Pdx1 Homeodomain Research Articles

Compromised Function of the Pancreatic Transcription Factor PDX1 in a Lineage of Desert Rodents

Gerbils are a subfamily of rodents living in arid regions of Asia and Africa. Recent studies have shown that several gerbil species have unusual amino acid changes in the PDX1 protein, a homeodomain transcription factor essential for pancreatic development and β-cell function. These changes were linked to strong GC-bias in the genome that may be caused by GC-biased gene conversion, and it has been hypothesized that this caused accumulation of deleterious changes. Here we use two approaches to examine if the unusual changes are adaptive or deleterious. First, we compare PDX1 protein sequences between 38 rodents to test for association with habitat. We show the PDX1 homeodomain is almost totally conserved in rodents, apart from gerbils, regardless of habitat. Second, we use ectopic gene overexpression and gene editing in cell culture to compare functional properties of PDX1 proteins. We show that the divergent gerbil PDX1 protein inefficiently binds an insulin gene promoter and ineffectively regulates insulin expression in response to high glucose in rat cells. The protein has, however, retained the ability to regulate some other β-cell genes. We suggest that during the evolution of gerbils, the selection-blind process of biased gene conversion pushed fixation of mutations adversely affecting function of a normally conserved homeodomain protein. We argue these changes were not entirely adaptive and may be associated with metabolic disorders in gerbil species on high carbohydrate diets. This unusual pattern of molecular evolution could have had a constraining effect on habitat and diet choice in the gerbil lineage.

Probing Disordered Region Influence on Pdx1 Binding to Natural Promoters and Near‐Consensus Sites

Pdx1 is a well‐known protein in the set of β‐cell‐specific transcription factors (TFs), involved in the production of insulin and islet amyloid polypeptide (IAPP) hormones. Like many TFs, Pdx1 contains regions of disorder whose function in transcription has not been adequately studied. It is known that the Pdx1 homeodomain (HD) binds to a core DNA recognition sequence containing the tetranucleotide TAAT, for which its consensus binding site is reported as 5´‐CTCTAAT(T/G)AG‐3´. Naturally, insulin is expressed in much higher levels than IAPP, perhaps owing to the affinity of Pdx1 to its target sites. Alignment of human insulin and iapp promoter elements highlights the single nucleobase substitution from 5´‐CTAAT‐3´ to 5´‐TTAAT‐3´, respectively. We hypothesize that the interactions mediated by the disordered tails provide additional target specificity by interacting with DNA sequences upstream of the TAAT motif. Here we report a combination of ITC, NMR spectroscopy, and multi‐microsecond MD calculations of Pdx1 variants that define its interactions with a panel of natural promoter and consensus‐derived sequences. Our results point to a small preference of the Pdx1 HD for 5´‐CTAAT‐3´. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. More significantly, our computational studies suggest that interactions between the disordered region of Pdx1 and the major groove of the DNA are responsible for sequence identification upstream of the core motif. Taken together, our results suggest a molecular mechanism where the disordered regions of Pdx1 are responsible for establishing differential affinity to elements from the insulin and iapp promoter sequences.

Thermodynamic and Structural Studies of Pdx1 Binding to Elements from Natural Promoters and Near-Consensus Sites

The production of insulin and islet amyloid polypeptide (IAPP) is limited to β-cells due to restricted expression of a set of tissue-specific transcription factors, the most well known of which is Pdx1. Naturally, insulin is expressed in much higher levels than IAPP, perhaps owing to the affinity of Pdx1 to its target sites. Like many transcription factors, Pdx1 contains regions of disorder whose function in transcription has not been adequately studied. It is well documented that the Pdx1 homeodomain binds to a core DNA recognition sequence containing the tetranucleotide TAAT, for which its consensus binding site is reported as 5′-CTCTAAT(T/G)AG-3′.

Binding Polymorphism in the DNA Bound State of the Pdx1 Homeodomain

The subtle effects of DNA-protein recognition are illustrated in the homeodomain fold. This is one of several small DNA binding motifs that, in spite of limited DNA binding specificity, adopts crucial, specific roles when incorporated in a transcription factor. The homeodomain is composed of a 3-helix domain and a mobile N-terminal arm. Helix 3 (the recognition helix) interacts with the DNA bases through the major groove, while the N-terminal arm becomes ordered upon binding a specific sequence through the minor groove. Although many structural studies have characterized the DNA binding properties of homeodomains, the factors behind the binding specificity are still difficult to elucidate. A crystal structure of the Pdx1 homeodomain bound to DNA (PDB 2H1K) obtained previously in our lab shows two complexes with differences in the conformation of the N-terminal arm, major groove contacts, and backbone contacts, raising new questions about the DNA recognition process by homeodomains. Here, we carry out fully atomistic Molecular Dynamics simulations both in crystal and aqueous environments in order to elucidate the nature of the difference in binding contacts. The crystal simulations reproduce the X-ray experimental structures well. In the absence of crystal packing constraints, the differences between the two complexes increase during the solution simulations. Thus, the conformational differences are not an artifact of crystal packing. In solution, the homeodomain with a disordered N-terminal arm repositions to a partially specific orientation. Both the crystal and aqueous simulations support the existence of different stable binding conformers identified in the original crystallographic data with different degrees of specificity. We propose that protein-protein and protein-DNA interactions favor a subset of the possible conformations. This flexibility in DNA binding may facilitate multiple functions for the same transcription factor.

Human Pdx1 Homeodomain in the Unbound State

Human Pdx1 Homeodomain in the Unbound State

A Novel Hypomorphic PDX1 Mutation Responsible for Permanent Neonatal Diabetes With Subclinical Exocrine Deficiency

OBJECTIVEGenes responsible for monogenic forms of diabetes have proven very valuable for understanding key mechanisms involved in β-cell development and function. Genetic study of selected families is a powerful strategy to identify such genes. We studied a consanguineous family with two first cousins affected by neonatal diabetes; their four parents had a common ancestor, suggestive of a fully penetrant recessive mutation.RESEARCH DESIGN AND METHODSWe performed genetic studies of the family, detailed clinical and biochemical investigations of the patients and the four parents, and biochemical and functional studies of the new mutation.RESULTSWe found a novel mutation in the pancreatic and duodenal homeobox 1 gene (PDX1, IPF1) in the two patients, which segregated with diabetes in the homozygous state. The mutation resulted in an E178G substitution in the PDX1 homeodomain. In contrast to other reported PDX1 mutations leading to neonatal diabetes and pancreas agenesis, homozygosity for the E178G mutation was not associated with clinical signs of exocrine pancreas insufficiency. Further, the four heterozygous parents were not diabetic and displayed normal glucose tolerance. Biochemical studies, however, revealed subclinical exocrine pancreas insufficiency in the patients and slightly reduced insulin secretion in the heterozygous parents. The E178G mutation resulted in reduced Pdx1 transactivation despite normal nuclear localization, expression level, and chromatin occupancy.CONCLUSIONSThis study broadens the clinical spectrum of PDX1 mutations and justifies screening of this gene in neonatal diabetic patients even in the absence of exocrine pancreas manifestations.

Pancreatic Ductal Morphogenesis and the Pdx1 Homeodomain Transcription Factor

Embryonic development of the pancreas is marked by an early phase of dramatic morphogenesis, in which pluripotent progenitor cells of the developing pancreatic epithelium give rise to the full array of mature exocrine and endocrine cell types. The genetic determinants of acinar and islet cell lineages are somewhat well defined; however, the molecular mechanisms directing ductal formation and differentiation remain to be elucidated. The complex ductal architecture of the pancreas is established by a reiterative program of progenitor cell expansion and migration known as branching morphogenesis, or tubulogenesis, which proceeds in mouse development concomitantly with peak Pdx1 transcription factor expression. We therefore evaluated Pdx1 expression with respect to lineage-specific markers in embryonic sections of the pancreas spanning this critical period of duct formation and discovered an unexpected population of nonislet Pdx1-positive cells displaying physical traits of branching. We then established a 3D cell culture model of branching morphogenesis using primary pancreatic duct cells and identified a transient surge of Pdx1 expression exclusive to branching cells. From these observations we propose that Pdx1 might be involved temporally in a program of gene expression sufficient to facilitate the biochemical and morphological changes necessary for branching morphogenesis.

T1900 Hepatocyte Growth Factor in Concert with PDX-1 Homeodomain Transcription Factor Regulate Pancreatic Ductal Cell Branching Morphogenesis

T1900 Hepatocyte Growth Factor in Concert with PDX-1 Homeodomain Transcription Factor Regulate Pancreatic Ductal Cell Branching Morphogenesis

Structural Basis for Induced Fit Mechanisms in DNA Recognition by the Pdx1 Homeodomain,

Pancreatic and duodenal homeobox 1 (Pdx1) is a homeodomain transcription factor belonging to the ParaHox family. Pdx1 plays an essential role in pancreatic endocrine and exocrine cell development and maintenance of adult islet beta-cell function. Mutations in the human pdx1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus, MODY-4. We demonstrate that the homeodomain reproduces the binding specificity of the full-length protein. We report the 2.4 A resolution crystal structure of the homeodomain bound to a target DNA. The two Pdx1/DNA complexes in the asymmetric unit display conformational differences: in the DNA curvature, the orientation of the homeodomain in the major groove, and the order of the N-terminal arm. Comparing the two complexes indicates invariant protein-DNA contacts, and variant contacts that are unique to each binding orientation. An induced fit model is proposed that depends on the DNA conformation and provides a mechanism for nonlocal contributions to binding specificity.

Insulin gene transcription is mediated by interactions between the p300 coactivator and PDX-1, BETA2, and E47.

Pancreatic beta-cell-type-specific expression of the insulin gene requires both ubiquitous and cell-enriched activators, which are organized within the enhancer region into a network of protein-protein and protein-DNA interactions to promote transcriptional synergy. Protein-protein-mediated communication between DNA-bound activators and the RNA polymerase II transcriptional machinery is inhibited by the adenovirus E1A protein as a result of E1A's binding to the p300 coactivator. E1A disrupts signaling between the non-DNA-binding p300 protein and the basic helix-loop-helix DNA-binding factors of insulin's E-element activator (i.e., the islet-enriched BETA2 and generally distributed E47 proteins), as well as a distinct but unidentified enhancer factor. In the present report, we show that E1A binding to p300 prevents activation by insulin's beta-cell-enriched PDX-1 activator. p300 interacts directly with the N-terminal region of the PDX-1 homeodomain protein, which contains conserved amino acid sequences essential for activation. The unique combination of PDX-1, BETA2, E47, and p300 was shown to promote synergistic activation from a transfected insulin enhancer-driven reporter construct in non-beta cells, a process inhibited by E1A. In addition, E1A inhibited the level of PDX-1 and BETA2 complex formation in beta cells. These results indicate that E1A inhibits insulin gene transcription by preventing communication between the p300 coactivator and key DNA-bound activators, like PDX-1 and BETA2:E47.

The Role of Hepatic Nuclear Factor 1α and PDX-1 in Transcriptional Regulation of the pdx-1 Gene

The PDX-1 homeodomain transcription factor regulates pancreatic development and adult islet beta cell function. Expression of the pdx-1 gene is almost exclusively localized to beta cells within the adult endocrine pancreas. Islet beta cell-selective transcription is controlled by evolutionarily conserved subdomain sequences (termed Areas I (-2839 to -2520 base pairs (bp)), II (-2252 to -2023 bp), and III (-1939 to -1664 bp)) found within the 5'-flanking region of the pdx-1 gene. Areas I and II are independently capable of directing beta cell-selective reporter gene activity in transfection assays, with Area I-mediated stimulation dependent upon binding of hepatic nuclear factor 3 beta (HNF3 beta), a key regulator of islet beta cell function. To identify other transactivators of Area I, highly conserved sequence segments within this subdomain were mutagenized, and their effect on activation was determined. Several of the sensitive sites were found by transcription factor data base analysis to potentially bind endodermally expressed transcription factors, including HNF1 alpha (-2758 to -2746 bp, Segment 2), HNF4 (-2742 to -2730 bp, Segment 4; -2683 to -2671 bp, Segment 7-8), and HNF6 (-2727 to -2715 bp, Segment 5). HNF1 alpha, but not HNF4 and HNF6, binds specifically to Area I sequences in vitro. HNF1 alpha was also shown to specifically activate Area I-driven transcription through Segment 2. In addition, PDX-1 itself was found to stimulate Area I activation. The chromatin immunoprecipitation assay performed with PDX-1 antisera also demonstrated that this factor bound to Area I within the endogenous pdx-1 gene in beta cells. Our results indicate that regulatory factors binding to Area I conserved sequences contribute to the selective transcription pattern of the pdx-1 gene and that control is mediated by endodermal regulators like HNF1 alpha, HNF3 beta, and PDX-1.

Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor α

Background & Aims: The progenitor cells responsible for transforming growth factor (TGF)-α–induced pancreatic ductal metaplasia and neoplasia remain uncharacterized. During pancreatic development, differentiated cell types arise from ductal progenitor cells expressing the Pdx1 homeodomain transcription factor. The aims of this study were, first, to evaluate the role of Pdx1-expressing stem cells in MT-TGFα transgenic mice, and second, to further characterize cell proliferation and differentiation in this model. Methods: To assess Pdx1 gene expression in normal and metaplastic epithelium, we performed in vivo reporter gene analysis using heterozygous Pdx1 lacZ/+ and bigenic Pdx1 lacZ/+/MT-TGFα mice. Results: Pdx1 lacZ/+/MT-TGFα bigenics showed up-regulated Pdx1 expression in premalignant metaplastic ductal epithelium. In addition to Pdx1 gene activation, TGF-α–induced metaplastic epithelium demonstrated a pluripotent differentiation capacity, as evidenced by focal expression of Pax6 and initiation of islet cell neogenesis. The majority of Pdx1-positive epithelial cells showed no expression of insulin, similar to the pattern observed during embryonic development. Conclusions: Overexpression of TGF-α induces expansion of a Pdx1-expressing epithelium characterized by focal expression of Pax6 and initiation of islet neogenesis. These findings suggest that premalignant events induced by TGF-α in mouse pancreas may recapitulate a developmental program active during embryogenesis. GASTROENTEROLOGY 1999;117:1416-1426

Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1

Pancreatic duodenal homeobox-containing transcription factor 1 (PDX-1) plays a crucial role in pancreas development and β-cell gene regulation. Absence of PDX-1 leads to pancreas agenesis and its malfunction causes MODY4 diabetes mellitus. PDX-1 has been suggested to be involved in the glucose-dependent regulation of insulin gene transcription. Whereas DNA-binding and transactivation domains of PDX-1 are in the process of being characterized, protein sequences responsible for its nuclear translocation remain unknown. By combining site-directed mutagenesis of putative phosphorylation sites and nuclear localization signal (NLS) motifs with on-line monitoring of GFP-tagged PDX-1 translocation, we demonstrate that the NLS motif RRMKWKK is necessary and in conjunction with the integrity of the ‘helix 3’ domain of the PDX-1 homeodomain is sufficient for the nuclear import of PDX-1. Furthermore, we show that there is no glucose-dependent cytoplasmic-nuclear cycling of PDX-1.

Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor

Glucose-stimulated expression of the insulin gene in β cells is mediated by the PDX-1 transcription factor. In this report, we show that stimulation results from effects on activation and DNA-binding potential. Thus, glucose specifically stimulated expression in MIN6 β cells from chimeras of PDX-1 and the GAL4 DNA-binding domain which spanned the N-terminal PDX-1 activation domain located between amino acids 1 to 79. GAL4:PDX activity was induced over physiological glucose concentrations and was also regulated by effectors of this response. The level of endogenous PDX-1 binding and phosphorylation were also induced under these conditions. We discuss how changes in PDX-1 phosphorylation may influence activity in glucose-treated β cells.

Functional characterization of the transactivation properties of the PDX-1 homeodomain protein.

Pancreas formation is prevented in mice carrying a null mutation in the PDX-1 homeoprotein, demonstrating a key role for this factor in development. PDX-1 can also bind to and activate transcription from cis-acting regulatory sequences in the insulin and somatostatin genes, which are expressed in pancreatic islet beta and delta cells, respectively. In this study, we compared the functional properties of PDX-1 with those of the closely related Xenopus homeoprotein XIHbox8. Analysis of chimeras between PDX-1, XIHbox8, and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that their transactivation domain was contained within the N-terminal region (amino acids 1 to 79). Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain A), 32 to 38 (subdomain B), and 60 to 73 (subdomain C). These sequences were also required by PDX-1 to synergistically activate insulin enhancer-mediated transcription with another key insulin gene activator, the E2A-encoded basic helix-loop-helix E2-5 and E47 proteins. These results indicated that N-terminal sequences conserved between the mammalian PDX-1 and Xenopus XIHbox8 proteins are important in transcriptional activation. Stable expression of the PDX-1 deltaABC mutant in the insulin- and PDX-1-expressing betaTC3 cell line resulted in a threefold reduction in the rate of endogenous insulin gene transcription. Strikingly, the level of the endogenous PDX-1 protein was reduced to very low levels in these cells. These results suggest that PDX-1 is not absolutely essential for insulin gene expression in betaTC3 cells. We discuss the possible significance of these findings for insulin gene transcription in islet beta cells.