RESUMO
Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced. This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. Downregulation of human NAP1L3 in umbilical cord blood (UCB) HSCs impairs the maintenance and proliferation of HSCs both in vitro and in vivo. NAP1L3 downregulation in UCB HSCs causes an arrest in the G0 phase of cell cycle progression and induces gene expression signatures that significantly correlate with downregulation of gene sets involved in cell cycle regulation, including E2F and MYC target genes. Moreover, we demonstrate that HOXA3 and HOXA5 genes are markedly upregulated when NAP1L3 is suppressed in UCB HSCs. Taken together, our findings establish an important role for NAP1L3 in HSC homeostasis and haematopoietic differentiation.
Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Sangue Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/metabolismo , Chaperonas de Histonas/genética , Humanos , Camundongos , Fase de Repouso do Ciclo Celular/genéticaRESUMO
Aberrant DNA methylation has been reported as an important phenotype in acute myeloid leukemia. However the clinical significance of methylation changes has not been clear yet. In this study methylation Specific Melting Curve Analysis (MS-MCA) and real time PCR was used to assess the CDKN2B promoter hyper-methylation and gene expression in 59 Iranian acute myeloid leukemia (AML) patients. The incidence of aberrant hyper methylation of CDKN2B gene and cytogenetic abnormalities were 37.3% (22 of 59 patients) and 35.6% (21 of 59) respectively in our patients. We observed that CDKN2B expression level was lower than normal mesenchymal stem cells. Our data revealed significant correlation between methylated CDKN2B promoter region and mRNA gene expression (P= 0.007). Also, our data indicated that AML patients with aberrant methylation of CDKN2B gene had a lower survival rates (P=0.043). In addition, they had a higher proportion of leukemic blast cells (P=0.022) and higher white blood cell count in peripheral blood (P=0.0123). Aberrant methylation of CDKN2B was observed to higher in M2 subtype and lower in M3 and M4 subtypes. Although, we observed a significant correlation between Methylation and survival, there was no significant correlation between CDKN2B methylation and treatment outcome of AML patients (P=0.187). Furthermore, our data didn't illustrated a significant correlation between CDKN2B expression and survival (P=0.93). In conclusion our study showed that the aberrant methylation is one of molecular mechanisms involved in CDKN2B gene expression, moreover we can consider the CDKN2B methylation, as a prognostic marker in predict AML patients' survival.
