RESUMO
Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
Assuntos
Reprogramação Celular/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/antagonistas & inibidores , Fator 1 de Modelagem da Cromatina/genética , Regulação da Expressão Gênica/genética , Heterocromatina/metabolismo , Camundongos , Nucleossomos/metabolismo , Interferência de RNA , Transdução GenéticaRESUMO
Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.
Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fator 1 de Modelagem da Cromatina/antagonistas & inibidores , Fator 1 de Modelagem da Cromatina/genética , Neoplasias do Colo/genética , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fatores de Transcrição , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Adulto JovemRESUMO
Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G2/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G2/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.