RESUMO
Estrogen (E2) is crucial for the development of breast cancer caused by BRCA1 mutation, and can increase the DNA damage in BRCA1-deficient cells. However, the mechanisms through which BRCA1 deficiency and E2 synergistically induce DNA damage remains unclear. In this study, we analyzed the distribution of DNA damage in E2-treated BRCA1-deficient cells. We detected DNA lesions in the vicinity of genes that are transcriptionally activated by estrogen receptor-α (ER). Loss of BRCA1 altered chromatin binding by ER, which significantly affected the distribution of DNA damage. Moreover, these changes were associated with the established mutations in BRCA1-mutant breast cancer. Taken together, our findings reveal a new mechanism underlying the DNA damage in breast cancer cells that is synergistically induced by BRCA1 deficiency and E2.
Assuntos
Neoplasias da Mama , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Dano ao DNA , Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Feminino , Humanos , MutaçãoRESUMO
Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).
Assuntos
Fenômenos Fisiológicos Celulares/genética , Clonagem Molecular/métodos , Técnicas Citológicas/métodos , Proteínas , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Vetores Genéticos/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Reorganization of topologically associated domain (TAD) is considered to be a novel mechanism for cell fate transitions. Here, we present a protocol to manipulate TAD via abscisic acid (ABA)-dependent genome linking. We use this protocol to merge two adjacent TADs and evaluate the influence on cell fate transitions. The advantages are that the manipulation does not change the genome and is reversible by withdrawing ABA. The major challenge is how to select linking loci for efficient TAD reorganization. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).
