Transcription factor occupancy of the insulin gene in vivo -: Evidence for direct regulation by Nkx2.2
Transcription factor occupancy of the insulin gene in vivo -: Evidence for direct regulation by Nkx2.2
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DOI：
10.1074/jbc.m205905200
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发表时间：
2003-01-10
影响因子：
4.8
通讯作者：
Stein, R
中科院分区：
文献类型：
--
作者：
Cissell, MA;Zhao, L;Stein, R
许多转录因子的结合位点是相对非选择性的，并且在基因组内以高频率发现。这就提出了一种可能性，即能够在体外与顺式作用元件结合并调节瞬时转染质粒转录的因子（不具有高级染色质结构）可能不占据内源基因内的该位点。封闭的染色质结构和来自具有相似核苷酸特异性的另一种DNA结合蛋白的竞争是两种可能的机制，通过这两种机制，转录因子可以被排除在体内潜在的结合位点之外。多种转录因子，包括Pdx-1、BETA-2和Pax6，已经涉及胰腺β细胞中胰岛素基因的表达。在这项研究中，染色质免疫沉淀试验已被用来表明，这些因素，事实上，结合胰岛素控制区序列在完整的β细胞。此外，另一个关键的胰岛富集的转录因子，Nkx2.2，被发现占据这个区域使用染色质免疫沉淀测定。体外DNA结合和瞬时转染试验确定了Nkx2.2如何影响胰岛素基因表达。Pdx-1也被证明在与体外对照相关的内源性胰岛淀粉样多肽、pax-4和葡萄糖激酶基因的区域内结合。由于Pdx-1不单独调节基因转录，因此检查这些序列是否被其他胰岛素转录调节因子占据。还发现BETA-2、Pax6和Nkx2.2在体内与淀粉样多肽、葡糖激酶和pax-4控制序列结合。这些研究揭示了Pdx-1、BETA-2、Pax6和Nkx2.2转录因子在调节胰岛β细胞中选择性表达的基因表达中的广泛应用。
Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. This raises the possibility that factors that are capable of binding to a cis-acting element in vitro and regulating transcription from a transiently transfected plasmid, which would not have higher order chromatin structure, may not occupy this site within the endogenous gene. Closed chromatin structure and competition from another DNA-binding protein with similar nucleotide specificity are two possible mechanisms by which a transcription factor may be excluded from a potential binding site in vivo. Multiple transcription factors, including Pdx-1, BETA-2, and Pax6, have been implicated in expression of the insulin gene in pancreatic beta cells. In this study, the chromatin immunoprecipitation assay has been used to show that these factors do, in fact, bind to insulin control region sequences in intact beta cells. In addition, another key islet-enriched transcription factor, Nkx2.2, was found to occupy this region using the chromatin immunoprecipitation assay. In vitro DNA-binding and transient transfection assays defined how Nkx2.2 affected insulin gene expression. Pdx-1 was also shown to bind within a region of the endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in vitro. Because Pdx-1 does not regulate gene transcription in isolation, these sequences were examined for occupancy by the other insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and pax-4 control sequences in vivo. These studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and Nkx2.2 transcription factors in regulating expression of genes selectively expressed in islet beta cells.