Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

origin. Referring to our own ChIP-Seq data (see below), we designed qPCR primers (5'-GACAGTGGGGAGACTCTTGC -3' and 5'-GACGAAGAAACCAGGGCTCA-3') to detect genomic DNA sequence (white box) that is ~10 kb apart from lamin B2 origin (black box) and is expected to be negative for pre-RC and GRWD1 bindings. (B) ChIP-qPCR assay for the indicated immunoprecipitates from asynchronously growing HeLa cells was performed with the above primers. Results are shown as the percent of input DNA. The means±S.D. are shown (n=3).
(C, D) Silencing of Cdt1 with another siRNA (siCdt1-4&5) also inhibits recruitment of GRWD1 and MCM7 onto replication origins at the lamin B2 and MCM4 loci in HeLa cells.
Cells transfected with control or Cdt1 (siCdt1-4&5) siRNAs for 48 hrs were subjected to immunoblotting (C) and ChIP-qPCR (D) with the indicated antibodies as described in Figure 2.
For ChIP data, the means±S.D. are shown (n=4). *, p<0.05. which may represent firing replication origins (39). Since MCM complexes are believed to be replicative helicases essential for firing, we analyzed the association of MCM7 peaks with SNS peaks. Even if we used a stringent overlap criterion of 0.5 kb to consider co-localization, approximately ~52% (~134,000 peaks) of total SNS peaks were identified as those closely associated with the MCM7 peaks, and ~33% (~115,000) of MCM7 peaks as those closely associated with SNS (left panel). The value of MCM7 overlapping with SNS (~115,000) is ~2.1-fold higher than that obtained with the randomized (shuffled) data sets, which contain the same number and length of the corresponding data sets but are randomly located, to estimate correlation by chance (right panel). *, p<0.001 by Chi-Square test. Remaining MCM7 peak not associated SNS peaks may include dormant origins that do not fire or fire at low frequency.
(C) Identification of the firing pre-RC sites (CDC_w0.5_MCM7_w0.5_SNS). With a stringent overlap criterion of 0.5 kb to consider co-localization, ~32,000 peaks were identified as strong candidates for the firing pre-RC sites.

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(D) Identification of GRWD1 binding peaks by ChIP-Seq. Genome-wide GRWD1 localization in asynchronous HeLa cells was examined by combining the analysis for endogenous GRWD1 using anti-GRWD1 antibody (see also Supplementary Figure S6) with that for exogenous HA-GRWD1 using anti-HA antibody. MACS2 broad peak caller identified ~320,000 GRWD1 peaks (p<0.001, q<0.05) and ~739,000 HA-GRWD1 peaks (p<0.001, q<0.05). We then analyzed the association of GRWD1 peaks with HA-GRWD1 peaks. Even when a stringent overlap criterion of 0.5 kb was used to consider co-localization, approximately ~65% (~208,000 peaks) of total GRWD1 peaks were identified as those closely associated with HA-GRWD1 peaks (left panel), the value being ~1.9-fold higher than that obtained with the shuffled data sets is the same as shown in Figure 5D).
Synchronization of T98G cells in the G0, G1/S, and late S/G2/M phases was achieved as described previously (15). To monitor S-phase progression, HeLa cells were synchronized in S phase by treatment with 2.5 mM hydroxyurea (HU) for 18hrs, released, and harvested at the indicated time points. Flow cytometry analysis was performed as described previously (15).
Double staining with anti-BrdU antibody and propidium iodide was carried out using BrdU Labeling and detection Kit I (Roche) and Cycletest Plus DNA reagent Kit (BO Biosciences).

Immunoprecipitation, Chromatin Binding Assay, and Immunoblotting
Immunoprecipitation from nuclear extracts and chromatin-binding assay were performed as described previously (15). Immunoblotting and quantification of the band signals were also performed as described previously (15).
For immunoprecipitation-re-immunoprecipitation experiments in Figure 2C, the first immunoprecipitates prepared with anti-Flag antibody (M2, Sigma) were eluted with NET gel buffer containing 3xFlag peptide at 150 µg/ml and then were re-immunoprecipitated with anti-T7 antibody (Novagen).

GST Pull-Down Assay
GST-Cdt1 or GST-CDC6 fusion proteins were bacterially produced and purified as described previously (28, 50). Recombinant GRWD1 was expressed in E. coli as a GST-fusion protein and purified on glutathione beads. If necessary, untagged GRWD1 was prepared with PreScission protease (Amersham BioScience), in accordance with the manufacturer's instructions.
GST pull-down assay was performed as described previously (15).

Histone Binding and Histone Chaperone Assay
To obtain highly purified recombinant GST-GRWD1-His, the protein was expressed in E. coli and purified sequentially on glutathione beads and Ni 2+ -chelating affinity beads. Core histones were purified from HeLa cells as described previously (51). Preparation of recombinant histone proteins (H2A/H2B and H3-H4 complexes) and a 195 bp DNA fragment containing the Lytechinus variegatus 5S ribosomal RNA gene was carried out as described previously (32, 33).
GST pull-down assay to test histone binding was performed essentially as described previously (51), except that bound proteins were eluted directly into SDS sample buffer.
Topological assay and nucleosome assembly assay were performed as described previously (32, 33), except that pGEX plasmid was used in the topological assay instead of circular "X174 dsDNA.