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. 2017 Mar 17;45(5):2490-2502.
doi: 10.1093/nar/gkw1211.

Temporal association of ORCA/LRWD1 to late-firing origins during G1 dictates heterochromatin replication and organization

Yating Wang  1 , Abid Khan  1 , Anna B Marks  2 , Owen K Smith  2 , Sumanprava Giri  1 , Yo-Chuen Lin  1 , Rachel Creager  3 , David M MacAlpine  3 , Kannanganattu V Prasanth  1 , Mirit I Aladjem  2 , Supriya G Prasanth  1
Affiliations

Temporal association of ORCA/LRWD1 to late-firing origins during G1 dictates heterochromatin replication and organization

Yating Wang et al. Nucleic Acids Res. .

Abstract

DNA replication requires the recruitment of a pre-replication complex facilitated by Origin Recognition Complex (ORC) onto the chromatin during G1 phase of the cell cycle. The ORC-associated protein (ORCA/LRWD1) stabilizes ORC on chromatin. Here, we evaluated the genome-wide distribution of ORCA using ChIP-seq during specific time points of G1. ORCA binding sites on the G1 chromatin are dynamic and temporally regulated. ORCA association to specific genomic sites decreases as the cells progressed towards S-phase. The majority of the ORCA-bound sites represent replication origins that also associate with the repressive chromatin marks H3K9me3 and methylated-CpGs, consistent with ORCA-bound origins initiating DNA replication late in S-phase. Further, ORCA directly associates with the repressive marks and interacts with the enzymes that catalyze these marks. Regions that associate with both ORCA and H3K9me3, exhibit diminished H3K9 methylation in ORCA-depleted cells, suggesting a role for ORCA in recruiting the H3K9me3 mark at certain genomic loci. Similarly, DNA methylation is altered at ORCA-occupied sites in cells lacking ORCA. Furthermore, repressive chromatin marks influence ORCA's binding on chromatin. We propose that ORCA coordinates with the histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of origins, which primes them for late replication.

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Figures

Figure 1.
Figure 1.
Dynamic binding of ORCA on chromatin during G1 phase. (A) Venn diagram summarizing the number of ORCA peaks at each time point during G1 phase and their overlaps. (B) Representative screenshots of regions with different ORCA binding pattern throughout G1 visualized with Integrated Genome Viewer (Ba, Bc, Be) and corresponding ChIP-qPCR validation of ORCA binding pattern during G1 (Bb, Bd, Bf). The horizontal bars below the peaks represent the called-peaks. ChIP was performed in the U2OS cell line stably expressing HA-ORCA. Error bars represent S.D., n = 3, *P < 0.05.
Figure 2.
Figure 2.
ORCA associates with replication origins. (A) Genome-wide co-localization of ORCA binding sites as a function of the distance from replication origin sites. (B) Percentage of ORCA binding sites that co-localize with replication origins at each time point of G1 phase. (C) Venn diagram summarizing the number of ORCA-associated origins at each time point during G1 phase and their overlaps. (D) Representative screenshots showing different ORCA-origin co-localization visualized with Integrated Genome Viewer.
Figure 3.
Figure 3.
ORCA binding regions are enriched for H3K9me3 and methylated CpG. (A) Co-localization of ORCA with H3K9me3. (Aa) Genome-wide co-localization of ORCA binding sites as a function of the distance from H3K9me3 sites. (Ab) Percentage of ORCA binding sites that co-localized with H3K9me3 at each time point of G1 phase. (Ac) Representative screenshots of ORCA and H3K9me3 co-localization visualized with Integrated Genome Viewer. (B) Co-localization of ORCA with methyl-CpG sites. (Ba) Genome-wide co-localization of ORCA binding sites as a function of the distance from methyl-CpG sites. (Bb) Percentage of ORCA binding sites that co-localized with methyl-CpG sites at each time point of G1 phase. (C) Percentage of H3K9me3 containing ORCA peaks that showed highly significant H3K9me3 reduction upon ORCA depletion. (D) MeDIP-qPCR at several ORCA binding regions in control and ORCA-depleted cells. Error bars represent S.D., n = 3, **P < 0.01.
Figure 4.
Figure 4.
ORCA-associated replication origins are enriched for H3K9me3 and methylated CpG. (A) Percentage of total origins that co-localize with H3K9me3 and methyl-CpG sites. (B) Percentage of ORCA-associated origins that co-localize with H3K9me3. (C) Percentage of ORCA-associated origins that co-localize with methyl-CpG sites. (D) Representative screenshots showing one ORCA-associated origin that co-localizes with H3K9me3 as well as methyl-CpG site visualized by Integrated Genome Viewer. Note the adjacent origins that do not co-localize with H3K9me3 and methyl-CpG site are not occupied by ORCA.
Figure 5.
Figure 5.
ORCA associates with methylated DNA sequence in vitro and interacts with DNA methyltransferases. (A) Electrophoretic mobility shift assay (EMSA) showing purified His-SUMO-ORCA protein preferentially interacts with methylated DNA substrate in vitro. His-SUMO is used as negative control. (B) EMSA showing purified His-SUMO-ORCA protein interacts with both hemi-methylated and fully methylated DNA substrate in vitro. (C) Immunoprecipitation (IP) in U2OS cells transiently expressing T7-ORCA and Myc-DNMT1 using T7 antibody. Results were analyzed by ORCA and Myc immunoblot. (D) IP in U2OS cell line stably expressing HA-ORCA using HA antibody. Results were analyzed by ORCA and DNMT3a immunoblot. (E) IP in U2OS cells transiently expressing Myc-DNMT3b using ORCA antibody. Results were analyzed by ORCA and Myc immunoblot. * denotes cross-reacting bands. (F) ORCA ChIP-qPCR in control and SUV39H1 and SUV39H2-depleted cells. Error bars represent S.D., n = 3, *P < 0.05, **P < 0.01.
Figure 6.
Figure 6.
Model depicting ORCA's role in licensing as well as heterochromatin organization. Model showing how ORCA functions during the licensing process (top) and heterochromatin organization (bottom).
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