Nucleotide supply, not local histone acetylation, sets replication origin usage in transcribed regions

Abstract

In eukaryotes, only a fraction of replication origins fire at each S phase. Local histone acetylation was proposed to control firing efficiency of origins, but conflicting results were obtained. We report that local histone acetylation does not reflect origin efficiencies along the adenosine monophosphate deaminase 2 locus in mammalian fibroblasts. Reciprocally, modulation of origin efficiency does not affect acetylation. However, treatment with a deacetylase inhibitor changes the initiation pattern. We demonstrate that this treatment alters pyrimidine biosynthesis and decreases fork speed, which recruits latent origins. Our findings reconcile results that seemed inconsistent and reveal an unsuspected effect of deacetylase inhibitors on replication dynamics.

Figure 1

Histone acetylation patterns do not correlate with origin efficiency. Chromatin immunoprecipitation experiments were performed with antibodies recognizing acetyl histone (A) H3 (06-599) or (B) H4 (06-598) and analysed by quantitative PCR using eight sets of primers (supplementary Tables S1 and S2 online). The acetylation level was normalized for each cell line to the median level along the locus (arbitrary units). Error bars represent s.e.m. values of three experiments. Grey boxes, genes; red circles, origins. (C) Schematic representation of origin efficiencies along the AMPD2 locus. Numbers represent origin efficiency in the indicated cell lines (data from the study by Anglana et al (2003) and circle colours symbolize this efficiency (orange, major origin; grey, minor origin; and tan, average efficiency). 474 versus 474AU and 474 versus 422 cells were compared for histone H3 and H4 acetylation profiles using Spearman and Wilcoxon's signed rank tests. In all cases, the correlation between mean values was significant (P<0.05) and the difference between medians of mean values was not significant, indicating that acetylation profiles are similar. Histone acetylation levels on oriGNAI3 were compared with those on oriA, oriB and oriC using the Mann–Whitney–Wilcoxon test. The levels of acetylation of both histones are significantly lower on oriGNAI3 (P=0.004 and P=5e6 for H3 and H4, respectively). Acetylation levels of oriA were similarly compared with those of oriGNAI3, oriB and oriC. The levels of acetylation of both histones are significantly higher on oriA (P=5e6 and P=6e6 for H3 and H4, respectively). See supplementary Table S3 online for details. AMPD2, adenosine monophosphate deaminase 2.

Figure 2

Histone acetylation patterns in growing or G1-arrested cells correlate neither with localization nor with efficiency of origins. Chromatin immunoprecipitation experiments were performed with antibodies recognizing acetyl histone (A,C,E,G,I) H3 or (B,D,F,H,J) H4 and analysed by microarray hybridization. Results are expressed as the ratio of immunoprecipited DNA signal to the input signal (arbitrary units). Mean values of two experiments are reported. Circles symbolize: orange, major origin; grey, minor origin; tan, average efficiency. Black star: rearrangement breakpoint. Cells were compared for histone acetylation profiles by using the Spearman test. In all cases, the correlation between mean values was significant (P<0.05). Comparison and P-value results are indicated at the top right corner of each graph.

Figure 3

Trichostatin A treatment induces global and locus-specific histone hyperacetylation. (A) Bulk histone H3 acetylation analysed by western blotting (left panel) with quantification of the signals (right panel). Similar results were obtained for histone H4 (data not shown). (B,C) Chromatin immunoprecipitation experiments were performed with antibodies recognizing acetyl histone (B) H3 or (C) H4 and analysed by quantitative PCR. Acetylation level was normalized for each condition to the median level along the locus at 0 h (Ctl). Mean values of two experiments are reported. Grey boxes, genes; red circles, origins. Control and TSA 24 h were compared for histone acetylation levels using the Wilcoxon's signed rank test. In all cases, the difference between medians of mean values was significant (P=0.008 and P=0.02 for H3 and H4, respectively) confirming that histone acetylation increases after TSA treatment. CT, cytidine and thymidine; TSA, trichostatin A.

Figure 4

Trichostatin-A-induced changes in replication dynamics of cells from cell line 422 are abrogated by exogenous precursors. (A) Relative frequency of initiation events at the genome-wide level as determined by the ratio of the number of initiation events relative to replication events (replication forks and initiations). Frequency of initiation was normalized to that of 0 h −CT. −CT and +CT, without and with cytidine and thymidine added in the medium. Values were normalized to that of 0 h. At least 100 DNA fibres were analysed for each time-point and culture condition. −CT and +CT conditions were compared by using a χ²-test. Densities of initiation are not significantly different between the two conditions at 0 h, whereas they are at 24 h (P=0.01). Similarly, densities of initiation are not significantly different between 0 h and 24 h in +CT conditions. (B) Localization of initiation events along the AMPD2 locus. At least 30 DNA fibres were analysed for each condition. Circles symbolize: orange, major origin; grey, minor origin; tan, average efficiency. Fisher's exact test indicated that initiation profiles are not significantly different between Ctl and TSA 6 h+CT, whereas they are different between Ctl and TSA 6 h (P=0.007). (C) Fork speed (left) and examples of combed DNA molecules labelled with IdU then with CldU (right). −CT and +CT conditions were compared using a Mann–Whitney–Wilcoxon test. Replication speeds are not significantly different between the two conditions at 0 h, whereas they are at 24 h (P<2e16). (D,E) CTP synthetase and TS messenger RNA levels. For each condition, levels were normalized to that of zero time point (arbitrary units). Errors bars represent standard error of the mean of two experiments. AMPD2, adenosine monophosphate deaminase 2; AU, arbitrary units; CldU, chlorodeoxyuridine; IdU, iododeoxyuridine; TS, thymidylate synthase.