肼屈嗪对多能性和染色质重塑基因的细胞类型依赖性调节。
Cell-type dependent regulation of pluripotency and chromatin remodeling genes by hydralazine.
机构信息
Departamento de Biología Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, 64720, Monterrey, Nuevo León, Mexico.
Depto. de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, 78216, San Luis Potosí, S.L.P., Mexico.
出版信息
Stem Cell Res Ther. 2023 Mar 16;14(1):42. doi: 10.1186/s13287-023-03268-w.
BACKGROUND
The generation of induced pluripotent stem cells has opened the field of study for stem cell research, disease modeling and drug development. However, the epigenetic signatures present in somatic cells make cell reprogramming still an inefficient process. This epigenetic memory constitutes an obstacle in cellular reprogramming. Here, we report the effect of hydralazine (HYD) and valproic acid (VPA), two small molecules with proven epigenetic activity, on the expression of pluripotency genes in adult (aHF) and neonatal (nbHF) human fibroblasts.
METHODS
aHF and nbHF were treated with HYD and/or VPA, and viability and gene expression assays for OCT4, NANOG, c-MYC, KLF4, DNMT1, TET3, ARID1A and ARID2 by quantitative PCR were performed. aHF and nbHF were transfected with episomal plasmid bearing Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and exposed to HYD and VPA to determine the reprogramming efficiency. Methylation sensitive restriction enzyme (MSRE) qPCR assays were performed on OCT4 and NANOG promoter regions. Immunofluorescence assays were carried out for pluripotency genes on iPSC derived from aHF and nbHF.
RESULTS
HYD upregulated the expression of OCT4 (2.5-fold) and NANOG (fourfold) genes but not c-Myc or KLF4 in aHF and had no significant effect on the expression of all these genes in nbHF. VPA upregulated the expression of NANOG (twofold) in aHF and c-MYC in nbHF, while it downregulated the expression of NANOG in nbHF. The combination of HYD and VPA canceled the OCT4 and NANOG overexpression induced by HYD in aHF, while it reinforced the effects of VPA on c-Myc expression in nbHF. The HYD-induced overexpression of OCT4 and NANOG in aHDF was not dependent on demethylation of gene promoters, and no changes in the reprogramming efficiency were observed in both cell populations despite the downregulation of epigenetic genes DNMT1, ARID1A, and ARID2 in nbHF.
CONCLUSIONS
Our data provide evidence that HYD regulates the expression of OCT4 and NANOG pluripotency genes as well as ARID1A and ARID2 genes, two members of the SWI/SNF chromatin remodeling complex family, in normal human dermal fibroblasts.
背景
诱导多能干细胞的产生开启了干细胞研究、疾病建模和药物开发领域的研究。然而，体细胞中的表观遗传特征使得细胞重编程仍然是一个效率低下的过程。这种表观遗传记忆构成了细胞重编程的障碍。在这里，我们报告了两种具有已知表观遗传活性的小分子肼（HYD）和丙戊酸（VPA）对成人（aHF）和新生儿（nbHF）人成纤维细胞中多能性基因表达的影响。
方法
用 HYD 和/或 VPA 处理 aHF 和 nbHF，并用定量 PCR 检测 OCT4、NANOG、c-MYC、KLF4、DNMT1、TET3、ARID1A 和 ARID2 的增殖基因表达。用携带 Yamanaka 因子（OCT4、SOX2、KLF4 和 c-MYC）的附加体质粒转染 aHF 和 nbHF，并暴露于 HYD 和 VPA 以确定重编程效率。用甲基敏感限制性内切酶（MSRE）qPCR 测定 OCT4 和 NANOG 启动子区域的甲基化。用免疫荧光法检测源自 aHF 和 nbHF 的 iPSC 的多能性基因。
结果
HYD 上调了 aHF 中 OCT4（2.5 倍）和 NANOG（4 倍）基因的表达，但对 nbHF 中所有这些基因的表达没有显著影响。VPA 上调了 aHF 中 NANOG（2 倍）和 nbHF 中 c-MYC 的表达，同时下调了 nbHF 中 NANOG 的表达。HYD 和 VPA 的组合取消了 HYD 诱导的 aHF 中 OCT4 和 NANOG 的过表达，同时增强了 VPA 对 nbHF 中 c-MYC 表达的影响。HYD 在 aHFDF 中诱导的 OCT4 和 NANOG 过表达不依赖于基因启动子的去甲基化，尽管 nbHF 中表观遗传基因 DNMT1、ARID1A 和 ARID2 的下调，但在两种细胞群中均未观察到重编程效率的变化。
结论
我们的数据提供了证据，证明 HYD 调节 OCT4 和 NANOG 多能性基因以及 ARID1A 和 ARID2 基因的表达，ARID1A 和 ARID2 基因是 SWI/SNF 染色质重塑复合物家族的两个成员，在正常人类真皮成纤维细胞中也是如此。
Zotero 插件