RESUMEN
The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.
Asunto(s)
Ciclo Celular , Replicación del ADN , Humanos , Células HeLa , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Imagen de Lapso de TiempoRESUMEN
Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.
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Ceramidas , Proteína Proto-Oncogénica c-fli-1 , Receptores Acoplados a Proteínas G , Sarcoma de Ewing , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Ceramidas/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Línea Celular Tumoral , Esfingomielina Fosfodiesterasa/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Fusión Oncogénica/genética , Animales , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Transducción de Señal , Dominios Proteicos , RatonesRESUMEN
HeLa cells are a cell line with two unique cellular features: a short-shouldered survival curve and two peaks of radioresistance during the cell cycle phase, while their underlying mechanisms remain unclear. We herein proposed that these radiobiological features are due to a common mechanism by which radiation suppresses homologous recombination repair (HRR) in a dose-dependent manner. This radio-suppression of HRR is mediated by an intra-S checkpoint and reduces survivals of cells in S phase, especially early S phase, resulting in both short shoulder and radioresistance with two peaks in the cell cycle. This new explanation may not be limited to HeLa cells since a similar close association of these features is also observed in other type of cells.
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Reparación del ADN , Hombro , Humanos , Células HeLa , Fase S , Ciclo Celular , Tolerancia a Radiación , Supervivencia CelularRESUMEN
OBJECTIVE: Ovarian cancer (OC) is a common malignant tumor in females with high recurrence and poor prognosis. Cisplatin is commonly used for OC clinical treatment, but its efficacy is usually challenged by the chemotherapy resistance of cancer cells. MicroRNAs (miRNAs), including miR-30a-5p, were identified to modulate drug resistance in numerous tumors. However, molecular mechanisms of miR-30a-5p in OC chemoresistance need more illumination. METHODS: MiR-30a-5p and Rap1 interacting factor 1 (RIF1) expression in OC tissues and cells were measured by qRT-PCR. The IC50 of cisplatin-resistant and cisplatin-sensitive OC cells was assessed by MTT assays. OC cell proliferation, apoptosis and migration were measured by EdU assays, TUNEL staining, and wound healing assays, respectively. The protein levels of EMT markers and RIF1 in OC cells were examined by western blotting. The binding capacity between miR-30a-5p and RIF1 was validated by luciferase reporter assays. RESULTS: Our study disclosed miR-30a-5p as a remarkably lowly-expressed miRNA in OC tissues in comparison to matched noncancerous tissues. Compared to parental cell lines, miR-30a-5p was also greatly downregulated in cisplatin-resistant OC cell lines. Additionally, functional assays indicated that miR-30a-5p suppressed malignant behaviors and cisplatin resistance of OC cells. Further, miR-30a-5p was revealed to target and negatively regulate RIF1 expression in OC. Moreover, it was validated that overexpressing RIF1 reverses the inhibitory influence of miR-30a-5p overexpression on malignant behaviors and cisplatin resistance of OC cells. CONCLUSION: MiR-30a-5p reduced cisplatin resistance in OC through downregulation of RIF1, which may be meaningful for targeting drug-resistant tumors.
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Cisplatino , MicroARNs , Neoplasias Ováricas , Proteínas de Unión a Telómeros , Femenino , Humanos , Apoptosis/genética , Cisplatino/farmacología , Fibrinógeno , MicroARNs/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Resistencia a AntineoplásicosRESUMEN
Rif1 mediates telomere length, DNA replication, and DNA damage responses in budding yeast. Previous work identified several posttranslational modifications of Rif1, however none of these was shown to mediate the molecular or cellular responses to DNA damage, including telomere damage. We searched for such modifications using immunoblotting methods and the cdc13-1 and tlc1Δ models of telomere damage. We found that Rif1 is phosphorylated during telomere damage, and that serines 57 and 110 within a novel phospho-gate domain (PGD) of Rif1 are important for this modification, in cdc13-1 cells. The phosphorylation of Rif1 appeared to inhibit its accumulation on damaged chromosomes and the proliferation of cells with telomere damage. Moreover, we found that checkpoint kinases were upstream of this Rif1 phosphorylation and that the Cdk1 activity was essential for maintaining it. Apart from telomere damage, S57 and S110 were essential for Rif1 phosphorylation during the treatment of cells with genotoxic agents or during mitotic stress. We propose a speculative "Pliers" model to explain the role of the PGD phosphorylation during telomere and other types of damage.
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Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión a Telómeros , Telómero , Replicación del ADN , Fosforilación , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
BACKGROUND: RAP1 interacting factor 1 (Rif1) is highly expressed in mice embryos and mouse embryonic stem cells (mESCs). It plays critical roles in telomere length homeostasis, DNA damage, DNA replication timing and ERV silencing. However, whether Rif1 regulates early differentiation of mESC is still unclear. METHODS: In this study, we generated a Rif1 conditional knockout mouse embryonic stem (ES) cell line based on Cre-loxP system. Western blot, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), RNA high-throughput sequencing (RNA-Seq), chromatin immunoprecipitation followed high-throughput sequencing (ChIP-Seq), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment. RESULTS: Rif1 plays important roles in self-renewal and pluripotency of mESCs and loss of Rif1 promotes mESC differentiation toward the mesendodermal germ layers. We further show that Rif1 interacts with histone H3K27 methyltransferase EZH2, a subunit of PRC2, and regulates the expression of developmental genes by directly binding to their promoters. Rif1 deficiency reduces the occupancy of EZH2 and H3K27me3 on mesendodermal gene promoters and activates ERK1/2 activities. CONCLUSION: Rif1 is a key factor in regulating the pluripotency, self-renewal, and lineage specification of mESCs. Our research provides new insights into the key roles of Rif1 in connecting epigenetic regulations and signaling pathways for cell fate determination and lineage specification of mESCs.
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Fibrinógeno , Células Madre Embrionarias de Ratones , Animales , Ratones , Fibrinógeno/metabolismo , Diferenciación Celular/genética , Línea Celular , Estratos Germinativos/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Circular RNAs have been identified as diagnostic and therapeutic targets for various tumors. The expression of circ_rac GTPase-activating protein 1 (circRACGAP1) is reported to drive the development of non-small cell lung cancer (NSCLC). This study further explored the potential mechanism of circRACGAP1-mediated development of NSCLC. The circRACGAP1 level was detected by quantitative RT-PCR. Sphere formation, CD133-positive cell percentage, and expression of octamer-binding transcription factor 4, Sox2, Nanog, and CD133 were detected to evaluate stemness of NSCLC. Migration and invasion were determined using wound healing and transwell assays. Protein expression was measured using Western blotting. The molecular mechanism was evaluated using RNA pull-down, RNA immunoprecipitation, and coimmunoprecipitation assays. In vivo tumor growth and metastasis were determined in nude mice. circRACGAP1 was highly expressed in NSCLC and was associated with stemness marker Sox2 expression. The stemness, metastasis, and epithelial mesenchymal transformation were repressed in circRACGAP1-depleted NSCLC cells. Mechanistically, circRACGAP1 recruited RNA-binding protein polypyrimidine tract-binding protein 1 to enhance the stability and expression of sirtuin-3 (SIRT3), which subsequently led to replication timing regulatory factor 1 (RIF1) deacetylation and activation of the Wnt/ß-catenin pathway. circRACGAP1 overexpression counteracted SIRT3 or RIF1 knockdown-mediated inhibition in stemness and metastasis of NSCLC cells. The in vivo tumor growth and metastasis were repressed by circRACGAP1 depletion. Patients with NSCLC with a higher serum exosomal circRACGAP1 level had a lower overall survival rate. In conclusion, circRACGAP1 facilitated stemness and metastasis of NSCLC cells through the recruitment of polypyrimidine tract-binding protein 1 to promote SIRT3-mediated RIF1 deacetylation. Our results uncover a novel regulatory mechanism of circRACGAP1 in NSCLC and identify circRACGAP1 as a promising therapeutic target.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Proteínas Activadoras de GTPasa , Neoplasias Pulmonares , MicroARNs , Sirtuina 3 , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Activadoras de GTPasa/genética , Neoplasias Pulmonares/patología , Ratones Desnudos , MicroARNs/genética , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN , Sirtuina 3/metabolismo , Células Madre NeoplásicasRESUMEN
53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Reparación del ADN , Neoplasias , Proteína 1 de Unión al Supresor Tumoral P53 , Humanos , Cromatina , Daño del ADN , Reparación del ADN por Unión de Extremidades , Mitosis , Neoplasias/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular TumoralRESUMEN
Resolution of ultrafine anaphase bridges (UFBs) must be completed before cytokinesis to ensure sister-chromatid disjunction. RIF1 is involved in UFB resolution by a mechanism that is not yet clear. Here, we show that RIF1 functions in mitosis to inhibit the formation of 53BP1 nuclear bodies and micronuclei. Meanwhile, RIF1 localizes on PICH-coated double-stranded UFBs but not on RPA-coated single-stranded UFBs. Depletion of RIF1 leads to an elevated level of RPA-coated UFBs, in a BLM-dependent manner. RIF1 interacts with all three isoforms of protein phosphatase 1 (PP1) at its CI domain in anaphase when CDK1 activity declines. CDK1 negatively regulates RIF1-PP1 interaction via the CIII domain of RIF1. Importantly, depletion of PP1 phenocopies RIF1 depletion, and phosphorylation-resistant mutant of PICH shows reduced interaction with the BTR complex and bypasses the need of RIF1 in preventing the formation of single-stranded UFBs. Overall, our data show that PP1 is the effector of RIF1 in UFB resolution.
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Anafase , Proteína Fosfatasa 1 , Humanos , Línea Celular , Cromátides , Citocinesis , MitosisRESUMEN
Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through the use of relatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880 kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicable to human cultured cells and specimens.
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Código de Histonas , Histonas , Animales , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Procesamiento Proteico-PostraduccionalRESUMEN
Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.
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Proteínas Portadoras , Proteínas de Drosophila , Drosophila melanogaster , Animales , Proteínas Portadoras/genética , Cromosomas , Replicación del ADN , ADN Satélite/genética , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Células Germinativas , Heterocromatina/genética , Cromosomas Politénicos/genéticaRESUMEN
The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.
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Proteínas de Drosophila , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Heterocromatina/genética , Cromosoma X/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Región Organizadora del Nucléolo , Proteínas Portadoras/genética , Proteínas de Drosophila/genéticaRESUMEN
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
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Replicación del ADN , Origen de Réplica , Escherichia coli/metabolismo , Proteínas de Unión al ADN/metabolismo , Replicón , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , ADN Bacteriano/genéticaRESUMEN
Mouse embryonic stem cells (mESCs) cycle in and out of a transient 2-cell (2C)-like totipotent state, driven by a complex genetic circuit involves both the coding and repetitive sections of the genome. While a vast array of regulators, including the multi-functional protein Rif1, has been reported to influence the switch of fate potential, how they act in concert to achieve this cellular plasticity remains elusive. Here, by modularizing the known totipotency regulatory factors, we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repressive complex PRC1.6. Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs. The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6, bolstering the intactness of the PRC1.6 complex. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state. Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352, but also derepresses a group of the endogenous retroviral element MERVL, a key marker for totipotency. Collectively, our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential, shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.
RESUMEN
In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.
Asunto(s)
Origen de Réplica , Proteínas de Unión a Telómeros , Animales , Replicación del ADN , Momento de Replicación del ADN , Fase S/genética , Proteínas de Unión a Telómeros/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. Here, we show that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a "community effect" counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. We propose a model in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins.
Asunto(s)
Proteínas Portadoras , Replicación del ADN , Proteínas de Drosophila , Drosophila melanogaster , Proteína Fosfatasa 1 , Origen de Réplica , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Fase S/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The 53BP1-RIF1-shieldin pathway maintains genome stability by suppressing nucleolytic degradation of DNA ends at double-strand breaks (DSBs). Although RIF1 interacts with damaged chromatin via phospho-53BP1 and facilitates recruitment of the shieldin complex to DSBs, it is unclear whether other regulatory cues contribute to this response. Here, we implicate methylation of histone H3 at lysine 4 by SETD1A-BOD1L in the recruitment of RIF1 to DSBs. Compromising SETD1A or BOD1L expression or deregulating H3K4 methylation allows uncontrolled resection of DNA ends, impairs end-joining of dysfunctional telomeres, and abrogates class switch recombination. Moreover, defects in RIF1 localization to DSBs are evident in patient cells bearing loss-of-function mutations in SETD1A. Loss of SETD1A-dependent RIF1 recruitment in BRCA1-deficient cells restores homologous recombination and leads to resistance to poly(ADP-ribose)polymerase inhibition, reinforcing the clinical relevance of these observations. Mechanistically, RIF1 binds directly to methylated H3K4, facilitating its recruitment to, or stabilization at, DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión a Telómeros , Proteína BRCA1/genética , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance.
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Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.
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Roturas del ADN de Doble Cadena , Replicación del ADN , Animales , ADN/metabolismo , Reparación del ADN , Ratones , Fosforilación , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The appearance of trophectoderm (TE) is a hallmark event in preimplantation development during murine embryogenesis. However, little is known about the mechanisms underlying TE specification. We find that the depletion of Rif1 breaks down the barrier to the transition from embryonic stem cells (ESCs) to trophoblast stem cells (TSCs). Rif1-null-induced TSCs show typical TE properties and the potential to differentiate into terminal trophoblast lineages. Global transcriptome analysis reveal that Rif1 deletion activates 2-cell embryo (2C)-related genes and induces a totipotent-like state. Chimeric assays further confirm that Rif1-null ESCs contribute to the functional placenta in addition to the fetus on embryonic day 12.5. Furthermore, we show overexpression of Hmgn3, one of the key upregulated gene in Rif1-null ESCs, facilitates the induction of TSCs. Therefore, we report two key genes regulating the conversion of TSCs and provide insights for investigating TE specification.