Genomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection - PubMed (original) (raw)

Genomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection

Neerja Karnani et al. Mol Biol Cell. 2010.

Abstract

DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, approximately 50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure.

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Figures

Figure 1.

Figure 1.

(A) Schematic of NS-BrIP and NS-LExo methods used to purify nascent strands and map replication origins. (B and C) NS peaks from array hybridizations. UCSC browser image showing MAT score for enrichment of nascent strand signal relative to control, and nascent strand hit positions identified for NS-BrIP and NS-Lexo on chromosome 11 (ENCODE region ENm009). The nascent strand hits had signal intensity that was enriched above the background at p ≤ 10−3. Nascent strand hits that overlapped across the NS-Lexo and NS-BrIP methods are indicated by asterisk.

Figure 2.

Figure 2.

Validation by qPCR confirms microarray data. Positive and negative sites detected by hybridization of nascent strands to microarrays were randomly selected from different chromosomal fragments and tested for enrichment (nascent strands over genomic) by qPCR by using two biological replicates of nascent strand preparations. qPCR for each biological replicate was performed in triplicate. Average Z scores of biological replicates were plotted for all the tested chromosomal regions as well as the known origins (b-globin and c-myc origins) and their respective background control regions (amylase and cymc background). The threshold for calling a microarray site positive was set at ≥15 standard deviations higher (vertical red line) than the background control regions (in this case amylase). Validation for NS-BrIP microarray data (primers to test array positives BP1 … BP15 and array negatives BN1 … BN11) (A) and validation of NS-LExo data set (primers to test array positives LP1 … LP15 and array negatives N1 LN11) (B). Asterisk, >150 Z score.

Figure 3.

Figure 3.

Concordance of origins across the methods. (A) Venn diagram showing the overlap between NS-LExo and NS-BrIP methods. The number of fragments found in each group is shown between brackets and the number of overlapping segments is given in the intersected regions. (B) Browser image showing a comparison of the origins identified by NS-BrIP and NS-LExo methods within a 400-kb region of chromosome 7 (ENCODE region ENm012).

Figure 4.

Figure 4.

Correlation of ORI with transcription initiation and transcription factors. (A) Frequency-histograms showing distribution of replication initiation sites with respect to distance from TSS. (B) Enrichment relative to random model of the localization of ORI with respect to TSS at 5 and 10 kb. (C) Origin activity at or within 5 kb of RNA pol II binding sites. (D) Origin activity relative to RFBR clusters. Data in B–D are expressed as percentage of enrichment (or depletion) in experimental data minus the percentage of enrichment (or depletion) in the random model. Asterisk indicates statistically significant enrichment/depletion relative to the random model (p range < 0.01–0.0001).

Figure 5.

Figure 5.

(A) Distribution of histone marks and DHS sites around ORIs. (B) Histograms showing whether origins were selectively enriched or depleted in chromosomal segments known to replicate in different parts of S phase: early, mid, late as defined previously (Karnani et al., 2007). (C) Correlation between replication origins and AT content. The horizontal line indicates the percent AT content of the ENCODE region (1% human genome). Data in A and B are expressed as percentage of enrichment (or depletion) in the experimental data minus that in the random model. Asterisk indicated significant enrichment/depletion relative to random model (p < 0.01–0.0001).

Figure 6.

Figure 6.

Validation of ORIs by ORC ChIP. (A) Fifteen ORIs were tested for ORC binding by performing ChIP assays with Orc2 and Orc3 antibodies. Rabbit IgG was used as a measure of nonspecific chromatin precipitation. b-globin, c-myc, and lamin B2 origins were used as positive controls. Origins that were positive for one or both ORC subunits are indicated with an asterisk. (B) Thirteen sites that were not called as ORIs by microarray data were used as negative controls. The ethidium bromide gel documentation images were stretched or reduced in width to make all panels the same size.

Figure 7.

Figure 7.

Comparison of genomic features specific to two methods of nascent stand purification. (A) Segregation of NS-method specific peaks and ORIs into different temporal classes of S phase (early, mid, and late). (B) Localization of NS-method–specific peaks and ORIs with respect to TSS at 5 and 10 kb. (C) NS-method peaks and ORIs at or within 5 kb of RNA pol II binding sites. (D) RFBR clusters. (E) Histone marks and DHS sites. Data in A–E are expressed as percent enrichment (or depletion) minus that in the random model. Asterisk indicated significant enrichment/depletion relative to random model (p range < 0.01–0.0001). (F) Validation of NS-BrIP– and NS-LExo–specific peaks by ORC ChIP. Six nascent strand peaks each from NS-BrIP– (Bsp1–6) and NS-LExo–specific (Lsp1–6) categories were tested for ORC binding by performing ChIP assays with Orc2 and Orc3 antibodies. Rabbit IgG was used as a negative control. Nascent strand peaks that were positive for one or both ORC subunits are indicated with an asterisk. The ethidium bromide gel documentation images were stretched or reduced in width to make all panels the same size.

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