Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors

Methylation of histone H3 on lysine 79 associates with a group of replication origins and helps limit DNA replication once per cell cycle.

Mammalian DNA replication starts at distinct chromosomal sites in a tissue-specific pattern coordinated with transcription, but previous studies have not yet identified a chromatin modification that correlates with the initiation of DNA replication at particular genomic locations. Here we report that a distinct fraction of replication initiation sites in the human genome are associated with a high frequency of dimethylation of histone H3 lysine K79 (H3K79Me2). H3K79Me2-containing chromatin exhibited the highest genome-wide enrichment for replication initiation events observed for any chromatin modification examined thus far (23.39% of H3K79Me2 peaks were detected in regions adjacent to replication initiation events). The association of H3K79Me2 with replication initiation sites was independent and not synergistic with other chromatin modifications. H3K79 dimethylation exhibited wider distribution on chromatin during S-phase, but only regions with H3K79 methylation in G1 and G2 were enriched in replication initiation events. H3K79 was dimethylated in a region containing a functional replicator (a DNA sequence capable of initiating DNA replication), but the methylation was not evident in a mutant replicator that could not initiate replication. Depletion of DOT1L, the sole enzyme responsible for H3K79 methylation, triggered limited genomic over-replication although most cells could continue to proliferate and replicate DNA in the absence of methylated H3K79. Thus, prevention of H3K79 methylation might affect regulatory processes that modulate the order and timing of DNA replication. These data are consistent with the hypothesis that dimethylated H3K79 associates with some replication origins and marks replicated chromatin during S-phase to prevent re-replication and preserve genomic stability

Depletion of H3K79 methyltransferase <i>DOT1</i>L does not change replication elongation and initiation rates.

<p>HCT116 cells were transfected with siRNA directed against <i>DOT1L</i> or scrambled siRNA control twice with a 48 h interval. A. Levels of H3K79Me2 in total cell proteins collected 72 hours after the second transfection. Actin was used as a loading control. B–D. Cells were labeled sequentially with ldU and CIdU as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#s4" target="_blank">Methods</a> section. Cells were then harvested and DNA extracted. The DNA was stretched on a silanized microscope coverslip, and visualized with antibodies against DNA containing ldU and CldU <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542-Conti2" target="_blank">[48]</a>. B. An example of combed DNA. Replication fork progression rates were calculated from the length of CldU (red replication tracks) and IdU (green replication tracks) signals. Inter-origin distance was measured by identifying replication initiation events (ori1 to ori3). C. A histogram of the distribution of replication fork speeds measured in DNA fibers from cells transfected with scrambled siRNA. D. A histogram of the distribution of replication fork speed measured in DNA fibers from cells transfected with siRNA targeting <i>DOT1</i>L. Depletion of <i>DOT1</i>L reduced the levels of H3K79Me2 but did not affect DNA replication fork velocity and inter-origin distance (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542.s005" target="_blank">Figure S5</a>).</p

Preferential enrichment of initiation events in H3K79Me2 containing chromatin at the G1 and G2 phases of the cell cycle.

<p>An asynchronously growing population of K562 cells was fractionated into separate populations of G1, S-phase and G2/M cells using centrifugal elutriation. Chromatin from each separate cell cycle phase population was isolated and analyzed by H3K79Me2 ChIP-Seq as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen-1003542-g001" target="_blank">Figure 1</a>. A. The distribution of H3K79Me2 and replication initiation events in the MYC locus during the different phases of the cell cycle. B. The distribution of H3K79Me2 and replication initiation events in the DBF4 locus during the different phases of the cell cycle. C. A box plot as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen-1003542-g001" target="_blank">Figure 1</a> legend showed the relative enrichment in H3K79Me2 for replication initiation events during the different phases of the cell cycle. Boxes indicate distributions of the second and third quartiles and whiskers, 95^th percentiles; values lower than a unit were converted into 1. All, replication initiation ratios in regions that were associated with H3K79Me2 in an asynchronous cell sample representing all stages of the cell cycle; G1, S and G2, regions associated with H3K79Me2 in samples from cells at the appropriate cell cycle phase; S-only, regions that were only associated with H3K79Me2 in S-phase but not in G1 and G2. D. The number of H3K79Me2 associated peaks on chromatin during subsequent stages of cell cycle progression. H3K4Me3 was used as a control. Chi-square test shows that the distributions of H3K79Me2 and H3K4Me3 in G1, S and G2 cells are statistically different (p<0.0001). H3K79 dimethylation was more abundant and exhibited a wider distribution during S-phase, but chromatin regions associated with H3K79Me2 solely in S-phase were not further enriched in replication initiation events. The association of H3K79Me2 with replication initiation events was re-established in G2.</p

H3K79 dimethylation accompanies replicator activity.

<p>A. Two transgenes containing sequences from the human beta-globin Locus Control Region (HS432), the human beta-globin promoter (GloPro) driving enhanced green fluorescent protein (EGFP) and two variants of the Rep-P replicator were inserted into a single location on murine chromosome 15 in murine erythroleukemia (RL4) cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542-Feng1" target="_blank">[11]</a>. Murine cells were utilized to facilitate detection of the exogenous sequences from the human beta globin locus; the murine locus control region was used as a control. One transgene variant (Rep-PWT) contains the native unaltered sequence of Rep-P-2 (starting 87 bp 5′ of the beta-globin promoter) that is essential for replication initiation. The other transgene variant (Rep-PAG1) harbors two point mutations at the Rep-P-2 sequence that prevent initiation of DNA replication. B. Chromatin immunoprecipitation with antibodies directed against H3K79Me2 in RL4 cells carrying wild-type (Rep-PWT) or mutant (Rep-PAG1) transgene cassettes. Each column represents the mean enrichment value inH3K79Me2 calcualted based on real-time PCR amplification of chromatin immunoprecipitation using the indicated primer pairs. Error bars indicate standard deviations. C. Nascent strand abundance analysis in RL4 cells carrying wild type (Rep-PWT) or mutant Rep-P (Rep-PAG1) transgene cassettes. Primers and probes used, listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen-1003542-t003" target="_blank">Table 3</a>, included GloPro (human beta-globin promoter), EGFP (the EGFP gene), bG59.8 (Rep-P 5′ end sequence), bG61.3 (Rep-P 3′ end sequence), AG (the AG region of Rep-P), and mLCR (murine Locus control region). All except mLCR are sequences from the transgene and their location is illustrated in the map shown in panel A. Sequences from transgenes harboring the active replicator were enriched in H3K79Me2 containing chromatin whereas sequences from the mutant transgene that did not initiate replication, were not.</p

H3K79Me2 containing chromatin is associated preferentially with replication initiation sites genome-wide.

<p>A–E. Screenshots of replication initiation data visualized with the integrated Genome Viewer (<a href="http://www.broadinstitute.org/igv/" target="_blank">http://www.broadinstitute.org/igv/</a>). A chromosome map is shown at the top, and the region-of-interest is delineated by a circle. The analyzed region is shown underneath the ideogram, with map coordinates indicated. The Replication panel shows the distribution of replication initiation events (ratio of reads obtained from a nascent strand preparation, and reads obtained from a corresponding control genomic DNA preparation) of the region-of-interest obtained from our published database, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542-Martin1" target="_blank">[3]</a> for details. Regions abundant in H3K79Me2 immunoprecipitated chromatin from H3K79Me2 ChIP-Seq in K562 cells are shown below the initiation profile as reads per kilobase per million aligned reads (RPKM). Ref-Seq genes are aligned above the experimental data. A. The distribution of H3K79Me2 and replication initiation events in the MYC locus. B. The distribution of H3K79Me2 and replication initiation events in the DBF4 locus. C. The distribution of H3K79Me2 and replication initiation events in the LCORL locus. D. The distribution of H3K79Me2 and replication initiation events in the BAG1 locus. E. The distribution of H3K79Me2 and replication initiation events in the UBAP1 locus. F. A box plot comparing the relative enrichment of replication initiation events in chromatin featuring H3K79Me2 obtained by ChIP-Seq as described in methods with various other chromatin features as reported in the UCSC genome browser (for details, see<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542-Martin1" target="_blank">[3]</a>. Boxes indicate distributions of the second and third quartiles and whiskers, 95^th percentiles. The horizontal lines in the boxes represent medians. Values lower than a unit were converted into 1. Methylation of H3K79 exhibits a marked enrichment in replication initiation events that was higher than any other measured histone modification or transcription factor. G. A histogram showing the replication enrichment ratio (calculated as in A, B) for genomic regions as a function of their distance from the closest H3K79Me2 interaction sites. A box plot version of the same histogram is shown as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003542#pgen.1003542.s001" target="_blank">Figure S1</a>.</p

Fraction of H3K79Me2 regions that are within 2000 bp from a 15% FDR replication peak.

*<p>Chromatin regions associated with H3K79Me2 solely in S-phase.</p

Primers and probes used in the study.

<p>Primers and probes used in the study.</p