MCM2–7-dependent cohesin loading during S phase promotes sister-chromatid cohesion
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
The MCM2–7 complex is required for cohesin loading during early S phase.
(A) DAPI (blue), anti-Myc (red), and anti-MCM2 (green) staining of HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in early S phase with …
Figure 1—figure supplement 1
MCM2–7 promotes cohesin loading in early S phase human cells.
(A) Quantification of the intensities of chromatin-bound RAD21-Myc in HeLa cells transfected with the indicated plasmids and siRNAs and synchronized in early S phase by thymidine. Each dot in the …
Figure 2 with 2 supplements
NIPBL and cohesin bind to MCM2–7 interdependently and co-localize in the genome.
(A) HeLa cells were transfected with the indicated siRNAs, arrested in early S phase with thymidine, and lysed in the presence of nuclease. The total lysates (input) and anti-MCM2 IP were blotted …
Figure 2—figure supplement 1
ChIP-seq analysis of cohesin, NIPBL, and MCM2 in human cells.
(A) The top consensus motif of RAD21 binding. (B) Annotated peaks and tracks of MCM2, RAD21, and NIPBL ChIP-seq samples in the selected region of chromosome six were visualized in the IGV browser. …
Figure 2—figure supplement 2
ChIP-seq analysis of cohesin, NIPBL, and MCM2 in human cells.
(A) Pie charts showing the genome distribution of the annotated NIPBL-enriched peaks in two independent ChIP-seq experiments. (B) Bar graphs showing the genome distribution of NIPBL and RAD21 in the …
Figure 3 with 4 supplements
DDK promotes the MCM–NIPBL–cohesin interaction.
(A) DAPI (blue) and anti-Myc (red) staining of HeLa cells that stably expressed RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in early S phase with thymidine. Scale bar, 5 …
Figure 3—figure supplement 1
DDK promotes cohesin loading in early S phase, but not in telophase.
(A) Quantification of the intensities of chromatin-bound RAD21-Myc of cells in Figure 3A. Each dot in the graph represents a single cell. Mean ± SD (siLuc, n = 101; siMCM2, n = 141; siCDC7, n = 102; …
Figure 3—figure supplement 2
DDK promotes the MCM–NIPBL–cohesin interaction in early S phase.
(A) HeLa cells were transfected with the indicated siRNAs, enriched in early S phase by thymidine, and lysed in the presence of Turbo nuclease. The total lysates (input) and anti-MCM2 …
Figure 3—figure supplement 3
The kinase activity of DDK is critical for cohesin loading in early S phase, but not in telophase.
(A) Quantification of the intensities of chromatin-bound RAD21-Myc of cells in Figure 3E. Each dot in the graph represents a single cell. Mean ± SD (DMSO, n = 72; XL413, n = 29). (B) Quantification …
Figure 3—figure supplement 4
CDC45 and GINS are dispensable for cohesin loading and the MCM–NIPBL–cohesin interaction.
(A) DAPI (blue) and anti-Myc (red) staining of HeLa cells stably expressing RAD21-Myc. Cells were transfected with the indicated siRNAs and arrested in ealy S phase by thymidine before fixation and …
Figure 4 with 1 supplement
Replisome components are required for interphase sister-chromatid cohesion.
(A) Representative images of G2-enriched HeLa cells transfected with the indicated siRNAs and stained with DAPI (blue in merge) and the FISH probe (red in merge). Cells were treated with thymidine …
Figure 4—figure supplement 1
Replisome components promote sister-chromatid cohesion at least in part through antagonizing WAPL.
(A) HeLa cells were transfected with the indicated siRNAs, arrested in early S phase by thymidine, lysed in the presence of Turbo nuclease and immunoprecipitated with anti-sororin antibody beads. …
Figure 5 with 1 supplement
Replisome components are required for cohesin loading and the MCM–NIPBL–cohesin interaction during early S phase.
(A) Flow cytometry analysis of HeLa cells transfected with the indicated siRNAs and treated with thymidine for 16–18 hr. The DNA content histograms were shown. (B) DAPI (blue) and anti-Myc (red) …
Figure 5—figure supplement 1
Chromatin binding of replisome components peaks in S phase.
(A) HeLa cells were arrested in mitosis with nocodazole and released from nocodazole treatment for the indicated times. Cells were harvested for flow cytometry analysis. The DNA content histograms …
Figure 6 with 4 supplements
RPA promotes interphase sister-chromatid cohesion, cohesin loading in early S phase, and the MCM–NIPBL–cohesin interaction.
(A) Representative images of G2-enriched HeLa cells transfected with the indicated siRNAs and stained with DAPI (blue in merge) and the early-replicating 466L19 FISH probe (red in merge). Selected …
Figure 6—figure supplement 1
RPA2 promotes sister-chromatid cohesion.
Lysates of cells in Figure 6C were blotted with the indicated antibodies.
Figure 6—figure supplement 2
RPA2 promotes sister-chromatid cohesion.
(A) Asynchronous cells or cells released from the thymidine arrest for the indicated times were harvested and stained with the 466L19 FISH probe. Quantification of the percentages of cells with two …
Figure 6—figure supplement 3
Depletion of RPA causes interphase cohesion defects in asynchronous cells.
(A) HeLa cells were transfected with the indicated siRNAs and stained with the 466L19 FISH probe. The percentages of cells with two unreplicated single FISH dots (red), cells with one unreplicated …
Figure 6—figure supplement 4
RPA2 promotes cohesin loading in S phase.
Quantification of the intensities of chromatin-bound STAG2 in HeLa cells transfected with the indicated siRNAs and synchronized in early S phase by thymidine. Each dot in the graph represents a …
Figure 7 with 1 supplement
MCM2–7-mediated cohesin loading and mobilization promote sister-chromatid cohesion.
In this speculative model, SCC2/4 (NIPBL/MAU2 in humans) associates with DDK and the dormant, phosphorylated MCM2–7, and promotes cohesin loading at the G1/S boundary. The loaded cohesin remains …
Figure 7––Figure Supplement 1
DDK-bound, potentiated MCM promotes cohesin loading and mobilization.
Based on this model, only the potentiated MCM complexes bound to DDK (which is limiting) can recruit cohesin and SCC2/4 (NIPBL/MAU2 in humans). These potentiated complexes are destined to fire early …
Additional files
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Supplementary file 1
siRNAs used in this study.
- https://doi.org/10.7554/eLife.33920.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.33920.024
