RESUMO
The WD40 domain-containing protein WRAP53ß (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53ß as an essential regulator of DNA double-strand break (DSB) repair. WRAP53ß rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53ß targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53ß facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53ß controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53ß impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53ß as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.
Assuntos
Reparo do DNA/fisiologia , Telomerase/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Telomerase/genética , Transativadores/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The cellular response to DNA double-strand breaks is orchestrated by the protein kinase ATM, which phosphorylates key actors in the DNA repair network. WRAP53ß is a multifunctional protein that controls trafficking of factors to Cajal bodies, telomeres and DNA double-strand breaks but what regulates the involvement of WRAP53ß in these separate processes remains unclear. Here, we show that in response to various types of DNA damage, including IR and UV, WRAP53ß is phosphorylated on serine residue 64 by ATM with a time-course that parallels its accumulation at DNA lesions. Interestingly, recruitment of phosphorylated WRAP53ß (pWRAP53ßS64) to sites of such DNA damage promotes its interaction with γH2AX at these locations. Moreover, pWRAP53ßS64 stimulates the accumulation of the repair factor 53BP1 at DNA double-strand breaks and enhances repair of this type of damage via homologous recombination and non-homologous end joining. At the same time, phosphorylation of WRAP53ß is dispensable for its localization to Cajal bodies, where it accumulates even in unstressed cells. These findings not only reveal ATM to be an upstream regulator of WRAP53ß, but also indicates that phosphorylation of WRAP53ß at serine 64 controls its involvement in the DNA damage response and may also restrict its other functions.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Corpos Enovelados/metabolismo , Dano ao DNA , Telomerase/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Radiação Ionizante , Raios UltravioletaRESUMO
X-chromosome inactivation and X-upregulation are the fundamental modes of chromosome-wide gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive and the X-upregulation dynamics are unknown. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling and finely dissect their separate effects on RNA levels during mouse development. Surprisingly, we uncover that X-upregulation elastically tunes expression dosage in a sex- and lineage-specific manner, and moreover along varying degrees of X-inactivation progression. Male blastomeres achieve X-upregulation upon zygotic genome activation while females experience two distinct waves of upregulation, upon imprinted and random X-inactivation; and ablation of Xist impedes female X-upregulation. Female cells carrying two active X chromosomes lack upregulation, yet their collective RNA output exceeds that of a single hyperactive allele. Importantly, this conflicts the conventional dosage compensation model in which naïve female cells are initially subject to biallelic X-upregulation followed by X-inactivation of one allele to correct the X dosage. Together, our study provides key insights to the chain of events of dosage compensation, explaining how transcript copy numbers can remain remarkably stable across developmental windows wherein severe dose imbalance would otherwise be experienced by the cell.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA Longo não Codificante , Alelos , Animais , Feminino , Masculino , Mamíferos/genética , Camundongos , RNA Longo não Codificante/genética , Regulação para Cima , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53ß, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.
Assuntos
Proteínas Quinases , RNA , DNA/genética , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteínas Quinases/metabolismoRESUMO
Ohno's hypothesis postulates that upregulation of X-linked genes rectifies their dosage imbalance relative to autosomal genes, which are present in two active copies per cell. Here we have dissected X-chromosome upregulation into the kinetics of transcription, inferred from allele-specific single-cell RNA sequencing data from somatic and embryonic mouse cells. We confirmed increased X-chromosome expression levels in female and male cells and found that the X chromosome achieved upregulation by elevated burst frequencies. By monitoring transcriptional kinetics in differentiating female mouse embryonic stem cells, we found that increased burst frequency was established on the active X chromosome when X inactivation took place on the other allele. Thus, our study provides mechanistic insights into X-chromosome upregulation.
Assuntos
Ativação Transcricional , Regulação para Cima , Cromossomo X/genética , Alelos , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Ligados ao Cromossomo X , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inativação do Cromossomo XRESUMO
Signal transducer and activator of transcription 3 (STAT3) is an oncogene and multifaceted transcription factor involved in multiple cellular functions. Its role in modifying anti-tumor immunity has been recently recognized. In this study, the biologic effects of STAT3 on immune checkpoint expression and anti-tumor responses were investigated in breast cancer (BC). A transcriptional signature of phosphorylated STAT3 was positively correlated with PD-L1 expression in two independent cohorts of early BC. Pharmacologic inhibition and gene silencing of STAT3 led to decreased Programmed Death Ligand 1 (PD-L1) expression levels in vitro, and resulted as well in reduction of tumor growth and decreased metastatic dissemination in a mammary carcinoma mouse model. The hampering of tumor progression was correlated to an anti-tumoral macrophage phenotype and accumulation of natural-killer cells, but also in reduced accrual of cytotoxic lymphocytes. In human BC, pro-tumoral macrophages correlated to PD-L1 expression, proliferation status and higher grade of malignancy, indicating a subset of patients with immunosuppressive properties. In conclusion, this study provides evidence for STAT3-mediated regulation of PD-L1 and modulation of immune microenvironment in BC.
RESUMO
We recently demonstrated that WRAP53ß acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53ß accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53ß, suggesting that WRAP53ß facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence.