Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-8029, USA.
Stem Cells. 2009 Nov;27(11):2703-11. doi: 10.1002/stem.229.
We previously characterized human islet-derived precursor cells (hIPCs) as a specific type of mesenchymal stem cell capable of differentiating to insulin (INS)- and glucagon (GCG)-expressing cells. However, during proliferative expansion, INS transcript becomes undetectable and then cannot be induced, a phenomenon consistent with silencing of the INS gene. We explored this possibility by determining whether ectopic expression of transcription factors known to induce transcription of this gene in beta cells, pancreatic and duodenal homeobox factor 1 (Pdx1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa), and neurogenic differentiation 1 (Neurod1), would activate INS gene expression in long-term hIPC cultures. Coexpression of all three transcription factors had little effect on INS mRNA levels but unexpectedly increased GCG mRNA at least 100,000-fold. In contrast to the endogenous promoter, an exogenous rat INS promoter was activated by expression of Pdx1 and Mafa in hIPCs. Chromatin immunoprecipitation (ChIP) assays using antibodies directed at posttranslationally modified histones show that regions of the INS and GCG genes have similar levels of activation-associated modifications but the INS gene has higher levels of repression-associated modifications. Furthermore, the INS gene was found to be less accessible to micrococcal nuclease digestion than the GCG gene. Lastly, ChIP assays show that exogenously expressed Pdx1 and Mafa bind at very low levels to the INS promoter and at 20- to 25-fold higher levels to the GCG promoter in hIPCs. We conclude that the INS gene in hIPCs is modified epigenetically ("silenced") so that it is resistant to activation by transcription factors.
我们之前已经将人胰岛前体细胞（hIPCs）鉴定为一种特定类型的间充质干细胞，其能够分化为胰岛素（INS）和胰高血糖素（GCG）表达细胞。然而，在增殖扩增过程中，INS 转录本变得无法检测，随后也无法被诱导，这一现象与 INS 基因的沉默一致。我们通过确定在长期 hIPC 培养物中异位表达已知能诱导该基因在β细胞、胰腺十二指肠同源盒因子 1（Pdx1）、V-maf 肌肉关节纤维肉瘤癌基因同源物 A（Mafa）和神经发生分化 1（Neurod1）中转录的转录因子，是否会激活 INS 基因表达，来探索这种可能性。三种转录因子的共表达对 INS mRNA 水平几乎没有影响，但出人意料的是，GCG mRNA 水平至少增加了 100,000 倍。与内源性启动子不同，在 hIPCs 中表达 Pdx1 和 Mafa 可激活外源大鼠 INS 启动子。使用针对翻译后修饰组蛋白的抗体进行的染色质免疫沉淀（ChIP）实验表明，INS 和 GCG 基因的区域具有相似水平的激活相关修饰，但 INS 基因具有更高水平的抑制相关修饰。此外，与 GCG 基因相比，INS 基因对微球菌核酸酶消化的可及性较低。最后，ChIP 实验表明，外源性表达的 Pdx1 和 Mafa 以非常低的水平结合到 hIPCs 中的 INS 启动子，而结合到 GCG 启动子的水平则高出 20-25 倍。我们得出结论，hIPCs 中的 INS 基因在表观遗传上被修饰（“沉默”），从而使其对转录因子的激活具有抗性。
