RESUMEN
Gene mutations play important roles in tumour development. In this study, we identified a functional histone H2B mutation H2BL-T11C, causing an amino acid variation from Leu to Pro (L3P, H2BL-L3P). Cells overexpressing H2BL-L3P showed stronger proliferation, colony formation, tumourigenic abilities, and a different cell cycle distribution. Meanwhile, the c-Myc expression was elevated as evident by RNA-seq. We further revealed that an H2BK5ac-H2BK120ubi crosstalk which regulates gene transcription. Moreover, EdU staining demonstrated an important role of c-Myc in accelerating cell cycle progression through the G1/S checkpoint, while treatment with 10058-F4, an inhibitor of the c-Myc/MAX interaction, alleviated the abnormal cell proliferation and cell cycle distribution in vitro and partially inhibited tumour growth in vivo. The mutation of amino acid L3P is associated with tumour progression, suggesting patients carrying this SNP may have higher risk of tumour development.
Asunto(s)
Proliferación Celular/fisiología , Variación Genética/genética , Histonas/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , Regulación hacia Arriba/fisiología , Animales , Línea Celular Tumoral , Células HEK293 , Histonas/metabolismo , Humanos , Lentivirus , Leucina/genética , Ratones , Ratones Desnudos , Mutación/genética , Neoplasias/metabolismo , Neoplasias/patología , Nucleótidos/genética , Prolina/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The regulation of histone deposits mediated by multi-chaperone complexes under physiological conditions remains to be further investigated. Here, we studied the function of nuclear autoantigenic sperm protein (NASP) in the regulation of liver cancer. We found that NASP levels in liver tumors were generally higher than in normal liver tissues and NASP down-regulation inhibited liver cancer cells from forming tumors. We further analyzed cellular responses and epigenetic mechanisms of the histone H3-H4 shortage induced by NASP knockdown in liver cancer cells. The results showed that the major effects of NASP knockdown were globally enhanced chromatin accessibility, which facilitates transcription release, and failure of replication initiation. Furthermore, we demonstrated that NASP depletion led to a global decrease of histone H3K9me1 modification associated with newly H3 processing, which occurred directly at the promoters of up-regulated anti-tumor genes BACH2 and RunX1T1. This also resulted in a synergistic effect on enhanced apoptosis with Myc and p53 decreases. Overall, our work provides new insights into the roles of NASP in tumorigenesis and cancer prevention.
Asunto(s)
Autoantígenos/metabolismo , Carcinoma Hepatocelular/patología , Cromatina/metabolismo , Histonas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Nucleares/metabolismo , Animales , Autoantígenos/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Trasplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-ß, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias Humanas/citología , Proteoma/metabolismo , Acetilación , Acilación/fisiología , Células Cultivadas , Hepatocitos/citología , Hepatocitos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Redes y Vías Metabólicas , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Ácido Succínico/metabolismoRESUMEN
RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.
Asunto(s)
Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapas de Interacción de Proteínas , Proteómica/métodos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Regulación hacia Abajo/genética , Regulación hacia Abajo/efectos de la radiación , Células HEK293 , Humanos , Modelos Biológicos , Proteínas/metabolismo , Proteolisis/efectos de la radiación , Receptores Citoplasmáticos y Nucleares/metabolismo , Ubiquitina/metabolismo , Rayos X , Receptor de Lamina BRESUMEN
Histone H2B monoubiquitination is a key histone modification that has significant effects on chromatin higher-order structure and gene transcription. Multiple biological processes have been suggested to be tightly related to the dynamics of H2B monoubiquitination. However, a comprehensive understanding of biological roles of H2B monoubiquitination is still poorly understood. In the present study, we developed an efficient tool to disrupt endogenous H2B monoubiquitination levels by using an H2BK120R mutant construct expressed in human cells. Genome-wide microarray analysis of these cells revealed a potential global view of biological functions of H2B monoubiquitination. Bioinformatics analysis of our data demonstrated that while H2B monoubiquitination expectedly affected a number of previously reported biological pathways, we also uncovered the influence of this histone modification on many novel biological processes. Therefore, our work provided valuable information for understanding the role of H2B monoubiquitination and indicated potential directions for its further studies.
