Functional regions of the homeodomain protein IDX-1 required for transactivation of the rat somatostatin gene.

Abstract

The insulin-, glucagon- and somatostatin-producing cells (beta, alpha, and delta, respectively) in the pancreatic islets derive from a common precursor stem cell and differentiate sequentially during embryonic development. The homeodomain protein islet duodenum HOX (IDX)-1 [insulin promoter factor (IPF)-1/somatostatin transactivating factor (STF)-1)] is a transcription factor critically required for both the development of the pancreas and the transcriptional expression of the insulin gene. IDX-1 may also act to determine the differentiation of the common pancreatic precursor to beta, alpha, and delta cells. Although IDX-1 is detected in most adult mouse islet beta-cells and regulates insulin gene transcription, it is also found in 15% of the delta-cells and transactivates the rat somatostatin gene. The roles of different domains of IDX-1 involved in the transactivation of the somatostatin gene are unclear. In this study, we have created a series of amino- and carboxy-terminal deletions, as well as point substitution mutations to delineate functional domains within the IDX-1 protein. We find that deletions amino-proximal to the homeodomain enhance DNA-binding to the TAAT-1 transcriptional control element within the somatostatin gene promoter. However, these amino-terminal deletions result in substantial decreases in transactivation of a transcriptional reporter containing the TAAT-1 element. Paradoxically, coexpression of the transcriptionally inactive, amino-terminally deleted IDX-1 mutant proteins, either with the wild-type IDX-1 or with themselves, results in a marked enhancement of transactivation of the transcriptional TAAT-1 element reporter. We provide evidence that this synergistic enhancement of transactivation is mediated by protein-protein interactions among the regions of IDX-1 located carboxyl-proximal to the homeodomain. Although successive deletions into the carboxy-terminal region do not alter DNA-binding, these deletions result in a biphasic enhancement and diminution of transactivation. The IDX-1 homeodomain mediates sequence- specific DNA-binding because substitution mutations within this region abolish DNA-binding. All of the amino- and carboxy-terminal deletion proteins were present in nuclear extracts of transfected cells, suggesting that nuclear localization signals reside within the IDX-1 homeodomain. The mapping of the functional domains of IDX-1 may facilitate understanding of IDX-1-mediated gene regulation and islet cell development.