Licensing of human DNA replication origins requires CDK4/6 activity
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE278714 | GEO | 2025/08/14
REPOSITORIES: GEO
Similar Datasets
Project description:Sumoylation is emerging as a post-translation modification important for chromosome duplication and stability. The origin recognition complex (ORC), which directs DNA replication initiation by loading the MCM replicative helicases onto origins, is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are largely unclear. Here we report the effects of hyper- and hypo-sumoylation of yeast ORC using multiple approaches. We show that ORC hyper-sumoylation preferentially reduces the activity of a subset of early origins, while Orc2 hypo-sumoylation has an opposing effect. Mechanistically, ORC hyper-sumoylation leads to reduced MCM loading in vitro and diminished MCM chromatin association in vivo. The importance of an appropriate level of ORC sumoylation is suggested by the data that either hyper- or hypo-sumoylation of ORC results in genome instability and a dependence on other genome maintenance factors for cell fitness. Thus, yeast ORC sumoylation status needs to be fine-tuned to achieve optimal origin activity control and genome stability.
2023-03-11 | PXD032977 | Pride
Project description:In eukaryotes, the hetero-hexameric origin recognition complex (ORC) is responsible for assembling Mcm2-7 complex into a head-to-head double hexamer (DH), forming pre-replicative complex (pre-RC) that licenses origin DNA for replication initiation. This process is tightly controlled throughout the cell cycle to ensure accurate duplication of the genome. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit plays a critical role in promoting pre-RC assembly. We found that removing a short segment (residues 175-200) from Orc2-IDR or mutating a key isoleucine (194) in this region significantly inhibits replication initiation across the genome and causes cell death. Although the Orc2-IDR mutants can still assemble the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate on DNA, the assembled mutant OCCM exhibits impaired ATP hydrolysis, preventing its conversion into Mcm2-7-ORC (MO) complex and subsequent DH formation. Interestingly, our in vitro assays showed that adding the Orc2-IDR peptide in the pre-RC reactions can rescue this defect. Furthermore, phosphorylation of this Orc2-IDR motif by S-cyclin dependent kinase (S-CDK) blocks its binding to Mcm2, leading to defective pre-RC assembly. Our findings provide crucial mechanistic insights into the multifaceted roles of ORC in modulating MCM loading to support origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.
2024-09-18 | GSE262693 | GEO
Project description:Cyclin dependent kinases (CDKs) lie at the heart of eukaryotic cell division, with different CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1-3. However, the principles on which cyclin-CDKs organise the cell cycle are still contentious, with current theories suggesting that either M-CDKs and S-CDKs are redundant with each other, and simply serve as a source of CDK activity, or that they are functionally specialised and execute distinct tasks. Here we reconcile these two views, showing that although S-CDKs are unable to drive mitosis, global CDK phosphorylation in vivo between S-CDK and M-CDK is surprisingly similar. Remarkably, S-CDK has cryptic mitotic activity which can be exposed by the removal of Protein Phosphatase 1 (PP1). In particular, removal of centrosomal PP1 allows S-CDK to execute mitosis indistinguishably from M-CDK, demonstrating their functional redundancy. Thus, we reconcile the two opposing views of cell cycle control, showing that although there is a minor level of specialisation between S-CDKs and M-CDKs, that the core cell cycle engine is based upon modulation of CDK activity, with cyclins providing refinements to this core system.
2022-03-22 | PXD029073 | Pride
Project description:Cyclin dependent kinase 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry, at least in part through phosphorylation of the retinoblastoma tumor suppressor protein (Rb). The CDK4/6-cyclin D-Rb pathway is commonly deregulated in human cancer, often through CDK4/6 or D-type cyclin overexpression, or inactivation of the CDK4/6 antagonist p16/CDKN2A. Importantly, a substantial fraction of cancers depend on continuous CDK4/6-cyclin D kinase activity and are sensitive to CDK4/6-specific inhibitors. Here, we investigate critical CDK4/6-cyclin D functions that may determine the sensitivity to CDK4/6 inhibitors, making use of the essential roles of CDK4/6 (CDK-4) and cyclin D (CYD-1) in the nematode C. elegans. In an unbiased screen, we found that simultaneous loss of C. elegans Rb (lin-35) and down-regulation of the APC/C substrate specificity factor FZR1/Cdh1 completely overcomes CDK-4/CYD-1 requirement. Furthermore, CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 N-terminus that correspond to inactivating phosphorylations of the human homologs. Thus, CDK-4/CYD-1 appears to promote cell cycle entry by antagonizing not only transcriptional repression by LIN-35 Rb but also protein degradation by APC/CFZR-1. Simultaneous knockdown of Rb and FZR1 in human breast cancer cells synergistically overcomes arrest by the CDK4/6-specific inhibitor PD 00332991. These results reveal APC/CFZR1 as a putative CDK4/6-Cyclin D target and important contributing factor in the response to CDK4/6-inhibitor treatment.
2016-07-05 | PXD001501 | Pride
Project description:The Origin Recognition Complex (ORC) is essential for the initiation of eukaryotic chromosome replication as it loads the replicative helicase, the minichromosome maintenance (MCM) complex, at replication origins. Origins display a stereotypic chromatin structure with nucleosome depletion at ORC binding sites and flanking arrays of regularly spaced nucleosomes. Although discovered long ago, it is unknown how this chromatin struture is established and if it matters for replication. By genome-scale biochemical reconstitution we screened ~300 origins and 17 purified budding yeast chromatin factors and found that ORC established nucleosome depletion over origins and flanking nucleosome arrays by orchestrating the chromatin remodelers INO80, ISW1a, ISW2 and Chd1. The functional importance of ORC’s chromatin organizing activity was directly demonstrated by ORC mutations that maintained classical MCM loader activity but lost array generation activity. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, besides its canonical role as MCM loader, has a second essential function as master regulator of the origin chromatin structure that is a crucial prerequisite for efficient chromosome replication.
2023-03-30 | GSE209681 | GEO
Project description:PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and arrayCGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort ovarian cancer patients. Concentration-dependent anti-proliferative effects of PD-0332991were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, Rb, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration and time dependent manner, and enhanced the effects of chemotherapy. Rb proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and associated with adverse clinical outcome (progression free survival, adjusted relative risk 1.49, 95%CI 0.99-2.22, p =0.054). PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Gene expression of 40 individual ovarian cell lines relative to an ovarian cell line reference mix containing equal amounts of 41 ovarian cell lines (including OCC-1 which was later identified as originating from mouse). The expression data was correllated with cell line growth response to CDK 4/6 inhibitor PD-0332991 to identify genes associated with drug sensitivity and resistance.
2011-01-22 | E-GEOD-26805 | biostudies-arrayexpress
Project description:The evolutionarily conserved Origin Recognition Complex (ORC), plays a key role in origin selection in eukaryotes. However, ORC is strikingly divergent in its DNA binding specificity, ranging from base-specific interactions in Saccharomyces cerevisiae to base-agnostic interactions in humans. The mechanisms underlying this distinct selectivity is unknown. Atomic model of the yeast ORC showed that base-specific interaction with the invariant thymines of the ARS consensus sequence (ACS) is encoded by a 19-amino acid insertion helix (IH) embedded in the winged helix domain (WHD) of Orc4. This IH is absent in the Orc4 of metazoans including humans, suggesting that removal of the IH might give the yeast ORC “human-like” properties. Indeed, yeast strain engineered with IH deficient Orc4 has completely altered ORC-binding sites enriched in poly-dT tracts located in larger nucleosome-depleted and intergenic open chromatin. In vivo and in vitro assays show that the mutant ORC loads MCM efficiently, in spite of its altered specificity in favor of binding patterns more characteristic of those observed in humans/metazoans. This work provides insights for understanding how ORC evolves to adopt a life cycle that requires plasticity in origin selection during development.
2020-10-18 | GSE149163 | GEO
Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.
2014-04-03 | E-GEOD-56453 | biostudies-arrayexpress
Project description:The selective CDK2 inhibitor BLU-222 was evaluated for mechanisms of response in the context of ovarian and breast cancer models. Using sensors of cellular CDK activity, it was found that sensitivity to either CDK4/6 or CDK2 inhibition is related to the differential dependence on a single CDK for G1/S transition. Unlike CDK4/6 inhibitors, BLU-222 was able to robustly inhibit proliferation through cell cycle inhibition in both G1 and G2 phases. However, it remained possible for cells to re-enter the cell cycle upon drug withdrawal. The anti-proliferative strength and impact on G1/S versus G2/M accumulation was found to be mediated by the RB tumor suppressor, as determined by functional perturbation strategies. To broaden the sensitivity to CDK2 inhibition, combinatorial drug screens were performed that identified both synergistic (e.g., CDK4/6 inhibitors) and antagonistic (e.g., WEE1 inhibitors) relationships. Models that were exceptionally sensitive to CDK2 inhibition displayed coordinate expression of Cyclin E1 and P16INK4A, an endogenous CDK4/6 inhibitor. Functional studies demonstrated that P16INK4A and CDK4/6 activity were key mediators of sensitivity to BLU-222. Clinical gene and protein expression analysis revealed a positive correlation between Cyclin E1 and P16INK4A and that ~25% of ovarian cancers exhibited coordinate expression of Cyclin E, P16INK4A, and RB, indicative of strong sensitivity to CDK2 inhibition. Together, this work advances a precision strategy for the use of CDK2 inhibitors in the context of ovarian and breast cancers.
2024-12-22 | GSE281746 | GEO
Project description:Methylation of histone H4 lysine 20 (H4K20), such as H4K20 mono-methylation (H4K20me1), regulates the biological processes of DNA replication, cell cycle progression, and DNA damage repair. Whereas H4K20me1 is knowingly written by SET8 (also known as PR-Set7) and erased by PHF8 (also known as KDM7B), the mechanism underlying H4K20me1-mediated regulation of DNA replication remains murky. Here, we report that a conserved tandem Tudor domain of BAHCC1 (BAHCC1TTD) specifically reads H4K20me1. Our biochemical, structural and cellular analyses demonstrated that the recognition of H4K20me1 by BAHCC1TTD facilitates the BAHCC1 recruitment onto the replication origins, where BAHCC1 interacts and recruits components of Minichromosome Maintenance (MCM) complex, a critical partner machinery of Origin Replication Complex (ORC) for mediating DNA replication. Combined actions of the H4K20me1-reading BAHCC1 and the H4K20me2-reading ORC ensure optimal genomic loading of MCM proteins. Depletion of BAHCC1, or disruption of BAHCC1TTD:H4K20me1 interaction, affected H4K20me1 homeostasis and MCM complex loading, leading to impaired DNA replication and defective cell cycle progression. Together, this study identifies a conserved BAHCC1TTD as an effector linking H4K20me1 to MCM recruitment for efficient DNA replication.
2025-06-03 | GSE213114 | GEO