nature structural & molecular biology: doi:10.1038/nsmb10.1038/nsmb.3363... · (a) boxplots...

Nature Structural & Molecular Biology: doi:10.1038/nsmb.3363

Supplementary Figure 1

Flow cytometry analysis of the mixed and pregastrula embryo populations.

(a) Nascent strand (NS) isolation schematic. 0.3-2 kb NS were isolated from total genomic DNA by denaturation and sucrose gradient centrifugation. NS enriched by lambda-exonuclease digestion were hybridized against total genomic DNA on high-density tiling arrays (see Methods for details). (b) Left panels show the scatter distribution (forward vs. side) of the analyzed events (upper panel, mixed C. elegans embryos; lower panel, pre-gastrula embryos). Middle panels show the cell cycle distribution of the events of the lefts panels. For the mixed embryo population, two windows are shown, large (1) and small (2) cell size, to confirm that both populations contain cells in S-phase. The pre-gastrula embryo middle panels represent the whole scatter distribution. PI, propidium iodide. The right panels show representative images of each embryo population analyzed by cell sorting. Embryos were labeled with EdU (green) and stained with DAPI (blue) (merged images on the right panels).



Replicates and validation of C. elegans replication origins.

(a) Correlation of the nascent strand microarray data in two of the three replicates for the pre-gastrula and mixed embryo populations. (b) Heatmap representing a significant set of probes of the three replicates (R1-R3) of nascent strand microarray data for pre-gastrula


(left) and mixed embryo (right) origins. Colors indicate the intensity of the microarray probe, from low (green) to red (high). (c) Alignment of three independent nascent strand microarray experiments (R1-R3) along C. elegans chromosome 1. The bottom panels show representative profiles of origins in the chromosome arms and center. The background signal from the L1-arrested larval sample was subtracted from each replicate (-L1). (d) Quantitative PCR validation of C. elegans replication origins detected by microarray analysis of the NS purification (n=3), together with the corresponding controls. The graph shows the microarray normalized log2-ratio data (samples compared with genomic DNA) as well as the position of inverted repeats.



Association of inverted repeats and operons with C. elegans replication origins.

(a) Boxplots showing the percentage of association of inverted repeats (IRs) with pre-gastrula and post-gastrula-specific origins (top graphs) and the reverse correlation (bottom panels), compared with random expectation, in chromosome arms and center (left and right panels respectively). *, P<10

-15 (according to the distribution function of the normal distribution); n.s., non-significant difference with

random. (b) Boxplots showing the number of pre-gastrula (green) and post-gastrula-specific (orange) origin summits in operons, compared with random data. *, P<10

-20 (according to the distribution function of the normal distribution). Data are shown as mean ± s.d.

(c) Distribution (%) of pre-gastrula (green) and post-gastrula-specific (orange) replication origins around operon transcription start sites (TSS) (mean percentage ± standard error) in chromosome arms and center.



C. elegans pregastrula origins are localized in open chromatin in transcriptional active regions.

(a) Distribution of the ChIP-Chip signals for the HTZ-1 histone variant and RNA pol II around ±2 kb from pre-gastrula replication origin summits (solid green line) or the centers of random regions (gray line; mean ± 2x s.d. for HTZ-1 and mean ± s.d. for RNA pol II) in chromosome arms (top panels) and centers (lower panels). Only autosomal chromosome were taken in account for HTZ-1 histone variant (b) Numbers of gap regions (left panels) or LEM-2 subdomains (right panels) inside chromosome arms that are overlapped by pre-gastrula origins versus random regions (number ± s.d.). The reverse correlations are also shown (bottom panels). *, P<0.05 (two-sided t-tests); n.s., non-significant differences with random expectation. (c) Mean distributions of pre-gastrula (green) and post-gastrula-specific (orange) origins in each chromosome (origin density/200 kb). Dashed black lines indicate the mean level of gene transcript (dcpm) at each chromosome. (d) Percentage of HOT regions associated with pre- (green) and post-gastrula-specific (orange) replication origins in chromosome arms and centers. * indicates P<10

-15 (Chi-squared test); n.s., non-significant difference with random

(mean ± s.d.). (e) Replication origin efficiency (total coverage of probes intensity; mean ± s.d.) relative to some genomic features. *, P<10

-8; **, P<10

-12 (two-sided t-tests) compared with the efficiency in all replication origins.



Distribution of C. elegans origins relative to genes and promoters.

(a) Percentage of pre-gastrula (green) and post-gastrula-specific (orange) replication origins around non-coding TSSs (±3000 bp) in the chromosome arms (left panel) or centers (right panel). Mean random profiles are represented by a full line (± standard error in dashed lines) in a lighter color than the one used for the real data. (b) Box plots showing the percentage of pre-gastrula (green) and post-gastrula-specific (orange) origin summits found in all non-coding gene promoters (total promoters) as well as in non-coding gene promoters in chromosome centers, left and right arms. *, P<10

-20 (according to the distribution function of the normal distribution); n.s.,

non-significant difference with random expectation (mean ± s.d.). (c) ChIP signals for histone H3K4me3 and RNA pol II around ±2 kb from the TSS of non-coding genes overlapped (left panel) or not (right panel) by post-gastrula-specific origins. The y-axis represents the average ChIP signal per 100 bp.



Sequence motifs associated with replication origins.

Scanning for the occurrence of GCTAAAA (a), [AC][GC]GC[CG]C (b) and CG[AC][GC]GCGC (c) motifs in 1-kb regions on both sides of origin summits. The x-axis indicates the position relative to the origin summit, the y-axis the percentage of pre-gastrula origins (green line), post-gastrula-specific origins (orange line) or 100 randoms (gray lines) with at least one occurrence per 100-bp window. Both Watson (sense) and Crick (antisense) chromosomal strands were considered. (d) Box plots showing the total percentage of pre-gastrula (green) and post-gastrula-specific (orange) origin summits overlapping with CGI-like sequences (for origins and random data) (mean ± s.d.) and in (e) chromosome arms and center, separately. *, P<10

-8 (according to the distribution function of the normal

distribution). (f) Results of bubble trap and 2D gel electrophoresis analysis for a region containing a post-gastrula-specific origin with a CGI-like (positive region; brown box; left panels) and a region without origin and CGI-like (negative region; right panel, same negative region already shown in Figure 2C). The raw data of these regions are on top (combination of three NS microarray replicates), the qPCR validation in the middle, and the 2D gels at the bottom. The black box indicates the position of the probe used for hybridization and the thin pink lines, the Cla1 sites. The results at the bottom are shown for both regions, comparing a mixed embryo sample and a negative control (L1-arrested larval sample). The arrow indicates the position of the signal representing the replication origin.


Supplemental_Table_S1:Tablesummarizingthesynchronizationresults(%)for15lculturesinbioreactor.Thedevelopmentalstagecorrespondingtoeachtimepointishighlightedinyellow.

Larvalstage 0 14 23 32 44 57L1 100 0.8 0.4 0.4 0.4 -L2 0 99.2 0.6 0.4 0.4 -

Dauer 0 0 0.8 0.8 0.8 -L3 0 0 98.2 0.2 0.4 -L4 0 0 0 98.1 0.6 -

Adults 0 0 0 0 97.4 -Adults(2-26cellsembryos) 0 0 0 0 0 93.2±2.6

Culturetime(hours)


Supplemental_Table_S2: Table indicating pre-gastrula and post-gastrula origin density for every chromosome and chromosome domain

Chromosome arms/Centre Length Origin number Origins/100 kb Mean origins/100 kb Origin number origins/100 kb Mean origins/100 kbChrI 17.3 7.8

Left arm 3500000 701 20.029 260 7.4Centre 7500000 1102 14.693 657 8.8

right arm 4072421 801 19.669 252 6.2

ChrII 13.8 8.5Left arm 4300000 613 14.2 318 7.4Centre 7700000 949 12.3 677 8.8

right arm 3279323 549 16.7 297 9.1

ChrIII 16.7 7.6Left arm 3800000 746 19.6 252 6.6Centre 7100000 987 13.9 599 8.4

right arm 2883681 575 19.9 202 7.0

ChrIV 13.4 8.3Left arm 4300000 763 17.7 325 7.6Centre 6400000 722 11.3 553 8.6

right arm 6793785 853 12.6 578 8.5

ChrV 10.4 7.5Left arm 5100000 622 12.2 397 7.8Centre 10400000 845 8.1 819 7.9

right arm 5419568 716 13.2 358 6.6

ChrX 11.5 9.4Left arm 2000000 267 13.3 169 8.5Centre 15718854 1770 11.3 1501 9.6

Mean left arms 3833333.3 618.0 16.1 268.8 7.5Mean right arms 4489755.6 698.8 15.6 337.4 7.5Mean arms 4131707.1 655.1 15.9 309.8 7.5Mean Centre 9136475.7 1062.5 11.6 801.0 8.8Mean all chrom 5898096.0 798.9 13.5 483.2 8.2

Pre-gastrula Post-gastrula specific


nature structural & molecular biology: doi:10.1038/nsmb10.1038/nsmb.3363... · (a) boxplots...

Documents