The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter

Abstract

Like expression of other cell-type-specific genes, expression of the insulin gene depends on the actions of a unique set of nuclear activators. These activators cooperate synergistically in building a transcriptional activation complex that binds to the regulatory domains of the gene and activates the basal RNA polymerase machinery (reviewed in reference 7). The complexity and specificity of the interactions among these activators limit the cell types capable of building a functional activation complex. Dissection of these interactions provides insight into the mechanism by which insulin expression is limited to the correct cell type.In adult mammals, activation of the insulin gene is tightly restricted to the ␤ cells in the pancreatic islets of Langerhans, where it is expressed at high levels. This specificity is reflected in the restricted function of the insulin promoter, the proximal few hundred base pairs of which can replicate the specificity of the intact gene (19,50). Because of the complexity of the intact promoter (9, 13, 24), a short portion of the rat insulin I promoter between bp Ϫ247 and Ϫ197 upstream from the transcription initiation site has been used as a model of the types of synergistic interactions that combine to give the characteristic activity of the full promoter (15). This 50-bp fragment contains at least three distinct DNA-binding sites named E2, A3, and A4 (13). The E and A elements synergize: neither has significant activity on its own, but in combination E and A elements produce ␤-cell specific transcriptional activation (15,23).The E2 element functions as a recognition site for dimers of basic helix-loop-helix (bHLH) proteins, including a heterodimer of the ubiquitous bHLH protein E47/Pan1 and the neuroendocrine specific bHLH protein BETA2/NeuroD1 (35). The A elements each contain the sequence TAAT and have been shown to bind to several homeodomain proteins found in ␤-cell nuclei (12,16,22, 32,37 (16,38,40).The LIM homeodomain protein Lmx1.1 contains two LIM domains that form zinc-binding structures in the amino end of the molecule. The second of these two LIM domains (LIM2) directly binds to the bHLH domain of E47/Pan1 and mediates the synergy between Lmx1.1 and E47/Pan1. This interaction is specific, since analogous domains from other LIM proteins and bHLH proteins cannot substitute for the LIM2 domain of Lmx1.1 or the bHLH domain of E47/Pan1, respectively (20).PDX-1 plays an important role both in the development of the pancreas and in maintaining ␤-cell function. Mice with a targeted disruption of the pdx1 gene selectively lack a pancreas (2, 21, 36). Similar pancreatic agenesis has been found in a human patient with a single nucleotide deletion in the pdx1 gene (46). If the pancreas is allowed to develop with an intact pdx1 gene, and the pdx1 gene is disrupted only in mature ␤ cells, diabetes ensues due to impaired ␤-cell function (3). This

Nuclear Factor of Activated T Cells Regulates Transcription of the Surfactant Protein D Gene (Sftpd) via Direct Interaction with Thyroid Transcription Factor-1 in Lung Epithelial Cells

“…Nonetheless, transcriptional synergism due to physical interaction of homeodomains with other transcription factors is well documented. For example, Pitx HD interacts with basic helix-loop-helix proteins on the proopiomelanocortin promoter, whereas Pdx1 HD interacts with Pan1 on the rat insulin promoter (8,73).…”

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…First, amino acid sequences outside of the homeodomain of Pdx1 itself may contribute to DNA binding either directly or though interactions with other proteins [120,121]. For example, the interaction of Pdx1 with the bHLH factor BETA2/NeuroD1 would allow for selective targeting to genes whose regulatory regions contain appropriately-spaced binding sites for both factors [122,123]. Second, the chromatin structure or conformation of DNA at specific genes within the nucleus could alter DNA site accessibility or binding affinity, respectively.…”

“…These interacting factors include bHLH proteins that bind to Eboxes (BETA2/NeuroD1 and E2A proteins) [122,123,140,141], the basic leucine zipper factor MafA that binds to the C-box [73,142], the homeodomain proteins Pbx1 and MEIS2 [47,48,143,144], and high mobility group (HMG) proteins that bind to the DNA backbone [123]. The free energy gained from these interactions may increase the likelihood that Pdx1 targets genes with binding sites for these factors [123,144]. Alternatively, these interactions may cause conformational changes in Pdx1 and/or DNA that enhance DNA binding affinity [125].…”

Regulation of Insulin Gene Transcription by ERK1 and ERK2 in Pancreatic β Cells

“…Expression of Bacterial His 6 -tagged Recombinant Proteins-The cDNAs encoding hamster E47 (the hamster protein is usually called Pan1), mouse Beta2 (also known as NeuroD1), hamster PDX-1 (PDX-1 is also known as IPF-1, IDX-1, and STF-1), and hamster Lmx1.1 (46,47) were subcloned into pRSET-His 6 (Invitrogen). His 6 -tagged proteins were purified as described (48).…”

“…It has been hypothesized that heterodimer complexes that bind to E box elements may synergize with complexes bound to A elements to cause transactivation of the insulin gene. In particular, E47 has been shown to synergistically interact with PDX-1 (47). In addition, ERK1/2 may have other substrates in ␤ cells, either direct or through protein kinase targets such as Rsk.…”