Cell-cycle regulated transcription associates with DNA replication timing in yeast and human

Abstract

Background: Eukaryotic DNA replication follows a specific temporal program, with some genomic regions consistently replicating earlier than others, yet what determines this program is largely unknown. Highly transcribed regions have been observed to replicate in early S-phase in all plant and animal species studied to date, but this relationship is thought to be absent from both budding yeast and fission yeast. No association between cell-cycle regulated transcription and replication timing has been reported for any species.

Results: Here I show that in budding yeast, fission yeast, and human, the genes most highly transcribed during S-phase replicate early, whereas those repressed in S-phase replicate late. Transcription during other cell-cycle phases shows either the opposite correlation with replication timing, or no relation. The relationship is strongest near late-firing origins of replication, which is not consistent with a previously proposed model—that replication timing may affect transcription—and instead suggests a potential mechanism involving the recruitment of limiting replication initiation factors during S-phase.

Conclusions: These results suggest that S-phase transcription may be an important determinant of DNA replication timing across eukaryotes, which may explain the well-established association between transcription and replication timing.

Figure 1

The transcription/T_rep association varies by cell-cycle stage. (A) Comparing mean T_rep of the top decile (10%) of most-induced versus most-repressed cell cycle-regulated genes reveals that genes highly expressed in G2 replicate early in both Sc and Sp, whereas those highly expressed in M/G1 (Sc) or G1 (Sp) replicate late (error bars represent the standard error). (B) The correlation between T_rep and expression levels of known cell cycle-regulated genes was calculated separately for expression levels from each time point of cell cycle-synchronized time courses [27,28]. An oscillation of the correlation coefficient (Pearson’s r) was observed for both budding yeast (all |r| > 0.107 are significant at P < 0.0025) and fission yeast (all |r| > 0.177 are at P < 0.0025). The approximate cell-cycle phase of each time point is shown [27,28]. Similar oscillations are observed for other methods of synchronization as well (Additional file 1: Figures S1 and S2). (C) A moving average of T_rep is shown for all cell cycle-regulated genes, arranged in order of their time of maximal expression, beginning immediately following mitosis. A similar pattern is observed for both yeast species, with the latest T_rep for genes with maximal transcript levels in G1, and the earliest T_rep for genes with maximal transcript levels in G2.

Figure 2

Asynchronous gene expression associates with T_rep in budding and fission yeast. Comparison of the 100 highest-expressed genes with the 100 lowest-expressed shows that highly expressed genes are replicated earlier in both budding yeast and fission yeast. Error bars represent the standard error.

Figure 3

Factors affecting the strength of the transcription/T_rep association. (A) Median G2-phase transcript levels (representing S-phase transcription; Additional file 1: Figure S4A) are shown for all cell cycle-regulated genes separated into 10 equally sized bins (deciles) by their T_rep. For both yeast species, no correlation is observed for the first five bins, whereas a strong relationship is present for later T_rep. (B) Consistent with the decile analysis, no oscillation is observed in the correlation between expression level and T_rep for early T_rep genes, while a strong oscillation is observed for late T_rep genes. (C) Only weak oscillation is observed in the correlation between expression level and T_rep for ORI-distal genes (>5 kb from the nearest ORI in budding yeast, or 10 kb in fission yeast), while a strong oscillation is observed for ORI-proximal genes.

Figure 4

Transcription and T_rep in human. (A) No oscillation is observed when comparing the T_rep versus expression levels of all cell cycle-regulated genes in HeLa cells (all |r| > 0.063 are significant at P < 0.05; the four time-points that exceed this are within the range expected by chance, given that 47 time-points were analyzed). (B) Significant oscillation is observed when comparing T_rep versus expression levels of cell cycle-regulated genes with late T_rep (red line; the final 50% of S phase; all |r| > 0.195 are significant), but not early T_rep (blue line). (C) Significant oscillation is observed when comparing T_rep versus expression levels of cell cycle-regulated genes within 10 kb of an ORI (blue line; all |r| > 0.197 are significant), but not further than 10 kb from an ORI (red line).

Figure 5

A model to explain these observations. Components: ORC and MCM2-7 are protein complexes comprising the pre-replicative complex. Blue cylinders represent nucleosomes, with dark blue indicating closed/repressive chromatin and light blue indicating open/accessible chromatin. Red proteins are limiting replication initiation factors (such as Cdc45 and Sld3). Txn = transcription. Sequence of events: in G1 (not depicted), the limiting replication initiation factors (red circles) associate with the earliest-firing ORIs (top row). When S phase begins, these early ORIs fire and release the factors, which are then free to associate with other ORIs (though note that Cdc45 is a component of the replication fork, so can only be recycled after fork termination). The relative affinities of the remaining ORIs for these factors - and thus their relative firing times - are determined by the chromatin state near the ORI during S-phase. ORIs near genes highly transcribed in S phase (middle row) have an accessible chromatin structure and thus high affinity, so will tend to fire earlier than those with little nearby S-phase transcription and thus less accessible chromatin (bottom row). Although not shown here, subnuclear positioning could help determine ORI accessibility, either by influencing chromatin structure or through other mechanisms. Figure adapted from [19].