RESUMO
The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.
Assuntos
Proteínas de Ligação a DNA , Ubiquitina , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.
Assuntos
Carcinoma/patologia , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Metástase Neoplásica/genética , Animais , Carcinogênese/genética , Carcinoma/genética , Linhagem Celular Tumoral , Cromatina/genética , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Progressão da Doença , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are activated by DNA double-strand breaks (DSBs). These DSBs occur in the context of chromatin but how chromatin influences the activation of these kinases is not known. Here we show that loss of the replication-dependent chromatin assembly factors ASF1A/B or CAF-1 compromises ATM activation, while augmenting DNA-PKcs activation, in response to DNA DSBs. Cells deficient in ASF1A/B or CAF-1 exhibit reduced histone H4 lysine 16 acetylation (H4K16ac), a histone mark known to promote ATM activation. ASF1A interacts with the histone acetyl transferase, hMOF that mediates H4K16ac. ASF1A depletion leads to increased recruitment of DNA-PKcs to DSBs. We propose normal chromatin assembly and H4K16ac during DNA replication is required to regulate ATM and DNA-PKcs activity in response to the subsequent induction of DNA DSBs.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteína Quinase Ativada por DNA/genética , Chaperonas de Histonas/genética , Proteínas Nucleares/genética , Acetilação , Linhagem Celular Tumoral , Cromatina/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA/genética , Células HCT116 , Células HeLa , Histonas/genética , Humanos , Chaperonas Moleculares , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Hypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown. RESULTS: We show that TET1 regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigates hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes. Cholesterol metabolic process genes are among the genes that showed high prevalence and statistical significance. We characterize one of the genes, INSIG1 (insulin induced gene 1), to confirm its expression and the 5hmC levels in its promoter. Knockdown of INSIG1 also mitigates hypoxia-induced EMT. Finally, TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity. TET1 acts as a co-activator to further enhance the expression of INSIG1 together with HIF-2α. We define the domain in HIF-1α that interacts with TET1 and map the domain in TET1 that confers transactivation to a 200 amino acid region that contains a CXXC domain. The TET1 catalytically inactive mutant is capable of rescuing hypoxia-induced EMT in TET1 knockdown cells. CONCLUSIONS: These findings demonstrate that TET1 serves as a transcription co-activator to regulate hypoxia-responsive gene expression and EMT, in addition to its role in demethylating 5mC.