RESUMO
The replication-stress response is essential to ensure the faithful transmission of genetic information to daughter cells. Although several stress-resolution pathways have been identified to deal with replication stress, the precise regulatory mechanisms for replication fork stability are not fully understood. Our study identified Methyl-CpG Binding Domain 1 (MBD1) as essential for the maintaining genomic stability and protecting stalled replication forks in mammalian cells. Depletion of MBD1 increases DNA lesions and sensitivity to replication stress. Mechanistically, we found that loss of MBD1 leads to the dissociation of Poly(ADP-ribose) polymerase 1 (PARP1) from the replication fork, potentially accelerating fork progression and resulting in higher levels of transcription-replication conflicts (T-R conflicts). Using a proximity ligation assay combined with 5-ethynyl-2'-deoxyuridine, we revealed that the MBD1 and PARP1 proteins were recruited to stalled forks under hydroxyurea (HU) treatment. In addition, our study showed that the level of R-loops also increased in MBD1-delated cells. Without MBD1, stalled replication forks resulting from T-R conflicts were primarily degraded by the DNA2 nuclease. Our findings shed light on a new aspect of MBD1 in maintaining genome stability and providing insights into the mechanisms underlying replication stress response.
Assuntos
Dano ao DNA , Replicação do DNA , Humanos , Animais , Instabilidade Genômica , Mamíferos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in Caenorhabditis elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.
Assuntos
Neoplasias da Próstata , Mutações Sintéticas Letais , Animais , Humanos , Masculino , Proteína BRCA2 , Caenorhabditis elegans/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mamíferos/metabolismo , Mutação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Ubiquitina Tiolesterase/genética , Proteína Fosfatase 2/metabolismoRESUMO
BRCA1 expression is highly regulated to prevent genomic instability and tumorigenesis. Dysregulation of BRCA1 expression correlates closely with sporadic basal-like breast cancer and ovarian cancer. The most significant characteristic of BRCA1 regulation is periodic expression fluctuation throughout the cell cycle, which is important for the orderly progression of different DNA repair pathways throughout the various cell cycle phases and for further genomic stability. However, the underlying mechanism driving this phenomenon is poorly understood. Here, we demonstrate that RBM10-mediated RNA alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD), rather than transcription, determines the periodic fluctuations in G1/S-phase BRCA1 expression. Furthermore, AS-NMD broadly regulates the expression of period genes, such as DNA replication-related genes, in an uneconomical but more rapid manner. In summary, we identified an unexpected posttranscriptional mechanism distinct from canonical processes that mediates the rapid regulation of BRCA1 as well as other period gene expression during the G1/S-phase transition and provided insights into potential targets for cancer therapy.
Assuntos
Neoplasias da Mama , Degradação do RNAm Mediada por Códon sem Sentido , Humanos , Feminino , Processamento Alternativo , Splicing de RNA , Neoplasias da Mama/genética , Instabilidade Genômica , Proteína BRCA1/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to ß-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.
