Abstract
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.
Highlights
Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome
BETA-2, Pax6, and Nkx2.2 were 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  cells
Pdx-1 antisera did not immunoprecipitate control sequences from the phosphoenolpyruvate carboxykinase (PCK) gene, which is not expressed in  cells (Fig. 1A)
Summary
Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. An extensive series of in vitro gel shift and cell transfection experiments indicates that regulation is mediated by islet-enriched DNA-binding transcription factors, including Pdx-1 [5,6,7], BETA-2 [8], and Pax6 [9] Each of these factors is a key regulator of pancreas development. Assays such as in vitro DNA binding, co-transfection of reporter constructs, and transgenic knock-out animals provide evidence for the involvement of a factor in transcriptional control Such experiments are indirect measures of regulation and do not account for conditions like competition with other binding proteins in vivo, chromatin structure of the endogenous gene, and cascades of regulatory interactions. Our findings that BETA-2, Pax, and Nkx2.2 bind within the transcription control region of other islet-enriched genes highlights how the ChIP assay can be used to identify targeted genes
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