Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1.: Importance of chromatin structure in directing promoter binding.
Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1.: Importance of chromatin structure in directing promoter binding.
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DOI：
10.1074/jbc.m111857200
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发表时间：
2002-04-12
影响因子：
4.8
通讯作者：
Mirmira, RG
中科院分区：
文献类型：
--
作者：
Chakrabarti, SK;James, JC;Mirmira, RG
转录因子Pdx 1在胰腺β细胞中表达，据信其调节几种β细胞特异性基因。尽管Pdx 1与β细胞基因元件的结合已在体外得到证实，但这些基因中几乎没有一个已被证明是细胞内Pdx 1的直接结合靶点（其中存在复杂的染色质结构）。为了确定哪些β细胞启动子在体内被Pdx 1结合，我们使用Pdx 1抗血清和来自β-TC 3细胞和Pdx 1转染的NIH 3 T3细胞的染色质进行染色质免疫沉淀测定，随后使用实时PCR定量共免疫沉淀的启动子。我们比较了这些在体内的研究结果，并行免疫沉淀，其中Pdx 1被允许结合到启动子片段在体外反应。我们的研究结果表明，在所有的细胞Pdx 1强烈结合胰岛素，胰岛淀粉样多肽，胰高血糖素，Pdx 1，和Pax 4启动子，而它不结合葡萄糖转运蛋白2型或白蛋白启动子。此外，在β细胞中未观察到Pdx 1与葡萄糖激酶启动子的结合。相反，在体外免疫沉淀，Pdx 1结合所有的启动子的程度大约成比例的Pdx 1结合位点的数量。我们的研究结果表明，在β细胞和非β细胞中，染色质结构在指导Pdx 1的启动子结合选择性方面起着关键作用。
The transcription factor Pdx1 is expressed in the pancreatic beta-cell, where it is believed to regulate several beta-cell-specific genes. Whereas binding by Pdx1 to elements of beta-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which beta-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from beta-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using realtime PCR. We compared these in vivo findings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in beta-cells. In contrast, in in vitro immunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in beta-cells and non-beta-cells.