Francis Joshua, Babu Daniella A, Deering Tye G, Chakrabarti Swarup K, Garmey James C, Evans-Molina Carmella, Taylor David G, Mirmira Raghavendra G
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Mol Endocrinol. 2006 Dec;20(12):3133-45. doi: 10.1210/me.2006-0126. Epub 2006 Aug 10.
The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in beta-cells (betaTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in betaTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the beta-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1.
胰腺十二指肠同源盒因子1（Pdx-1）是一种类似Hox的转录因子，负责激活胰岛素基因。先前的研究已经证明，Pdx-1在体外与对应于胰岛素启动子A盒的短（20-40个核苷酸）DNA片段相互作用。然而，Pdx-1在染色质复杂环境中如何与DNA结合从未被研究过。在本研究中，我们探讨了β细胞（βTC3）与胰腺导管细胞（mPAC）中胰岛素基因的染色质可及性如何影响Pdx-1与DNA的相互作用。我们证明，Pdx-1占据βTC3细胞中的内源性胰岛素启动子，但不占据mPAC细胞中的内源性胰岛素启动子，这一发现与细胞内Pdx-1蛋白浓度无关。基于微球菌核酸酶保护分析，两种细胞类型之间启动子结合的差异似乎是由于胰岛素启动子-126至-296碱基对（相对于转录起始位点）之间预测的Pdx-1结合位点处的染色质可及性所致。使用纯化的Pdx-1和体外重组染色质的结合研究表明，启动子这一关键区域内核小体的定位可能解释了染色质可及性的差异。与这些观察结果一致，荧光共定位研究表明，Pdx-1不占据细胞核内紧密的、富含核小体的染色质区域。我们的研究结果提出了一个模型，即β细胞中的胰岛素转录至少部分是由-126至-296碱基对之间关键调控区域内染色质可及性增强所促进的，从而允许诸如Pdx-实 1等转录激活因子占据。
