RESUMO
DNA damage can induce a tumor suppressive response termed cellular senescence. Damaged senescent cells permanently arrest growth, secrete inflammatory cytokines and other proteins and harbor persistent nuclear foci that contain DNA damage response (DDR) proteins. To understand how persistent damage foci differ from transient foci that mark repairable DNA lesions, we identify sequential events that differentiate transient foci from persistent foci, which we term 'DNA segments with chromatin alterations reinforcing senescence' (DNA-SCARS). Unlike transient foci, DNA-SCARS associate with PML nuclear bodies, lack the DNA repair proteins RPA and RAD51, lack single-stranded DNA and DNA synthesis and accumulate activated forms of the DDR mediators CHK2 and p53. DNA-SCARS form independently of p53, pRB and several other checkpoint and repair proteins but require p53 and pRb to trigger the senescence growth arrest. Importantly, depletion of the DNA-SCARS-stabilizing component histone H2AX did not deplete 53BP1 from DNA-SCARS but diminished the presence of MDC1 and activated CHK2. Furthermore, depletion of H2AX reduced both the p53-dependent senescence growth arrest and p53-independent cytokine secretion. DNA-SCARS were also observed following severe damage to multiple human cell types and mouse tissues, suggesting that they can be used in combination with other markers to identify senescent cells. Thus, DNA-SCARS are dynamically formed distinct structures that functionally regulate multiple aspects of the senescent phenotype.
Assuntos
Ciclo Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Cromatina/metabolismo , Citocinas/metabolismo , Dano ao DNA/efeitos da radiação , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Citocinas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios XRESUMO
Numerous in vitro as well as genetic studies have demonstrated that the activities of the E2A proteins are regulated at multiple levels, including modulation of DNA binding by the Id proteins, association with the transcriptional modulators p300 and ETO, and posttranslational modifications. Here, we use affinity purification of tagged E47 combined with mass spectrometry in order to show that E47 interacts with the entire ensemble of Id proteins, namely, Id1, Id2, Id3, and Id4. Furthermore, we find that the lysine-specific histone demethylase 1 (LSD1), the protein arginine N-methyltransferase 5 (PRMT5), the corepressor CoREST, and the chaperones of the 14-3-3 family associate with affinity-purified E47. We also identify a spectrum of amino acid residues in E47 that are phosphorylated, including an AKT substrate site. We did, however, find that mutation of the identified AKT substrate site by itself did not perturb B cell development. In sum, these studies show that the entire ensemble of Id proteins has the ability to interact with E47, identify factors that associate with E47, and reveal a spectrum of phosphorylated residues in E47, including an AKT substrate site.