RESUMO
DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Cromatina/química , Cromatina/efeitos da radiação , Montagem e Desmontagem da Cromatina/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação/efeitos da radiação , Fator B de Elongação Transcricional Positiva/metabolismo , Isoformas de Proteínas/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/química , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.
Assuntos
Proteínas de Ciclo Celular/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Estruturas R-Loop/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Fase S , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Effective treatment for glioblastoma (GBM) is limited by the presence of the blood-brain barrier (BBB) and rapid resistance to single agent therapies. To address these issues, we developed a transferrin-functionalized nanoparticle (Tf-NP) that can deliver dual combination therapies. Using intravital imaging, we show the ability of Tf-NPs to traverse intact BBB in mice as well as achieve direct tumor binding in two intracranial orthotopic models of GBM. Treatment of tumor-bearing mice with Tf-NPs loaded with temozolomide and the bromodomain inhibitor JQ1 leads to increased DNA damage and apoptosis that correlates with a 1.5- to 2-fold decrease in tumor burden and corresponding increase in survival compared to equivalent free-drug dosing. Immunocompetent mice treated with Tf-NP-loaded drugs also show protection from the effects of systemic drug toxicity, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to gliomas and other central nervous system tumors.