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. 2015 Oct 15;43(18):8746-61.
doi: 10.1093/nar/gkv766. Epub 2015 Jul 30.

DNA Sequence Templates Adjacent Nucleosome and ORC Sites at Gene Amplification Origins in Drosophila

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Free PMC article

DNA Sequence Templates Adjacent Nucleosome and ORC Sites at Gene Amplification Origins in Drosophila

Jun Liu et al. Nucleic Acids Res. .
Free PMC article

Abstract

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

Figures

Figure 1.
Figure 1.
Sequences important for amplicon function are depleted of nucleosomes in stage 10 follicle cells. (A) Amplicon origin activity is restricted to follicle cells during late oogenesis. DAFC-66D is bound by ORC and active specifically during stages 10B-11 of oogenesis. Below is a stage 10 egg chamber showing the location of the somatic follicle cells (pink) that surround the germline oocyte and nurse cells. (B) Size distribution of all DNA fragments after MNase digestion of stage 10 follicle cell genomic DNA from Oregon-R (OR_s10.1) as determined by paired end sequencing. The filtered size range of 127–168 was used to map nucleosome positions. (CF) Nucleosome positions at the two major amplicons on the 3rd (DAFC-66D) and X (DAFC-7F) chromosomes. In each panel, the genomic coordinates and organization of the amplicons are shown above, and the corresponding signal graph for nucleosome occupancy shown in blue below. Transcription units are indicated by boxes, with translated regions in orange. Each locus has multiple chorion protein (cp) genes. Preferred initiation sites are indicated by black hashed lines and preferred ORC binding sites as red lines, but both amplicons reside within a larger zone of distributed ORC binding. (C) Nucleosome position at DAFC-66D during stage 10. Amplification enhancing elements (AERs a–d) stimulate origin activity and were defined by deletion mapping (87). The ACE3 and Ori-β are essential for origin activity, with the regions most important for origin function that correspond to ORC binding sites (red lines). (D) Enlargement of the ACE3 region. The green line demarcates an A-T rich region in ACE3 that is highly conserved at the amplicon in the genus Drosophila. (E) Enlargement of Ori-β region, the preferred site of replication initiation. (F) Nucleosome position at DAFC-7F. ACE1 is important for origin function, binds ORC, and is shown as a box, with the central region that has the strongest effect on origin function shown as a black box.
Figure 2.
Figure 2.
Nucleosome depleted regions (NDRs) at the amplicons are not exceptionally large. NDRs at DAFC-66D (A) and DAFC-7F (B) are demarcated by magenta lines and numbered. (C) Comparison of amplicon NDR sizes, defined by internucleosome distance, to the distribution of all NDRs genome-wide. The orange line represents the distribution of all NDR sizes in the follicle cell genome during stage 10. The dots represent the size of the different NDRs from DAFC-66D (66D) and DAFC-7F (7F) as defined in panels (A) and (B).
Figure 3.
Figure 3.
Nucleosome positions at DAFC-66D are similar among different stages and cell types. (A) Distribution of internucleosomal distances in Mnase-Seq data for follicle cells from different stages of oogenesis and embryonic S2 cells in culture. Follicle cells from stages 1–8 (OR_s8.1, OR_s8.2), stage 10 (OR_s10.1, OR_s10.2), or stage 12–13 (OR_s12.1), or stage 10 in which replication was inhibited by expressing dacapo (dap_s10). Numbers after the decimal points represent biological replicates. Internucleosomal distance for replicates of S2 cells in culture (S2cell.1, S2cell.2, S2cell.3) represents analysis of published MNase reads (79). (B and C) Nucleosome occupancy at DAFC-66D Ori-β (B) and ACE3 (C) are similar in follicle cells from different stages of oogenesis and S2 cultured cells.
Figure 4.
Figure 4.
NDR sizes at DAFC-66D and DAFC-7F are similar among different stages and cell types. (A and B) Nucleosome occupancy in follicle cells during different stages of oogenesis and in S2 cells in culture at DAFC-66D (A) and DAFC-7F (B). The different NDRs at the origins are indicated by magenta lines and numbers. (C) Comparison of NDR sizes in follicle cells at stages 1–8 (OR_s8.1), stage 10 (OR_s10.1), and in S2 cultured cells (S2cell.1) (79). The different NDR's at DAFC-66D (66D) and DAFC-7F (7F) correspond to the numbers shown in (A) and (B).
Figure 5.
Figure 5.
ORC binding sites are nucleosome depleted in S2 cells and ovarian follicle cells. Average nucleosome occupancy at ORC sites genome wide (y-axis) is plotted around the center of the ORC binding sites (x-axis) for follicle cells at different stages (OR_s8.1, OR_s8.2, OR_10.1, OR_10.2, dap_s10, OR_s12.1) and S2 cells in culture (S2cell.1, S2cell.2, S2cell.3). ORC sites with binding score >7.5 and S2 cell MNase-Seq data were from published sources (36,79).
Figure 6.
Figure 6.
Nucleosome position at DAFC-66D is largely determined by primary DNA sequence. (A) Comparison of observed nucleosome occupancy at DAFC-66D in follicle cells in stage 10 (dark blue) with predicted nucleosome occupancy (light blue) based on nucleosome DNA sequence preferences (104,105). Red asterisks indicate three predicted nucleosome positions that were less occupied in vivo than predicted. (B and C) Expanded view of predicted and observed nucleosome occupancy at ACE3 (B) and Ori-β (C), with nucleotide composition plotted above (see color key). Nucleosome occupied sites are relatively GC rich (black and blue), while nucleosome depleted regions in ACE3 and Ori-β contain extended poly A:T tracts (red and green) that correspond to ORC binding sites.
Figure 7.
Figure 7.
ORC binding to DAFC-66D is influenced by DNA sequence and hyperacetylation of positioned nucleosomes. The DAFC-66D 3.8 kb minimal origin with nucleosome occupancy in vivo (blue) shown above and relative ORC binding to different naked DNA fragments in vitro indicated below (bar graph) (12). The preferred nucleosome and ORC occupancy distributions are inverse of each other. Regions of GC rich DNA (blue line) and conserved, bent, poly A:T DNA (red box) are indicated (94,124). The red box above indicates the region of greatest hyperacetylation on histones H3 and H4 (H3Ac / H4Ac) that occurs exclusively during stages 10–11 when ORC is bound and the origin is active (70). This acetylated nucleosome resides between the sites of high ORC occupancy, with one corresponding to Ori-β, the preferred replication initiation zone (black dotted line) (89,90). This region lies at the center of a diminishing gradient of ORC occupancy and histone acetylation that extends outwards over ∼20 kb epigenome domain (red arrows below) (66,70). These data lead to a model wherein both DNA sequence and nucleosome acetylation contribute to the location and developmental timing of pre-RC assembly at the amplicons.

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