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

ABSTRACT Activation of insulin gene transcription specifically in the pancreatic β cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-β cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3987 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3987-3996 ◽

Cited By ~ 65

Author(s):

M Peshavaria ◽

E Henderson ◽

A Sharma ◽

C V Wright ◽

R Stein

Keyword(s):

Amino Acids ◽

Gene Transcription ◽

Transcriptional Activation ◽

Functional Characterization ◽

Insulin Gene ◽

Regulatory Sequences ◽

Homeodomain Protein ◽

Transactivation Domain ◽

Endogenous Insulin ◽

Insulin Gene Transcription

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.

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Insulin Gene Transcription Is Mediated by Interactions between the p300 Coactivator and PDX-1, BETA2, and E47

Molecular and Cellular Biology ◽

10.1128/mcb.22.2.412-420.2002 ◽

2002 ◽

Vol 22 (2) ◽

pp. 412-420 ◽

Cited By ~ 127

Author(s):

Yi Qiu ◽

Min Guo ◽

Suming Huang ◽

Roland Stein

Keyword(s):

Dna Binding ◽

Rna Polymerase Ii ◽

Gene Transcription ◽

Present Report ◽

Amino Acid Sequences ◽

Insulin Gene ◽

Homeodomain Protein ◽

Β Cells ◽

Β Cell ◽

Insulin Gene Transcription

ABSTRACT Pancreatic β-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 β-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-β cells, a process inhibited by E1A. In addition, E1A inhibited the level of PDX-1 and BETA2 complex formation in β 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.

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Regulation of Insulin Gene Transcription by the Immediate-Early Growth Response Gene Egr-1

Endocrinology ◽

10.1210/en.2005-1336 ◽

2006 ◽

Vol 147 (6) ◽

pp. 2923-2935 ◽

Cited By ~ 26

Author(s):

Kazuhiro Eto ◽

Varinderpal Kaur ◽

Melissa K. Thomas

Keyword(s):

Transcriptional Activation ◽

Growth Response ◽

Insulin Gene ◽

Β Cells ◽

Pancreatic Β Cells ◽

Expression Levels ◽

Promoter Sequences ◽

Early Growth Response ◽

Insulin Promoter ◽

Insulin Gene Expression

Abstract Changes in extracellular glucose levels regulate the expression of the immediate-early response gene and zinc finger transcription factor early growth response-1 (Egr-1) in insulin-producing pancreatic β-cells, but key target genes of Egr-1 in the endocrine pancreas have not been identified. We found that overexpression of Egr-1 in clonal (INS-1) β-cells increased transcriptional activation of the rat insulin I promoter. In contrast, reductions in Egr-1 expression levels or function with the introduction of either small interfering RNA targeted to Egr-1 (siEgr-1) or a dominant-negative form of Egr-1 decreased insulin promoter activation, and siEgr-1 suppressed insulin gene expression. Egr-1 did not directly interact with insulin promoter sequences, and mutagenesis of a potential G box recognition sequence for Egr-1 did not impair the Egr-1 responsiveness of the insulin promoter, suggesting that regulation of insulin gene expression by Egr-1 is probably mediated through additional transcription factors. Overexpression of Egr-1 increased, and reduction of Egr-1 expression decreased, transcriptional activation of the glucose-responsive FarFlat minienhancer within the rat insulin I promoter despite the absence of demonstrable Egr-1-binding activity to FarFlat sequences. Notably, augmenting Egr-1 expression levels in insulin-producing cells increased the mRNA and protein expression levels of pancreas duodenum homeobox-1 (PDX-1), a major transcriptional regulator of glucose-responsive activation of the insulin gene. Increasing Egr-1 expression levels enhanced PDX-1 binding to insulin promoter sequences, whereas mutagenesis of PDX-1-binding sites reduced the capacity of Egr-1 to activate the insulin promoter. We propose that changes in Egr-1 expression levels in response to extracellular signals, including glucose, can regulate PDX-1 expression and insulin production in pancreatic β-cells.

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The Homeoprotein Alx3 Expressed in Pancreatic β-Cells Regulates Insulin Gene Transcription by Interacting with the Basic Helix-Loop-Helix Protein E47

Molecular Endocrinology ◽

10.1210/me.2005-0472 ◽

2006 ◽

Vol 20 (11) ◽

pp. 2876-2889 ◽

Cited By ~ 11

Author(s):

Mercedes Mirasierra ◽

Mario Vallejo

Keyword(s):

Gene Transcription ◽

Insulin Gene ◽

Β Cells ◽

Pancreatic Β Cells ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Insulin Gene Transcription

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c-jun inhibits transcriptional activation by the insulin enhancer, and the insulin control element is the target of control

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.655-662.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 655-662

Author(s):

E Henderson ◽

R Stein

Keyword(s):

Skeletal Muscle ◽

Beta Cells ◽

Gene Transcription ◽

Transcriptional Activation ◽

Pancreatic Beta Cells ◽

Insulin Gene ◽

Control Element ◽

Basic Leucine Zipper ◽

Insulin Control ◽

Insulin Gene Transcription

Selective transcription of the insulin gene in pancreatic beta cells is regulated by its enhancer, located between nucleotides -340 and -91 relative to the transcription start site. One of the principal control elements within the enhancer is found between nucleotides -100 and -91 (GCCATCTGCT, referred to as the insulin control element [ICE]) and is regulated by both positive- and negative-acting transcription factors in the helix-loop-helix (HLH) family. It was previously shown that the c-jun proto-oncogene can repress insulin gene transcription. We have found that c-jun inhibits ICE-stimulated transcription. Inhibition of ICE-directed transcription is mediated by sequences within the carboxy-terminal region of the protein. These c-jun sequences span an activation domain and the basic leucine zipper DNA binding-dimerization region of the protein. Both regions of c-jun are conserved within the other members of the jun family: junB and junD. These proteins also suppress ICE-mediated transcription. The jun proteins do not appear to inhibit insulin gene transcription by binding directly to the ICE. c-jun and junB also block the trans-activation potential of two skeletal muscle-specific HLH proteins, MyoD and myogenin. These results suggests that the jun proteins may be common transcription control factors used in skeletal muscle and pancreatic beta cells to regulate HLH-mediated activity. We discuss the possible significance of these observations to insulin gene transcription in pancreatic beta cells.

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Short-term regulation of insulin gene transcription

Biochemical Society Transactions ◽

10.1042/bst0300312 ◽

2002 ◽

Vol 30 (2) ◽

pp. 312-317 ◽

Cited By ~ 18

Author(s):

B. Leibiger ◽

T. Moede ◽

S. Uhles ◽

P.-O. Berggren ◽

I. B. Leibiger

Keyword(s):

Insulin Secretion ◽

Gene Transcription ◽

Insulin Gene ◽

Current View ◽

Β Cells ◽

Pancreatic Β Cells ◽

Short Term ◽

Major Nutrient ◽

Glucose Metabolites ◽

Insulin Gene Transcription

Short-term regulation of insulin gene transcription is still a matter of debate. However, an increasing body of evidence shows that insulin gene transcription is affected by signals, such as incretins, glucose metabolites, intracellular Ca2+, and by insulin secreted from pancreatic β-cells, all supporting the concept of an immediate response resulting in insulin gene transcription following food-uptake. The present review aims to summarize the current view on the mechanisms underlying the up-regulation of insulin gene transcription in response to glucose, the major nutrient factor in insulin secretion and biosynthesis.

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c-jun inhibits transcriptional activation by the insulin enhancer, and the insulin control element is the target of control.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.655 ◽

1994 ◽

Vol 14 (1) ◽

pp. 655-662 ◽

Cited By ~ 24

Author(s):

E Henderson ◽

R Stein

Keyword(s):

Skeletal Muscle ◽

Beta Cells ◽

Gene Transcription ◽

Transcriptional Activation ◽

Pancreatic Beta Cells ◽

Insulin Gene ◽

Control Element ◽

Basic Leucine Zipper ◽

Insulin Control ◽

Insulin Gene Transcription

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

Journal of Biological Chemistry ◽

10.1074/jbc.m301198200 ◽

2003 ◽

Vol 278 (35) ◽

pp. 32969-32977 ◽

Cited By ~ 111

Author(s):

Shih Khoo ◽

Steven C. Griffen ◽

Ying Xia ◽

Richard J. Baer ◽

Michael S. German ◽

...

Keyword(s):

Gene Transcription ◽

Insulin Gene ◽

Β Cells ◽

Pancreatic Β Cells ◽

Insulin Gene Transcription

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DREAM regulates insulin promoter activity through newly identified DRE element

Open Life Sciences ◽

10.2478/s11535-013-0123-3 ◽

2013 ◽

Vol 8 (2) ◽

pp. 97-106

Author(s):

Teodora Daneva ◽

Shina Pashova ◽

Radoslava Emilova ◽

Plamen Padeshki ◽

Hristo Gagov ◽

...

Keyword(s):

Islets Of Langerhans ◽

Gene Transcription ◽

Promoter Activity ◽

Regulatory Element ◽

Transcriptional Start Site ◽

Gene Promoter ◽

Insulin Gene ◽

Β Cells ◽

Insulin Promoter ◽

Direct Binding

AbstractDownstream regulatory element antagonist modulator (DREAM) protein is a 31 kDa Ca2+-regulated transcriptional repressor. It functions as a silencer of the gene transcription. In low intracellular free Ca2+ concentration DREAM tightly binds to the downstream regulatory element (DRE) of gene promoter and impedes the transcription. In higher Ca2+ concentrations DREAM binds Ca2+ and disconnects from DRE of the gene promoter enabling transcription. We report that DREAM is expressed in different human tissues including the pancreas, where it is located in the islets of Langerhans. Location of DREAM in RIN-F5 cells in cultures is restricted to the nucleus and membranes and changes after increased Ca2+-levels. The proteins dissociate from dimmers to monomers and translocate out of the nucleus. The expression of DREAM in β-cells in the islets of Langerhans regulates the promoter activity of the insulin gene by directly interacting with the sequence located between +52 bp and +81 bp downstream of the transcriptional start site of the promoter. Our results provide evidence for the existence of DRE sequence in the insulin gene promoter. It is suggested that DREAM is a repressor of insulin gene transcription, whose effect is mediated by direct binding to DRE sequence.

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