Palbociclib interferes with origin firing in a pRb independent manner
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ABSTRACT:
PROVIDER: PRJNA748597 | ENA |
REPOSITORIES: ENA
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Project description:Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, promoting needs to understand more about the mechanism of inhibitors beyond pRb. Our study demonstrates here for the first time how palbociclib impairs the origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA without any induced cell cycle arrest. Furthermore, palbociclib treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of proteins of the Pre-initiation complex (Pre-IC) on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and on its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib to target genomic instability towards pRb deficient patient
2023-07-01 | GSE180530 | GEO
Project description:Accurate genome duplication requires a tightly regulated DNA replication program, which relies on the fine regulation of origin firing. While the molecular steps involved in origin firing have been determined predominantly in budding yeast, the complexity of this process in human cells has yet to be fully elucidated. Here, we describe a straightforward proteomics approach to systematically analyse protein recruitment to the chromatin during induced origin firing in human cells. Using a specific inhibitor against CHK1 kinase, we induced a synchronised wave of dormant origin firing (DOF) and assessed the S phase chromatin proteome at different time points. We provide time-resolved loading dynamics of 3,269 proteins, including the core replication machinery and origin firing factors. This dataset accurately represents known temporal dynamics of proteins on the chromatin during the activation of replication forks and the subsequent DNA damage due to the hyperactivation of excessive replication forks. Finally, we used our dataset to identify the condensin II subunit NCAPH2 as a novel factor required for efficient origin firing and replication. Overall, we provide a comprehensive resource to interrogate the protein recruitment dynamics of replication origin firing events in human cells.
2025-01-24 | PXD058340 | Pride
Project description:Accurate genome duplication requires a tightly regulated DNA replication program, which relies on the fine regulation of origin firing. While the molecular steps involved in origin firing have been determined predominantly in budding yeast, the complexity of this process in human cells has yet to be fully elucidated. Here, we describe a straightforward proteomics approach to systematically analyse protein recruitment to the chromatin during induced origin firing in human cells. Using a specific inhibitor against CHK1 kinase, we induced a synchronised wave of dormant origin firing (DOF) and assessed the S phase chromatin proteome at different time points. We provide time-resolved loading dynamics of 3,269 proteins, including the core replication machinery and origin firing factors. This dataset accurately represents known temporal dynamics of proteins on the chromatin during the activation of replication forks and the subsequent DNA damage due to the hyperactivation of excessive replication forks. Finally, we used our dataset to identify the condensin II subunit NCAPH2 as a novel factor required for efficient origin firing and replication. Overall, we provide a comprehensive resource to interrogate the protein recruitment dynamics of replication origin firing events in human cells.
2025-01-24 | PXD047108 | Pride
Project description:Initiation of eukaryotic chromosome replication follows a spatiotemporal program. Current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by Dbf4 C-terminus and was successfully reconstituted in vitro. An interaction defective mutant dbf4ΔC phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain of Fkh1 to Dbf4 restores the Fkh-dependent origin firing, but specifically interferes with the pericentromeric origin activation. Furthermore, Dbf4 directly interacts with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of non-centromeric origins to promote early replication, in a manner that is reminiscent to the recruitment of Dbf4 to pericentromeric origins by Ctf19.
2018-01-19 | GSE103073 | GEO
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157470 | GEO
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157469 | GEO
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157468 | GEO
Project description:Mrc1 is a conserved checkpoint mediator protein that transduces replication-stress signal to downstream effector kinase. Loss of mrc1 checkpoint activity results in aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early-origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 Bypass Segment) on Mrc1. ∆HBS does not suppress late/dormant origin firing in the presence of hydroxyurea but causes precocious and enhanced activation of weak early-firing origins during normal S-phase progression, and bypasses the requirement of Hsk1 for growth. This may be caused by disruption of intramolecular binding between HBS and NTHBS (N-terminal-Target-of-HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting intramolecular interaction. We propose that Mrc1 exerts “brake” on initiation (through intra-molecular interaction) and this brake can be released (upon loss of intra-molecular interaction) by either Hsk1-mediated phosphorylation of Mrc1 or deletion of HBS (or phosphomimic mutation) which can bypass the function of Hsk1 for growth. The “brake” mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.
2017-01-06 | GSE93178 | GEO
Project description:Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, underlining a need to characterize the effects of CDK4/6 inhibitors beyond pRb. Our study demonstrates how palbociclib impairs origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA while showing no cell cycle arrest. Furthermore, this CDK4/6 inhibitor treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of the Pre-initiation complex (Pre-IC) proteins on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and of its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib in combination with other drugs to target genomic instability in pRB-deficient cancers.
| S-EPMC10341899 | biostudies-literature