All buffers for chromatin preparation and ChIP were supplied with 10 µg/mL of leupeptin,

10 µg/mL of pepstatin, 10 µg/mL of aprotonin and 1 mM PMSF. For H4K5ac SCAR-seq,

15 chromatin preparation and ChIP buffers were additionally supplied with trichostatin A (Sigma) at

final concentration 1 µg/mL.

Mononucleosomal-sized DNA fragments were dual size-selected with AMPure® XP reagent

(Beckman Coulter) with a bead ratio 0,8-3:1 according to manufacturer protocol and eluted into

50 µl of water. EdU DNA fragments were biotinylated in 100 µl of click reaction for 45 minutes

20 supplied with 1x Click-it reaction buffer (Invitrogen), 0,5 mM Biotin-TEG-azide

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(Berry&associates), 0,5 mM THPTA (Sigma), 0,1 mM CuSO4 (Invitrogen), 10 mM Sodium

ascorbate (Jena Bioscience) for 45 minutes at room temperature (modified from (32))

Sequencing adapter ligation. End repair, A-tailing and adapter ligation was performed using

KAPA Hyper prep kit (Roche) supplied with 15 µM Illumina TruSeq adapters purchased from

5 Pentabase.

Isolation of replicated double-stranded DNA fragments from library. Biotinylated fragments

were captured with 200 µg of Dynabeads® MyOne™ Streptavidin T1 according to the

manufacturer's protocol (Thermo) and washed sequentially with 200 µl of the following buffers

at room temperature: 4 times with 1x B&WT buffer (5 mM Tris HCl pH 7,5, 0,5 mM EDTA, 1

10 M NaCl, 0,05 % Tween 20), once with 2x B&WT buffer.

Isolation of nascent EdU strands. Dynabeads with captured biotinylated library fragments were

sequentially washed 3 times with alkaline buffer (0.1 M NaOH, 0,05 % Tween 20) for 1 minute,

twice with 1x B&WT, twice with EBT (10 mM Tris-HCl pH 8,5, 0,05 % Tween 20) and

resuspended in 20 µL of EB.

15 Isolation of replicated parental strands. The pH of the first alkaline wash supernatant was

neutralized by adding acetic acid to a final concentration of 0.1 M and 2 mM EDTA pH 8.0,

following by purification on Amicon Ultra 10k centrifuging filters (Millipore).

Library amplification and sequencing. Libraries were amplified using KAPA Hyper prep kit in

11 cycles of PCR using either bead suspension or purified parental strand as a template.

20 Mononucleosomal-sized library fragments were dual size-selected with AMPure® XP reagent

(Beckman Coulter) with a bead ratio 0,77-0,95:1 according to manufacturer protocol and eluted

into 15 µl of EB. Libraries were quantified with qBIT High-sensitivity DNA kit (Thermo) and

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run on Bioanalyzer High-sensitivity DNA kit chip for quality control. Sequencing was performed

on NextSeq 500 (Illumina) using NextSeq 500/550 High Output v2 kits (FC-404-2005).

Input samples. As inputs, the nascent EdU strands were purified from MNase-digested

chromatin, following SCAR-seq protocol but omitting the histone ChIP step. Briefly, MNase-

5 digested mononucleosomal-sized fragments were purified. EdU-labeled DNA fragments were

biotinylated in click-it reaction. Subsequently end repair, A-tailing and adapter ligation were

performed, followed by streptavidin capture and alkaline isolation of nascent EdU-strands.

Libraries were amplified in PCR and size-selected. Inputs were prepared for each cell line and

EdU pulse duration as SCAR-seq.

10 QPCR test of EdU-labeled strand isolation and antibody validation. Efficiency of EdU strand

isolation by alkaline denaturation was verified in qPCR with a hemi-EdU-nylated library. Briefly

equimolar quantities of oligonucleonucleotides 1 and 2 were mixed and heated for 30 seconds at

94°C, followed by incubation for 45 s at 55 °C and 30 minutes at 72 °C for annealing and second

strand synthesis with Taq polymerase (NEB) in presence of 0,2 mM of each dATP, dCTP, dGTP

15 (Thermo), 0,06 mM dTTP (Thermo), and 0,14 mM EdUTP (Jena Bioscience). Hemi-EdU-

nylated double stranded DNA fragment was purified with Minelute Reaction Clean-up column

(Qiagen) and library constructed followed SCAR-seq protocol except that a half of the sample

was processed without alkaline washes (no NaOH). QPCR was performed with primer pairs

(table S2) in triplicates with Sybrgreen I Mastermix in 480 Lightcycler (Roche) according to

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manufacturer’s recommendations. Partition was calculated as a ratio of quantification of qPCR

products as indicated (Fig. S1B)

H4K20me2 (Diagenode, C15200205) dot blots were performed with 1 µg of peptides (JPT)

(antibody dilution 1:2000) according to standard protocols.

OK-seq

Purification of Okazaki fragments was performed essentially as described previously (8) except

that Click-it reaction conditions were set up as in SCAR-seq. Sequencing was performed on

10 NextSeq 500 (Illumina) using NextSeq 500/550 High Output v2 kits (FC-404-2005).

Genome editing

The histone-binding domain of the endogenous MCM2 gene was disabled by mutation of two

15 codons that changed tyrosine 81 and 90 to alanine residues (Fig. S6A) (25) using a CRISPR-

Cas9 protocol modified from (24). Briefly, cells were reverse transfected with SpCas9-sgRNA-

2A-GFP (addgene # 48138) and a single-stranded oligonucleotide donor (table S2) (Integrated

DNA Technologies) using Lipofectamine 2000 reagent (Invitrogen). GFP-positive single cells

were sorted into 96-well plates 48 h post-transfection (BD FACSAria I Cell Sorter) and cultured

20 for 10 days. Individual clones were expanded in 96-well plates and 24-well plates. After 2 days

genomic DNA from cells in 24-well plates was extracted using QuickExtract DNA Extraction

Solution (Epicentre) and used for genotyping. The genomic region around the mutated sites was

amplified by PCR using Taq 2x Master Mix (NEB) and primers 3 and 4 (TAG Copenhagen)

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(table S2). PCR products were digested with restriction enzyme AatII (NEB) and analyzed by

agarose gel electrophoresis. Positive clones were verified by sequencing. Cells were tested

negative for mycoplasma contamination.

5 Protein extraction and co-immunoprecipitation

For extracts, cells from four 60-80 % confluent 15 cm dishes were washed in PBS and pelleted.

For high salt extracts, cells were resuspended in 800 µL NP40-NaCl buffer (300 mM NaCl, 0,5

% NP-40, 50 mM Tris, pH 7,8, 0,2 mM EDTA, 5 % glycerol, 1 mM DTT, 10 µg/mL leupeptin,

10 10 µg/mL pepstatin, 0,1 mM PMSF, 0,2 mM Na3VO4, 5 mM Na fluoride, 10 mM β-

glycerolphosphate) and proteins were extracted for 15 minutes on ice. Insoluble material was

removed by centrifugation at 11000 g for 10 minutes at 4 °C. For DNase I extracts, cell pellets

were resuspended in 800 µL CSK-T buffer (10 mM PIPES, pH 7, 100 mM NaCl, 300 mM

Sucrose, 3 mM MgCl2, 0.5 % Triton X-100, 1 mM ATP, 1 mM DTT, 10 µg/mL leupeptin, 10

15 µg/mL pepstatin, 0,1 mM PMSF, 0,2 mM Na3VO4, 5 mM Na fluoride, 10 mM β-

glycerolphosphate) and soluble proteins removed by extraction 10 minutes on ice. Chromatin

was collected by centrifugation at 1500 g for 5 minutes at 4 °C, resuspended in 800 µL CSK-T

buffer (as above but with 0.1 % Triton X-100) and incubated with DNase I (1000 U/mL, Roche)

at 25 °C for 30 minutes. DNase I-released material (solubilized chromatin) was collected after

20 centrifugation at 16000 g for 10 minutes at 4 °C. For immunoprecipitation, extracts (4 mg high

salt extracts or 2 mg of solubilized chromatin) were pre-cleared with 50 µL BSA-blocked (10

mg/mL BSA in 1,5 mM Tris, pH 7.8) Protein A-Sepharose beads (GE Healthcare) for 40 minutes

at 4 °C, followed by incubation with 50 µL antibody-coupled BSA-blocked beads (0,25 µg

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MCM2 antibody per 1 mg extract, table S1) for 4 hours at 4 °C. Control rabbit IgGs (1 µg, Santa

Cruz) were coupled for the control pull-down. The beads were washed five times with 800 µL

wash buffer (300 mM NaCl, 0,2 % NP-40, 50 mM Tris, pH 7,8, 0.2 mM EDTA, 5 % glycerol, 1

mM DTT, 10 µg/mL leupeptin, 10 µg/mL pepstatin, 0,1 mM PMSF, 0,2 mM Na3VO4, 5 mM Na

5 fluoride, 10 mM β-glycerolphosphate) and protein complexes were eluted by boiling in 25 µL

Laemmli sample buffer for 20 minutes. For immunoprecipitation of solubilized chromatin, the

NaCl and Triton X-100 concentrations were adjusted to 300 mM and 0,25 %, respectively, by

diluting 2 mg of extract 1:1 with high salt CSK-T buffer (as above with 500 mM NaCl and 0,4 %

Triton X-100) before pre-clearing. Western blotting was performed as described previously (25).

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Cell cycle analysis by EdU-FACS

2x105 cells were seeded in a 6-well plate and two days later pulsed with EdU (20 µM) for 20

minutes. Cells were trypsinized, washed in cold PBS and collected by centrifugation at 300 g for

15 5 minutes at 4 °C. Cell pellets were resuspended in 750 µL cold PBS and cells were fixed by

adding 1,75 mL of cold 100 % ethanol slowly and drop-wise, while vortexing at low speed (final

concentration 70 %). Cells without EdU pulse were fixed as a negative control. Samples were

stored at 4 °C for minimum 1 hour. Cells were washed in 1 mL of 1 % BSA in PBS,

permeabilized in 200 µL of 0,25 % Triton X-100 in PBS for 10 minutes at room temperature and

20 resuspended in 1 mL of 1 % BSA in PBS (all centrifugation steps at 500 g for 5 minutes at RT).

5x105 cells were pelleted, resuspended in 200 µL Click-iT reaction mix (Invitrogen; Alexa Fluor

647 azide) and incubated for 30 minutes, shaking at 300 rpm at room temperature, protected

from light. Cells were washed twice in 1 mL of 1 % BSA in PBS and DNA was stained with

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Propidium Iodide (final concentration 10 µg/mL) in 400µL of PBS supplemented with 20 µg/mL

of RNase A for 30 minutes, shaking at 300 rpm at room temperature, protected from light.

Samples were stored protected from light at 4 °C until analysis by flow cytometry (10000 cells

per sample, BD FACSCalibur, CellQuest Pro software). Data were processed in FlowJo

5 (v.10.4.2).

Data analysis

Raw data processing. SCAR-seq samples were demultiplexed in Illumina Basespace (trimmed

10 >30bp) and processed according to ENCODE (phase-3) histone ChIP-seq specifications in

single-end mode. In brief, raw fastq files were trimmed and mapped to ENCODE mm10 by

BWA aln version 0.7.13 (quality threshold = 5, seed length = 32, maximum differences in seed =

2, MAPQ = 30). Duplicated reads were marked and removed by picard-tools (version 2.8.2) and

ENCODE mm10 blacklist regions were masked.

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For the OK-seq, a custom sequencing primer was used as previously described (8). The reads

were de-multiplexed with Illumina bcl2fastq (version v2.18), trimmed with Trim Galore!

(Galaxy version 0.4.2), filtered FASTQ (Galaxy version 1.0.0) 10-70bp and mapped with Bowtie

for Illumina (Galaxy Version 1.1.5) to mouse (Mus musculus) mm10. Duplicated reads were

20 marked and removed by picard-tools (version 2.8.2) and ENCODE mm10 blacklist regions were

masked.

Read counts were computed by samtools flagstat (version 1.5), before and after removal of

duplicated reads. For quality control of H3K36me3 SCAR-seq samples, normalized strand cross-

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correlation coefficients (NSC) and relative strand cross-correlation coefficients (RSC) were

computed by phantompeakqualtools (version 1.2) (33).

Processed read files were split into forward and reverse strand according to the SAM flag, using

samtools view (version 1.5) -F 20 and -f 16, respectively. Read coverage for all H3K36me3

5 SCAR-seq samples and ENCODE H3K36me3 (ENCSR253QPK) ChIP-seq were computed from

filtered bam files using deepTools multiBamSummary (34) (version 2.5.3) in bins of 1kb and

within Gencode vM17 annotated genes. Data were passed on to deepTools plotCorrelation for

pairwise Pearson correlations. Correlations were visualized with pheatmap.

10 Histone partition signal. Histone partition was computed from mapped, trimmed and de-

duplicated SCAR-seq reads within book-ended 1 kb bins as follows:

Partition = (F - R)/(F + R)

F and R correspond to the number of mapped reads to forward and reverse strand, respectively.

Partition value relate to the ratio of histones with a specific modification being segregated to the

15 nascent forward (above 0) and nascent reverse (below 0) strand within each window,

respectively. The histone partition was normalized by the total read count.

SCAR-seq replicates were analyzed separately for statistical testing to ensure robustness within

marks, while average signal was used for most visualizations.

20 Replication fork directionality. RFD was computed from mapped, trimmed and de-duplicated

OK-seq data in book-ended bins of 1kb according to (8):

RFD = (R - F)/(F + R)

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F and R correspond to reads mapped on forward and reverse strand respectively. RFD value

reveals the proportions of forks moving rightward (>0, replicating forward strand by leading

strand mechanism) and leftward (<0, replicating reverse strand by leading strand mechanism)

within each window, respectively.

Data smoothing. Partition signal from SCAR-seq and RFD signal from OK-seq were smoothed

similarly. Smoothing was computed across consecutive book-ended 1 kb bins, performing a

uniform blur considering the neighboring 30 bins on each side. Smoothing radius was increased

to 80 bins for genome views of SCAR-seq H4K5ac, H4K20me2 and H3K36me3 WT replicate 1

10 samples. Bins from chromosome 1 – 19 with normalized read-counts CPM > 0.3 were included

in downstream analysis.

Similar to C. elegans embryos (12), the RFD indicated less replication directionality in mESC

than in human somatic cells (8). This reduction in RFD amplitude may reflect increased

15 stochasticity in the replication program in mESC with high chromatin plasticity (35), but is also

likely affected by the smoothing of RFD signal.

Input signal. To deduct input nucleosome partition signal and potential technical biases from

SCAR-seq signal, samples were paired with an input sample to evaluate the signal above input

20 ratio. This was done separately for each strand by feeding a pileup signal with fragment

extension of 75 to MACS2 bdgcmp (36) (version 2.1.1) and evaluating the log10 likelihood

between ChIP-enriched model and open chromatin model (-m logLR). The logLR signal

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averaged across 1 kb windows was used to reproduce partition skews and evaluate significance

levels.

Annotation. Bins from each sample which were included for analysis, were annotated using

5 ChIPseeker annotatePeak (37) with Mmusculus.UCSC.mm10.knownGene as TxDb object,

org.Mm.eg.db as annotation package, tssRegion = -1,1 and overlap = all.

A simplified annotation was also performed by merging all intron, exon, UTR, and Promoter

annotations into “gene”, leaving the rest of the bins as “intergenic”.

10 To compare the annotation distributions from OK-seq and SCAR-seq samples to a genome-wide

background distribution, chromosomes 1-19 were binned into 1 kb regions. Bins at position 0 – 3

Mb and those overlapping the mm10 ENCODE blacklist (29) were excluded from the

annotation.

15 Initiation zone and partition skew detection. A nonparametric derivative estimation (38) of RFD

signal were computed on non-filtered bins to detect sharp transitions between neighboring bins,

defining the replication initiation zones (ascending slopes). These initiation zones were defined

as bins with RFD derivative above the 0.9 quantile and zero-crossing. For initiation zones less

than 3 bins apart, the bin with the highest RFD derivative was selected.

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Partition and RFD rates around initiation zones were computed up to 100 kb upstream and

downstream of each initiation zone by averaging values within each bin position. Average values

in windows of 150 kb upstream and downstream if each initiation zone were plotted in heatmaps

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using pheatmap, where initiation zone windows with a proportion of NA values (bins with

insufficient read coverage of a specific mark) above the 0.9 quantile were excluded.

Furthermore, extreme partition and RFD ratios (values > 0.9999 quantile or < 00001 quantile)

were set to the quantile value.

Initiation zone edges, where the RFD reach local extrema, were determined within 100 kb

upstream and downstream of the initiation zone, by selecting the location with minimum and

maximum RFD value, respectively. A window size of 200 kb around each initiation zone was

chosen based on the average initiation zone size (from upstream to downstream initiation zone

10 edge) (mean size = 112 kb) and the distances to neighboring initiation zones (mean distance =

359 kb).

The difference in SCAR-seq partition ratios at initiation zone edges were tested with a paired

Wilcoxon signed-rank test. The test was performed for each SCAR-seq sample, while initiation

15 zones with insufficient read coverage at one or both initiation zone edges were excluded from

this test.

To evaluate the partition skew around initiation zones after input restrictions (see section Input

signal), only regions with positive average logLR on either strand were considered. If a bin with

20 partition signal at a specific initiation zone edge did not pass the logLR test, then partition signal

from the closest bin with a positive average logLR on either strand were considered instead.

Thus, partition ratios for regions that passed the input restriction were tested similar to before

with a Wilcoxon signed-rank test.

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RFD and partition within TADs and U-domains. Histone partition and RFD rates were

computed within TADs and U-domains by averaging values across the relative distance in breaks

of 0.01. For TADs, average signal was split into A and B compartments.

Enrichment of initiation zones in active TADs compared to inactive TADs was tested by Fisher’s

exact test, using the number of bins with OK-seq signal above the coverage threshold as a test

background set.

10 Feature overlap at initiation zones. The fraction of bins with an annotated mESC active

promoter (as defined by RNA-seq, see section Transcription) and the fractions of bins

overlapping active or inactive enhancers (21) (as defined by CAGE, see section Transcription)

were computed in 100 kb windows upstream and downstream of initiation zones. The fraction of

bins overlapping a TAD border was calculated in windows of 500 kb upstream and downstream

15 of initiation zones at a resolution of 10 kb. Random feature fractions were computed around

sampled non-initiation zone bins with sufficient OK-seq signal permuted 10 times.

Partition breakpoint and initiation zone overlap. Sharp transitions in partition ratios for

H4K20me2 samples were detected similarly to initiation zones, to mark partition breakpoints

20 genome-wide. Co-localization of initiation zones and partition breakpoints were computed for

each sample, determining the nearest distance from an initiation zone to a partition breakpoint.

Distances from initiation zones to random bins with H4K20me2 SCAR-seq signal were also

computed for each sample, permuted 10 times. Fractions of co-localization were computed in a

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distance of -500 to 500 kb in bins of 10 kb, reporting the number of pairs at a specific distance

apart, out the total number of initiation zone and partition breakpoint neighbor pairs. H4K20me2

WT and MCM2-2A#1 mutant replicate 1 is shown in Fig. 3E.

5 External data analysis

Transcription. Raw PRO-seq data from mESCs (GEO GSM2284185) were processed as

described (39) except that reads were mapped by BWA aln (version 0.7.13) to (ENCODE)

mm10. Paired-end read files were split according to the SAM flag by samtools view (version

10 1.5), where forward and reverse reads were marked by 163, 83 and 99, 147 respectively,

according to dUTP treatment. Data was summarized in bins of 1 kb and a PRO-seq directionality

score was calculated and smoothed for each genomic bin similarly to SCAR-seq partition.

Associations between PRO-seq directionality and both SCAR-seq partition and RFD were

evaluated using Spearman rank correlation coefficients.

15

Gene activity in mESC was determined from processed gene quantifications based on RNA-seq

data (ENCSR000CWE), where FPKM values were averaged over mESC replicates and active

gene transcription was determined at FPKM>0.5.

20 FANTOM5 enhancer annotations (PMC5215096) expressed above noise levels (40) were used to

assess mESC specific enhancer activity from six mESC CAGE libraries (CNhs14098,

CNhs14099, CNhs14100, CNhs14091, CNhs14092, CNhs14093). FANTOM5 enhancers

expressed above 0.5 TPM in at least three out of the six libraries were determined as active

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enhancers (n = 3316), while inactive enhancers refer to the remaining set of FANTOM5

enhancers (n = 36830).

Coordinates of mESC super-enhancer annotations (22) were lifted to build mm10 using liftOver

5 with default settings from build mm9 using the UCSC liftover chain.

Histone partition and RFD rates were calculated around enhancers in 100 kb windows

upstream and downstream of the enhancer midpoint. Average signal was split by enhancer

type and activity. Significant differences in histone partition amplitude between active and

10 inactive enhancers, calculated as the difference between partition from enhancer upstream and

downstream extrema, were tested by Mann Whitney U test.

Replication timing profiles. mESC RT (41) data were downloaded from

https://www.replicationdomain.com/database.php# (accession Int14787930) and lifted from

15 mouse mm8 to mouse build mm10 using liftOver. Processed signal was binned into 1 kb regions

and used in connection with RFD and partition signal within U-domains.

U-domains The coordinates of replication timing U-domains of human ESC BG01 (13) were

lifted from human build hg18 to mouse build mm10 using liftOver.

20

Chromatin confirmation data. Genomic coordinates and compartment identities of topological

associated domains (TADs) in mESC were retrieved from (16). From the same source, observed

inter-chromosomal Hi-C contact matrices were downloaded through the Juicer dump Tool

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(version 1.8.9) (42) at 10 kb resolution. Contact matrices were used to calculate genome-wide

directionality index (DI) (15).

DI = ((B-A) /(|B - A|)) (((A-E2) / E) + ((B-E)2 / E))

A and B are the number of reads that map from a given 10 kb bin to the upstream and

5 downstream 1 Mb region, respectively. E is equal to (A+B)/2, referring to the expected number

of reads under the null hypothesis.

ChIP-seq data. ENCODE ChIP-seq signal for histone modifications H3K27ac, p300, H3K4me1,

H3K4me3, H3K36me3, and H3K27me3 (accession numbers in table S3) were used as is, in fold

10 change over control signal. Average fold change signal was computed with bwtool (version 1.0)

in regions of 150 kb upstream and downstream of initiation zones. For comparison, fold change

signal was also computed around an equal number of random bins having OK-seq signal.

DNase signal. DNase signal was retrieved from (ENCSR000CMW) and average signal around

15 initiation zones and random regions with OK-seq signal was calculated similar to ChIP-seq

signal.

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Supplementary Figures
Fig. S1
A B
Partition=(p5,R-p5,F)/(p5,R+p5,F)
K20me0 qPCR: 1.0
+ NaOH
P5 F

Partition
yes 0.5
no
K20me1 R
0.0
no NaOH P5 + NaOH no NaOH
-0.5
K20me2 P5 yes
R
yes -1.0
H4 aa14-33
F

H4K5ac-parental
0.4 H4K5ac
Fraction

H4K20me2-parental
H4K20me2
0.2 Input
Genome

0.0
Downstream <3kb

Promoter

5' UTR

exon

intron

3' UTR

Distal Intergenic

Fig. S1. SCAR-seq technique and quality controls. (A) Verification of H4K20me2 antibody specificity
(Diagenode # C15200205). Dot-blot of H4 tail peptides (14-33 amino acid) with K20 unmodified
(H4k20me0), mono-methylated (H4K20me1) and di-methylated (H4K20me2). (B) Test of EdU-labeled
strand isolation strategy. Left: Scheme of control library prepared with hemi-EdU-nylated DNA fragment
(Methods). Right: qPCR test to calculate partition. Library fragments containing only isolated EdU strand
(+NaOH) or hemi-EdU-nylated double-stranded DNA (no NaOH) were tested for strandedness with
indicated primers. (C) Validation of genome-wide signal by annotation fractions of bins with sufficient read
counts within SCAR-seq samples of H4K20me2 nascent (orange) and parental (light orange) strand, and
H4K5ac nascent (green) and parental (light green) strand, compared to input samples (sand) and genomic
background (grey) annotation fractions.

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Fig. S2
A derivative D
0.005
RFD

Downstream <3kb
0.000

Promoter
0.2
RFD

0.0 5' UTR

0.2 Genome
OK-seq
0 Chr10 exon Initiation
zone
B C
intron

density
3' UTR
density

Distal Intergenic

0.0e+00 0.0e+00
1 2 3 (Mb) 50 100 150 200 (kb) 0.0 0.2 0.4
Distance to nearest initiation zone Initiation zone size Fraction

E p300 H3K4me1 DNase

0.24
0.55 0.8 0.8
0.21

Signal
0.50
Fold Change (signal / input)

0.18
0.45
0.15
0.40 0.5 0.5
0.12
-100 0 100 (kb) -100 0 100 (kb) -100 0 100 (kb) -100 0 100 (kb)
Position relative to
H3K4me3 initiation zone center
1.50 1.4
1.25 1.2
Random
1.00 1.0 Initiation zone

0.8

-100 0 100 (kb) -100 0 100 (kb)

Position relative to initiation zone center

Fig. S2. Detection and characterization of initiation zones. (A) Identification of initiation zones in a
representative genomic region by detecting sharp transitions between neighboring bins based on RFD
derivative (top) and RFD (bottom) profiles. Black vertical lines mark initiation zone centers identified as the
breakpoints in RFD derivative and red chevrons indicate initiation zone edges (identified as max. and min.
RFD). (B) Density plot (y-axis) of distance in kilobase (x-axis) to the nearest scored initiation zone. (C)
Density plot (y-axis) of initiation zone size (x-axis) (distance in kb between upstream and downstream
edges). (D) Annotation fractions of OK-seq bins (blue) with sufficient read counts, initiation zone scored
bins (light blue) and genomic background (grey). (E) Average of ChIP-seq signal enrichment (ChIP signal
above input) (y-axis) of histone modification marks, DNase I hyper-sensitive sites and the histone
acetyltransferase p300 around initiation zones with distances (x-axis) in kilobases. For DNase, raw signal is
reported (y-axis).

20
Fig. S3
A

0.15
0.010 OK-seq
0.10 H4K20me2

Partition
0.05 0.005 H4K5ac
RFD

0.00 0.000 Replicate

1
2
3
0 0 100 (kb) 0 0 100 (kb)
Distance from initiation zone center Distance from initiation zone center

B C 5.3x10-14 2.8x10-46 3.6x10-64 8.2x10-60 1.2x10-13

RFD H4K20me2 H4K5ac
0.3 0.1 0.1
0.05
0.10
0.1
0 0 0 Strand
Initiation zone size

-0.1 -0.05 0.05 leading

Partition
-0.3 -0.1 -0.1
0.00
lagging
-0.05

-0.10

rep1 rep2 rep1 rep2 rep3

150 0 150 150 0 150 150 0 150 (kb) H4K5ac H4K20me2
Distance from initiation zone center

4.6x10-34 0.0474
E
D EdU strand 0.10
Parental strand
0.05 Strand
0.008 H4K20me2-EdU leading

Partition
0.010
H4K20me2-parental
0.004
Partition

0.005 H4K5ac-EdU 0.00

H4K5ac-parental lagging
0.000 0.000
Replicate
-0.004 1
2
-0.008 3
0 0 100 0 0 100 (kb)
H4K5ac H4K20me2
Distance from initiation zone center Distance from initiation zone center parental parental

F
RFD H4K20me2 H4K5ac
0.010 0.010
0.1 0.005 0.005
Partition

Partition

Initiation zone
RFD

0.0 0.000 0.000 Top

Bottom
-0.005 -0.005
-0.1
-0.010 -0.010
-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100 (kb)
Distance from initiation zone center Distance from initiation zone center Distance from initiation zone center

21
Fig. S3. Partition of old (H4K20me2) and new (H4K5ac) H4 around initiation zones.
(A) Average profile of RFD (left) and partition of H4K20me2 (orange) and H4K5ac (green) shown for all
replicates around initiation zones (right). Top: Scheme indicating sister chromatids with the newly-replicated
forward (magenta) and reverse (blue) strand switching from leading strand synthesis (full line) to lagging
strand synthesis (dashed line) and histone partition strand preference H4K20me2 (orange) and H4K5ac
(green) at initiation zones. (B) Heatmap of RFD and partition of H4K20me2 and H4K5ac centered at
initiation zones (x-axis 0), and ordered by descending initiation zone size. (C) Histone PTM partition in bins
with signal above input (positive logLR on either strand) at or proximal to initiation zone downstream (leading
strand replication, dark shaded) or upstream edge (lagging strand replication, light shaded) with significant
partition differences in each replicate (paired Wilcoxon signed-rank test P < 1.3x10-13). (D) Average
partition profiles of H4K20me2 (brown) and H4K5ac (light green) measured on the parental DNA strand
(Methods) in regions centered on initiation zones. Top: Scheme illustrating leading and lagging replication
on the new DNA strand along with the parental (chalk line) DNA strands with histone partition indicated by
color. (E) Partition measured by SCAR-seq of the parental strand at initition zone edges (as in Fig. 1C) with
significant partition differences for both marks (paired Wilcoxon signed-rank test of H4K5ac parental strand:
P = 4.6x10 -34, and H4K20me2 parental strand: P = 0.047). (F) RFD and histone PTM partition around initiation
zones, spit by initiation zone strength (top: RFD derivative > median RFD derivative, bottom: RFD derivative <
median RFD derivative).

22
Fig. S4

A B
RFD H4K20me2 H4K5ac
0.1
0.05 0.2 0.06 0.06
0 0
RFD

0
0.00 -0.06 0.06
-0.2 -0.1
-0.05

0.010
Partition

0.005
0.000 −1,2 U center 1,2 (Mb) −1,2 U center 1,2 (Mb) −1,2 U center 1,2 (Mb)
-0.005 Distance from U-domain center
RT, log2(E/L)

0.02 OK-seq
0.00 H4K20me2
-0.02 H4K5ac
-0.04 RT
-0.06
border mid border
Relative position within U-domain

Fig. S4. RFD and partition of old (H4K20me2) and new (H4K5ac) H4 within U-domains. (A) Average
RFD (top), histone PTM partition (middle) and log2(early/late) replication timing (42) (bottom) across scaled
U-domains (13). (B) Heatmaps of RFD (left) and partition of H4K20me2 (middle) and H4K5ac (right)
centered at U-domain midpoints and ordered according to U-domain descending size (y-axis).

23
Fig. S5

A B Upstream border TAD Downstream border

Center Initiation zone Random
0.025 0.025

RFD
TAD border fraction

0.008 0.000 0.000

0.025 0.025

0.005 0.005 0.005

Partition
0.000 0.000

-500 -250 0 250 500 (kb) -0.005 -0.005

Distance from center -0.010 -0.010 OK-seq
H4K20me2
H4K20me2
C 0.002 0.002 H4K5ac

Partition
0.001 0.001
1
0.5 0.000 0.000
ENCODE
0 -0.001 -0.001
-0.5 -0.002 -0.002
-1
H4K5ac

0.004 0.004
Partition
ENCODE

0.000 0.000

-0.004 -0.004

-400 -200 0 200 400 -400 -200 0 200 400 (kb)

Distance from TAD border Distance from TAD border

D Chr3 33 mb 35 mb
E 3.9x10-74 1.1x10-42 2.4x10-45
34 mb
OK-seq
0.2
RFD

0.2
0.0
0.2 Strand
Partition

leading
Direction Partition

0.1 0.0
0.0 lagging
0.1

PRO-seq
1.0 -0.2
0.5
0.0

initiation zones
Genes Active rep1 rep2 rep3
Enhancers Inactive

F
Enhancer fraction

Promoter fraction

0.030
0.02 Center
0.025 Initiation zone
0.020 Random
0.01 0.015

0.010
-100 -50 0 50 100 (kb) -100 -50 0 50 100 (kb)
Distance from center Distance from center
24
Fig. S5. Histone partition within TADs, regulatory regions and active genes.
(A) Enrichment of TAD borders in windows of 1 Mb centered at initiation zones (blue) and random OK-
seq bins with sufficient signal (grey). Border fractions (y-axis) are calculated in distance bins of 20 kb (x-
axis). (B) Heatmap of pairwise Pearson correlations of SCAR-seq H3K36me3 sample read coverage in bins
of 1 kb and ENCODE H3K36me3 ChIP-seq. (C) RFD (blue) and histone PTM partition (y-axis) centered
around upstream TAD border (left panel) and downstream TAD border (right panel). (D) Example of RFD
(blue), H3K36me3 partition (red) and PRO-seq (lilac) transcription directionality score over a genomic region.
In bottom panel, mESC active (black) and mESC inactive (grey) gene annotations, where arrowheads show
gene direction, and blocks show enhancer annotations. (E) Partition ratios for all H3K36me3 samples at
initiation zone downstream edges (leading strand replication, dark red) and at upstream edges (lagging
strand replication, light red) with significant partition differences in each replicate (paired Wilcoxon
signed-rank test P < 1.0x10-42). (F) Enrichment of mESC active enhancers (left) and mESC active
promoters (right) in windows of 200 kb centered at initiation zones (blue) and random OK-seq bins with
sufficient signal (grey). Fractions (y-axis) are calculated in distance bins of 2 kb (x-axis).

25
Fig. S6
A
81 90
Wild type: GPMEEEEDGEELIGDGMERDYRPIPELDVYEAEGLALDDEDVEELTASQREAAERTMRQRDREAGRGLGR
MCM2-2A: GPMEEEEDGEELIGDGMERDARPIPELDVAEAEGLALDDEDVEELTASQREAAERTMRQRDREAGRGLGR

MCM2-2A #1: [Y81A;Y90A];[Y81A;Y90A]

MCM2-2A #2: [Y81A;Y90A];[Y90A]

B
#1

#2

#1

#2
2A

2A

2A

2A
2-

2-

2-

2-
MW
M

M
T
C

C
trl

T
W

(kDa)
M

M
C

98 MCM2
High salt extracts
17 Histone H3

98 MCM2
Solubilized chromatin
17 Histone H3
Input MCM2 IP

C D
WT MCM2-2A #1 MCM2-2A #2 33 Mb 35 Mb
Chr3
34 Mb 36 Mb
OK-seq
0.3
RFD

0.0
-0.3
H4K20me2 WT
0.04
0.00
-0.04
H4K20me2 MCM2-2A
0.50
80 0.00
Partition

0 -0.50
H4K5ac WT
0 0.05
% cells

50 WT 0.00
40 MCM2-2A #1 -0.05
30 H4K5ac MCM2-2A
MCM2-2A #2 0.3
20
10 0.0
0 -0.3
G1 S G2/M initiation zones
Genes
Cell cycle phase
Enhancers

Fig. S6. Mutation of the MCM2 histone-binding domain. (A) Overview of MCM2 HBD mutations
generated by genome editing. Amino acid sequence of the mouse MCM2 HBD (top) highlighting Y81 and
Y90 altered to alanine residues by genome editing. Two independent clones were used in this study with the
indicated genotype (bottom). (B) Co-immunoprecipitation (IP) analysis of histone H3 with MCM2 in high
salt extracts and solubilized chromatin, verifying loss of MCM2 histone binding in MCM2-2A mutants. (C)
Cell cycle analysis of WT and MCM2-2A mutants. Scatterplot of EdU (y-axis) versus Propidium iodide
(PI, x-axis) intensities of one representative experiment (top). Bar-diagram showing relative numbers of cells
in G1-, S- and G2/M-phase (mean and SD of three biological replicates) (bottom). (D) RFD and partition of
H4K20me2 and H4K5ac at a genomic region in WT and MCM2-2A, as in Fig. 3B but shown on different
y-axis scales. Initiation zone centers (black lines), active gene orientation (arrowheads) and active enhancers
(bars) are shown.
Fig. S7
A 0.10
H4K5ac
H4K20me2
0.05 0.1 0.1

Partition
Partition

Partition
MCM2-2A #1
0.00 0.0 0.0 MCM2-2A #2
Wild type
replicate
-0.05 -0.1 -0.1 1
2
3
-0.10
-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100
Distance (kb) from initiation zone center
B
H4K5ac leading lagging H4K20me2 leading lagging leading lagging

*** *** *** *** *** *** *** *** *** *** *** ***

0.3 0.3 0.3

Partition

Partition
Partition

0.0 0.0 0.0

-0.3 -0.3 -0.3

rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2
MCM2-2A #1 MCM2-2A #2 MCM2-2A #1 MCM2-2A #2 MCM2-2A #1 MCM2-2A #2

C
H4K5ac MCM2-2A #1 H4K20me2 MCM2-2A #1
0.10 0.2
Initiation zone
0.1
Partition

Partition

Partition

0.05 0.1 rank

0.00 0.0 0.0 Top
-0.05 -0.1 -0.1 Bottom
-0.10 -0.2 -0.2
-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100
Distance (kb) from initiation zone center

D MCM2-2A #2 MCM2-2A #2 MCM2-2A #2

count count
H4K20me2 partition

H4K5ac partition

K20me2 Partition

count
1000 1000 2000
1500
0.0 500 0.0 500 0.0
1000
0 0 500

0.0 0.0 0.0

RFD RFD H4K5ac Partition
WT MCM2-2A #1 MCM2-2A #2

count
400

0.0 0.0 0.0 200

0.0 0.0 0.0

RFD RFD RFD
Fig. S7. Recycling of parental histones to the leading strand in MCM2 histone-binding mutants. (A)
Average partition of H4K5ac, H4K20me2 and H3K36me3 around initiation zones in WT (dotted line),
MCM2-2A #1 (dashed line), and MCM2-2A #2 (full line) cells. Replicates are shown in different color
shades. (B) Partition ratios of H4K5ac (green), H4K20me2 (orange) and H3K36me3 (red) in MCM2-2A #1
(left) and MCM2-2A #2 (right) at initiation zone edges with significant partition differences (***, paired
Wilcoxon signed-rank test P < 1.28 x10-45). (C) Partition of H4K5ac, H4K20me2 and H3K36me3 in
MCM2-2A #1 around initiation zones, split by initiation zone strength (top: RFD derivative > median RFD
derivative, bottom: RFD derivative < median RFD derivative). (D) Scatterplots of RFD and histone PTM
partition in the WT and MCM2-2A as indicated. Spearman's rank correlation coefficient shown in top left
corner.

28
 Fig. S8
A
rep1 wt rep1 wt 1
rep2 MCM2 #2 rep2 wt
0.98
rep2 wt rep3 wt
rep3 wt rep2 MCM2-2A #1
rep2 MCM2-2A #1 rep1 MCM2-2A #1
0.94
rep1 MCM2-2A #2 rep1 MCM2-2A #2
rep1 MCM2-2A #1 rep2 MCM2-2A #2 0.92
rep1 wt
rep2 MCM2-2A #2
rep2 wt
rep3 wt
rep2 MCM2 -2A #1
rep1 MCM2-2A #2
rep1 MCM2-2A t#1

rep1 wt
rep2 wt
rep3 wt
rep2 MCM2-2A #1
rep1 MCM2-2A #1
rep1 MCM2-2A #2
rep2 MCM2-2A #2
B Pro-Seq

0.4
Fraction

H4K20me2 MCM2-2A #1
0.2 H4K20me2 MCM2-2A #2
H4K20me2 wt
H4K5ac MCM2-2A #1
0.0 H4K5ac MCM2-2A #2
H4K5ac
Downstream <3kb

Promoter

5' UTR

exon

intron

3' UTR

Distal Intergenic
Genome

C D E
Correlation with PRO-Seq

0.8 0.4

Correlation with Hi-C

directionality index
0.2 RFD
MCM2-2A #1 H4K20me2
Density

0.4 0.0 H3K36me3

4 Gene H4K5ac
Annotation 0.2 0.2 PRO-seq
2 Active +
Active -
0.0 0.4
0
MCM2-2A #1
MCM2-2A #2

MCM2-2A #1
MCM2-2A #2

MCM2-2A #1
MCM2-2A #2

MCM2-2A #1
MCM2-2A #2

MCM2-2A #1
MCM2-2A #2
PRO-seq
wild type

wild type

wild type

wild type

wild type
RFD

RFD

-0.3 0.0 0.3

Partition

F H4K5ac MCM2-2A H4K20me2 MCM2-2A

0.10
Compartment
0.10 0.1
0.05 A
0.05
Partition

B
0.00 0.0
0.00
MCM2-2A
-0.05
-0.1 -0.05 #2
-0.10 #1
border mid border border mid border border mid border
Relative position within TADs Relative position within TADs Relative position within TADs
G H4K5ac MCM2-2A #1 H4K20me2 MCM2-2A #1

0.10 0.10
0.05 Enhancer
0.05 0.05 type
Partition

0.00 Active
0.00 0.00
Inactive
-0.05 -0.05 Super enhancer
-0.05
-0.10
-0.10
-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100 (kb)
Distance from enhancers Distance from enhancers Distance from enhancers
Fig. S8. Genomic scale sister chromatid histone PTM asymmetry in MCM2 histone-binding mutants.
(A) Recycling accuracy of H3K36me3 in MCM2-2A #1 and MCM2-2A #2 compared to WT shown as
pairwise Pearson correlations of H3K36me3 sample read coverage in bins of 1 kb (left heatmap) and within
annotated genes (right heatmap). (B) Annotation distribution of bins with sufficient read coverage of
SCAR-seq (H3K36me3, H4K20me2 and H4K5ac) in WT (dark red, orange, green), MCM2-2A #1 (light
red, orange, green) and MCM2-2A #2 (medium red, orange, green), PRO-seq (lilac) and genomic
background (grey) annotations. The fraction of bins (y-axis) are calculated within each SCAR-seq mark and
MCM2 genotype. (C) Average partition of H4K5ac, H4K20me2 and H3K36me3 (y-axis) in MCM2-2A #1
(dotted line) and #2 (full line) across scaled TADs, split by A (full color) and B (shaded color) compartment
identity (17). Signal averaged in breaks of 0.01 relative distance. (D) Average partition of H4K5ac,
H4K20me2 and H3K36me3 in MCM2-2A #1 around enhancers (colors as in Fig 2D) (21). (E) Density
distribution of H3K36me3 partition in active forward (red) and reverse (blue) genes of WT (full) and
MCM2-2A (dash-dotted). (F) Spearman correlation of PRO-seq directionality score (29) with RFD and partition
of H4K20me2 and H3K36me3 in WT and MCM2-2A mutants.(G) Spearman correlation of Hi-C directionality
index (16) with RFD, partition of H4K5ac, H4K20me2 and H3K36me3 in WT and MCM2-2A mutants, and
PRO-seq directionality score (29).

30
Fig. S9

A Hox B cluster Hox D cluster

OK-seq OK-seq
0.3 0.2

RFD
0.1 0.1
RFD

0.0 0.0
-0.1 -0.1
-0.3
H4K20me2 wt H4K20me2 wt
0.3 0.2
0.1 0.1
0.0 0.0
-0.1 -0.1
-0.3
H4K20me2 MCM2-2A#1 H4K20me2 MCM2-2A#1
0.3 0.2
0.1 0.1 CPM > 0.3
0.0 0.0
CPM < 0.3
Partition

Partition
-0.1 -0.1
-0.3
H4K5ac wt H4K5ac wt
0.3 0.2
0.1 0.1
0.0 0.0
-0.1 -0.1
-0.3
H4K5ac MCM2-2A#1 H4K5ac MCM2-2A#1
0.3 0.2
0.1 0.1
0.0 0.0
-0.1 -0.1
-0.3
Chr11 Chr2 5 0 5 0

Gm53 Hoxb9 Hoxb8 Hoxb5os Mir10a Hoxb3 Hoxb2 Hoxb1 Evx2 Hoxd13 Hoxd11 Hoxd9 Hoxd8 Hoxd3 Hoxd1

TAD
border Enhancer
Gene

WT
sister F

sister R

MCM2-2A Old histones

Fig. S9. Epigenomic sister chromatid asymmetry landscapes in WT and MCM2-2A mutants. (A) RFD
(blue) and partition of H4K20me2 (orange) and H4K5ac (green) in WT cells and MCM2-2A #1 in the
genomic region of Hox B and Hox D gene clusters. Genes are shown in the bottom panel, arrowheads
demonstrate gene direction. Bins with read coverage below the CPM threshold are included in 50%
transparency. (B) Schematic of how genomic regions could be affected by sister chromatid asymmetries in
histone PTM inheritance. TADs (lilac triangles). Black dotted lines indicate long-range frequently
interacting regions, detected by Hi-C. Initiation zones (blue/red arrows), parental histone PTM levels
(orange), sisters (F, forward nascent; R, reverse nascent). Not drawn to scale, see (A) for a direct
comparison of WT and MCM2-2A.

31
 Submitted Manuscript: Confidential

Table S1. Antibodies used in this study.

Name Supplier Catalog number

H4K20me2 monoclonal antibody Diagenode C15200205

Anti-Histone H4 (acetyl K5) antibody Abcam ab51997

[EP1000Y]
Anti-Histone H3 (tri methyl K36) Abcam ab9050

MCM2 (D7G11) (used for IP) Cell Signaling 3619

MCM2 (anti-BM28) (used for WB) BD Transduction 610701
Laboratories
H3 ChIP Grade Abcam ab1791
Mouse TrueBlot Rockland Inc 18-8817-31
ProteinA-HRP GE Healthcare NA9120

32
 Submitted Manuscript: Confidential

Table S2. Oligonucleotides used in this study.

Name Experiment Supplier Sequence

1 qPCR test for TAG GTG AAG TGA AGA GAC CAG TGA
strandedness Copenhagen A/S AGA GTC ACT GGA GTA GAC ATT
TTA CCC CTA CTT GGC ACT CGG
AGT GCA CCC TGC TGG GCT ACT
GGG ACG TAG GTG GGG T
2 qPCR test for TAG ACC CCA CCT ACG TCC CAG
strandedness Copenhagen A/S
F qPCR test for TAG GTG CAC TCC GAG TGC CAA GT
strandedness Copenhagen A/S
R qPCR test for TAG ACT TGG CAC TCG GAG TGC AC
strandedness Copenhagen A/S
P5 qPCR test for TAG AAT GAT ACG GCG ACC ACC GA
strandedness Copenhagen A/S
gRNA MCM2 TAG GCTCGATGTCTACGAGGCCG
mutant Copenhagen A/S
generation
ssODN* MCM2 Integrated DNA CTTAAGTGGGTTGTTTGGGGATTCAT
mutant Technologies TGTCCACTGTTGGTCTCTTGCAGAGA
generation CGCCCGTCCCATTCCGGAGCTCGAC
GTCGCCGAGGCCGAAGGATTGGCCC
TGGATGATGAAGATGTGGAGGAGCT
GACAGCCAGTCAGAGAGAGGCAGCC
GAGCGGACCATGAGGCAGCGGGACC
GTGAGGCTGGCAGAGGC
3 MCM2 TAG CCACCCAGATTCACATGGCT
mutant Copenhagen A/S
genotyping
4 MCM2 TAG AGTTCTTGAAGCGGTGGTGG
mutant Copenhagen A/S
genotyping
* 7 point mutations are in bold: 4 missense mutations (to introduce Y81A, Y90A), 3 silent
mutations (to introduce AatII site, disrupt PvuII site and mutate PAM).

33
 Submitted Manuscript: Confidential

Table S3. Source and accession number of published datasets used in this study.

Data Source Accession

H3K27ac ENCODE ENCSR000CGQ
EP300 ENCODE ENCSR000CCD
K4me1 ENCODE ENCSR000CGN
K4me3 ENCODE ENCSR212KGS
K36me3 ENCODE ENCSR000CGR
K27me3 ENCODE ENCSR059MBO
DNAse-seq ENCODE ENCSR000CMW
Replication Timing Hiratani et al 2008 Int14787930
TADs Bonev et al. 2017 GSE96107
U-domains Baker et al, 2012 U-domain BG02
PRO-seq Engreitz et al. 2016 GSE85798
CAGE FANTOM5 FANTOM5
RNA-seq ENCODE ENCSR000CWC

34
 Submitted Manuscript: Confidential

Additional Data table S1. Sequencing samples generated in this study.

The file contains information about all data generated in this study from SCAR-seq and OK-seq.

For K36me3_r2 and K36me3_r3, two rounds of sequencing were performed, marked in column

4 as S1 and S2.

Column description:

1. Sample ID (identical to GEO sample ID)

2. Short experiment name

3. Length of EdU pulse (in minutes)

10 4. Biological replicate ID

5. Number of mapped reads

6. Fraction of reads mapped

7. Number of mapped reads after quality filtering and de-duplication

8. Fraction of duplicated reads

15 9. Non-redundant fraction (NRF) for library complexity QC

10. Normalized strand cross-correlation coefficients (NSC) for enrichment QC

11. Relative strand cross-correlation coefficients (RSC) for enrichment QC

20

35
References and Notes
1. C. D. Allis, T. Jenuwein, The molecular hallmarks of epigenetic control. Nat. Rev. Genet. 17,
487–500 (2016). doi:10.1038/nrg.2016.59 Medline
2. R. P. Halley-Stott, J. B. Gurdon, Epigenetic memory in the context of nuclear reprogramming
and cancer. Brief. Funct. Genomics 12, 164–173 (2013). doi:10.1093/bfgp/elt011
Medline
3. C. Alabert, A. Groth, Chromatin replication and epigenome maintenance. Nat. Rev. Mol. Cell
Biol. 13, 153–167 (2012). doi:10.1038/nrm3288 Medline
4. E. I. Campos, J. M. Stafford, D. Reinberg, Epigenetic inheritance: Histone bookmarks across
generations. Trends Cell Biol. 24, 664–674 (2014). doi:10.1016/j.tcb.2014.08.004
Medline
5. C. Alabert, T. K. Barth, N. Reverón-Gómez, S. Sidoli, A. Schmidt, O. N. Jensen, A. Imhof, A.
Groth, Two distinct modes for propagation of histone PTMs across the cell cycle. Genes
Dev. 29, 585–590 (2015). doi:10.1101/gad.256354.114 Medline
6. V. Pospelov, G. Russev, L. Vassilev, R. Tsanev, Nucleosome segregation in chromatin
replicated in the presence of cycloheximide. J. Mol. Biol. 156, 79–91 (1982).
doi:10.1016/0022-2836(82)90460-0 Medline
7. A. T. Annunziato, Split decision: What happens to nucleosomes during DNA replication? J.
Biol. Chem. 280, 12065–12068 (2005). doi:10.1074/jbc.R400039200 Medline
8. N. Petryk, M. Kahli, Y. d’Aubenton-Carafa, Y. Jaszczyszyn, Y. Shen, M. Silvain, C. Thermes,
C.-L. Chen, O. Hyrien, Replication landscape of the human genome. Nat. Commun. 7,
10208 (2016). doi:10.1038/ncomms10208 Medline
9. O. Hyrien, in The Initiation of DNA Replication in Eukaryotes, D. Kaplan, Ed. (Springer,
2016), pp. 65–85.
10. G. Saredi, H. Huang, C. M. Hammond, C. Alabert, S. Bekker-Jensen, I. Forne, N. Reverón-
Gómez, B. M. Foster, L. Mlejnkova, T. Bartke, P. Cejka, N. Mailand, A. Imhof, D. J.
Patel, A. Groth, H4K20me0 marks post-replicative chromatin and recruits the TONSL–
MMS22L DNA repair complex. Nature 534, 714–718 (2016). doi:10.1038/nature18312
Medline
11. Z. Jasencakova, A. N. D. Scharf, K. Ask, A. Corpet, A. Imhof, G. Almouzni, A. Groth,
Replication stress interferes with histone recycling and predeposition marking of new
histones. Mol. Cell 37, 736–743 (2010). doi:10.1016/j.molcel.2010.01.033 Medline
12. E. Pourkarimi, J. M. Bellush, I. Whitehouse, Spatiotemporal coupling and decoupling of
gene transcription with DNA replication origins during embryogenesis in C. elegans.
eLife 5, e21728 (2016). doi:10.7554/eLife.21728 Medline
13. A. Baker, B. Audit, C.-L. Chen, B. Moindrot, A. Leleu, G. Guilbaud, A. Rappailles, C.
Vaillant, A. Goldar, F. Mongelard, Y. d’Aubenton-Carafa, O. Hyrien, C. Thermes, A.
Arneodo, Replication fork polarity gradients revealed by megabase-sized U-shaped
replication timing domains in human cell lines. PLOS Comput. Biol. 8, e1002443 (2012).
doi:10.1371/journal.pcbi.1002443 Medline

36
14. B. D. Pope, T. Ryba, V. Dileep, F. Yue, W. Wu, O. Denas, D. L. Vera, Y. Wang, R. S.
Hansen, T. K. Canfield, R. E. Thurman, Y. Cheng, G. Gülsoy, J. H. Dennis, M. P.
Snyder, J. A. Stamatoyannopoulos, J. Taylor, R. C. Hardison, T. Kahveci, B. Ren, D. M.
Gilbert, Topologically associating domains are stable units of replication-timing
regulation. Nature 515, 402–405 (2014). doi:10.1038/nature13986 Medline
15. J. R. Dixon, S. Selvaraj, F. Yue, A. Kim, Y. Li, Y. Shen, M. Hu, J. S. Liu, B. Ren,
Topological domains in mammalian genomes identified by analysis of chromatin
interactions. Nature 485, 376–380 (2012). doi:10.1038/nature11082 Medline
16. B. Bonev, N. Mendelson Cohen, Q. Szabo, L. Fritsch, G. L. Papadopoulos, Y. Lubling, X.
Xu, X. Lv, J.-P. Hugnot, A. Tanay, G. Cavalli, Multiscale 3D genome rewiring during
mouse neural development. Cell 171, 557–572.e24 (2017).
doi:10.1016/j.cell.2017.09.043 Medline
17. E. Lieberman-Aiden, N. L. van Berkum, L. Williams, M. Imakaev, T. Ragoczy, A. Telling, I.
Amit, B. R. Lajoie, P. J. Sabo, M. O. Dorschner, R. Sandstrom, B. Bernstein, M. A.
Bender, M. Groudine, A. Gnirke, J. Stamatoyannopoulos, L. A. Mirny, E. S. Lander, J.
Dekker, Comprehensive mapping of long-range interactions reveals folding principles of
the human genome. Science 326, 289–293 (2009). doi:10.1126/science.1181369 Medline
18. A. Loyola, T. Bonaldi, D. Roche, A. Imhof, G. Almouzni, PTMs on H3 variants before
chromatin assembly potentiate their final epigenetic state. Mol. Cell 24, 309–316 (2006).
doi:10.1016/j.molcel.2006.08.019 Medline
19. J. C. Black, C. Van Rechem, J. R. Whetstine, Histone lysine methylation dynamics:
Establishment, regulation, and biological impact. Mol. Cell 48, 491–507 (2012).
doi:10.1016/j.molcel.2012.11.006 Medline
20. C. Cayrou, B. Ballester, I. Peiffer, R. Fenouil, P. Coulombe, J.-C. Andrau, J. van Helden, M.
Méchali, The chromatin environment shapes DNA replication origin organization and
defines origin classes. Genome Res. 25, 1873–1885 (2015). doi:10.1101/gr.192799.115
Medline
21. R. Andersson, C. Gebhard, I. Miguel-Escalada, I. Hoof, J. Bornholdt, M. Boyd, Y. Chen, X.
Zhao, C. Schmidl, T. Suzuki, E. Ntini, E. Arner, E. Valen, K. Li, L. Schwarzfischer, D.
Glatz, J. Raithel, B. Lilje, N. Rapin, F. O. Bagger, M. Jørgensen, P. R. Andersen, N.
Bertin, O. Rackham, A. M. Burroughs, J. K. Baillie, Y. Ishizu, Y. Shimizu, E. Furuhata,
S. Maeda, Y. Negishi, C. J. Mungall, T. F. Meehan, T. Lassmann, M. Itoh, H. Kawaji, N.
Kondo, J. Kawai, A. Lennartsson, C. O. Daub, P. Heutink, D. A. Hume, T. H. Jensen, H.
Suzuki, Y. Hayashizaki, F. Müller, A. R. R. Forrest, P. Carninci, M. Rehli, A. Sandelin,
An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461
(2014). doi:10.1038/nature12787 Medline
22. W. A. Whyte, D. A. Orlando, D. Hnisz, B. J. Abraham, C. Y. Lin, M. H. Kagey, P. B. Rahl,
T. I. Lee, R. A. Young, Master transcription factors and Mediator establish super-
enhancers at key cell identity genes. Cell 153, 307–319 (2013).
doi:10.1016/j.cell.2013.03.035 Medline
23. A. Groth, A. Corpet, A. J. L. Cook, D. Roche, J. Bartek, J. Lukas, G. Almouzni, Regulation
of replication fork progression through histone supply and demand. Science 318, 1928–
1931 (2007). doi:10.1126/science.1148992 Medline
37
24. M. Foltman, C. Evrin, G. De Piccoli, R. C. Jones, R. D. Edmondson, Y. Katou, R. Nakato, K.
Shirahige, K. Labib, Eukaryotic replisome components cooperate to process histones
during chromosome replication. Cell Rep. 3, 892–904 (2013).
doi:10.1016/j.celrep.2013.02.028 Medline
25. H. Huang, C. B. Strømme, G. Saredi, M. Hödl, A. Strandsby, C. González-Aguilera, S. Chen,
A. Groth, D. J. Patel, A unique binding mode enables MCM2 to chaperone histones H3-
H4 at replication forks. Nat. Struct. Mol. Biol. 22, 618–626 (2015).
doi:10.1038/nsmb.3055 Medline
26. R. Georgescu, Z. Yuan, L. Bai, R. de Luna Almeida Santos, J. Sun, D. Zhang, O. Yurieva, H.
Li, M. E. O’Donnell, Structure of eukaryotic CMG helicase at a replication fork and
implications to replisome architecture and origin initiation. Proc. Natl. Acad. Sci. U.S.A.
114, E697–E706 (2017). doi:10.1073/pnas.1620500114 Medline
27. M. E. Douglas, F. A. Ali, A. Costa, J. F. X. Diffley, The mechanism of eukaryotic CMG
helicase activation. Nature 555, 265–268 (2018). doi:10.1038/nature25787 Medline
28. V. Tran, C. Lim, J. Xie, X. Chen, Asymmetric division of Drosophila male germline stem
cell shows asymmetric histone distribution. Science 338, 679–682 (2012).
doi:10.1126/science.1226028 Medline
29. ENCODE Project Consortium, An integrated encyclopedia of DNA elements in the human
genome. Nature 489, 57–74 (2012). doi:10.1038/nature11247 Medline
30. Q. L. Ying, J. Wray, J. Nichols, L. Batlle-Morera, B. Doble, J. Woodgett, P. Cohen, A.
Smith, The ground state of embryonic stem cell self-renewal. Nature 453, 519–523
(2008). doi:10.1038/nature06968 Medline
31. Z. Tseng, T. Wu, Y. Liu, M. Zhong, A. Xiao, in Cancer Genomics and Proteomics (Springer,
2014), pp. 11–22.
32. S. I. Presolski, V. P. Hong, M. G. Finn, Copper-catalyzed azide-alkyne click chemistry for
bioconjugation. Curr. Protoc. Chem. Biol. 3, 153–162 (2011). Medline
33. P. V. Kharchenko, M. Y. Tolstorukov, P. J. Park, Design and analysis of ChIP-seq
experiments for DNA-binding proteins. Nat. Biotechnol. 26, 1351–1359 (2008).
doi:10.1038/nbt.1508 Medline
34. F. Ramírez, D. P. Ryan, B. Grüning, V. Bhardwaj, F. Kilpert, A. S. Richter, S. Heyne, F.
Dündar, T. Manke, deepTools2: A next generation web server for deep-sequencing data
analysis. Nucleic Acids Res. 44 (W1), W160–W165 (2016). doi:10.1093/nar/gkw257
Medline
35. E. Meshorer, D. Yellajoshula, E. George, P. J. Scambler, D. T. Brown, T. Misteli,
Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev.
Cell 10, 105–116 (2006). doi:10.1016/j.devcel.2005.10.017 Medline
36. Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C. Nusbaum, R.
M. Myers, M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIP-Seq (MACS).
Genome Biol. 9, R137 (2008). doi:10.1186/gb-2008-9-9-r137 Medline

38
37. G. Yu, L. G. Wang, Q. Y. He, ChIPseeker: An R/Bioconductor package for ChIP peak
annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015).
doi:10.1093/bioinformatics/btv145 Medline
38. K. De Brabanter, J. A. K. Suykens, B. De Moor, Nonparametric regression viaStatLSSVM.
J. Stat. Softw. 55, 1–21 (2013). doi:10.18637/jss.v055.i02
39. J. M. Engreitz, K. Sirokman, P. McDonel, A. A. Shishkin, C. Surka, P. Russell, S. R.
Grossman, A. Y. Chow, M. Guttman, E. S. Lander, RNA-RNA interactions enable
specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell
159, 188–199 (2014). doi:10.1016/j.cell.2014.08.018 Medline
40. S. Rennie, M. Dalby, L. van Duin, R. Andersson, Transcriptional decomposition reveals
active chromatin architectures and cell specific regulatory interactions. Nat. Commun. 9,
487 (2018). doi:10.1038/s41467-017-02798-1 Medline
41. I. Hiratani, T. Ryba, M. Itoh, T. Yokochi, M. Schwaiger, C.-W. Chang, Y. Lyou, T. M.
Townes, D. Schübeler, D. M. Gilbert, Global reorganization of replication domains
during embryonic stem cell differentiation. PLOS Biol. 6, e245 (2008).
doi:10.1371/journal.pbio.0060245 Medline
42. N. C. Durand, M. S. Shamim, I. Machol, S. S. P. Rao, M. H. Huntley, E. S. Lander, E. L.
Aiden, Juicer provides a one-click system for analyzing loop-resolution Hi-C
experiments. Cell Syst. 3, 95–98 (2016). doi:10.1016/j.cels.2016.07.002 Medline

39