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

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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Analysis of the Role of E2A-Encoded Proteins in Insulin Gene Transcription

Molecular Endocrinology ◽

10.1210/mend.11.11.0004 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1608-1617 ◽

Cited By ~ 11

Author(s):

Arun Sharma ◽

Eva Henderson ◽

Laura Gamer ◽

Yuan Zhuang ◽

Roland Stein

Keyword(s):

Gene Transcription ◽

Present Report ◽

Insulin Gene ◽

Apparent Difference ◽

Transactivation Domain ◽

Β Cell ◽

Helix Loop Helix ◽

Encoded Proteins ◽

Gene Enhancer ◽

Insulin Gene Transcription

Abstract Pancreatic β-cell type-specific transcription of the insulin gene is mediated, in part, by factors in the basic helix-loop-helix (bHLH) family that act on a site within the insulin enhancer, termed the E1-box. Expression from this element is regulated by a heteromeric protein complex containing ubiquitous (i.e. the E2A- and HEB-encoded proteins) and islet-enriched members of the bHLH family. Recent studies indicate that the E2A- and HEB-encoded proteins contain a transactivation domain, termed AD2, that functions more efficiently in transfected β-cell lines. In the present report, we extend this observation by demonstrating that expression of full-length E2A proteins (E47, E12, and E2/5) activates insulin E element-directed transcription in a β-cell line-selective manner. Stimulation required functional interactions with other key insulin gene transcription factors, including its islet bHLH partner as well as those that act on the RIPE3b1 and RIPE3a2 elements of the insulin gene enhancer. The conserved AD2 domain in the E2A proteins was essential in this process. The effect of the E2A- and HEB-encoded proteins on insulin gene expression was also analyzed in mice lacking a functional E2A or HEB gene. There was no apparent difference in insulin production between wild type, heterozygote, and homozygous mutant E2A or HEB mice. These results suggest that neither the E2A- or HEB-encoded proteins are essential for insulin transcription and that one factor can substitute for the other to impart normal insulin E1 activator function in mutant animals.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.900-911.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 900-911 ◽

Cited By ~ 138

Author(s):

Kinuko Ohneda ◽

Raghavendra G. Mirmira ◽

Juehu Wang ◽

Jeffrey D. Johnson ◽

Michael S. German

Keyword(s):

Gene Transcription ◽

Transcriptional Activation ◽

Insulin Gene ◽

Nuclear Proteins ◽

Bhlh Protein ◽

Homeodomain Protein ◽

Β Cells ◽

Activation Domains ◽

Insulin Promoter ◽

Insulin Gene Transcription

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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The Islet β Cell-enriched MafA Activator Is a Key Regulator of Insulin Gene Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m409475200 ◽

2005 ◽

Vol 280 (12) ◽

pp. 11887-11894 ◽

Cited By ~ 124

Author(s):

Li Zhao ◽

Min Guo ◽

Taka-aki Matsuoka ◽

Derek K. Hagman ◽

Susan D. Parazzoli ◽

...

Keyword(s):

Gene Transcription ◽

Insulin Gene ◽

Β Cell ◽

Insulin Gene Transcription

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PIASy is a SUMOylation-independent negative regulator of the insulin transactivator MafA

Journal of Molecular Endocrinology ◽

10.1530/jme-19-0172 ◽

2019 ◽

Vol 63 (4) ◽

pp. 297-308

Author(s):

Suzuka Onishi ◽

Kohsuke Kataoka

Keyword(s):

Transcription Factors ◽

Gene Transcription ◽

Gene Promoter ◽

Terminal Region ◽

Insulin Gene ◽

Negative Regulator ◽

Β Cell ◽

E3 Ligases ◽

Amino Terminal ◽

Insulin Gene Transcription

Insulin plays a central role in glucose homeostasis and is produced exclusively by pancreatic islet β-cells. Insulin gene transcription is regulated by a set of β-cell-enriched transcription factors that bind to cis-regulatory elements within the promoter region, and regulation of the insulin gene promoter is closely linked to β-cell functionality. PIASy, a member of the PIAS family of SUMO E3 ligases, is thought to affect insulin gene transcription, but its mechanism of action is not fully understood. Here, we demonstrate that PIASy interacts with MafA and represses insulin gene promoter activity. MafA is a β-cell-restricted basic leucine-zipper transcriptional activator that binds to the C1 element of the insulin gene promoter. In line with previous studies showing the transactivator domain of MafA is SUMOylated, PIASy enhanced the SUMOylation of MafA. However, a SUMOylation-deficient mutant of MafA was still repressed by PIASy, indicating that this modification is dispensable for repression. Using a series of MafA and PIASy mutants, we found that the basic domain of MafA and the amino-terminal region of PIASy containing the SAP domain are necessary for their interaction. In addition, SUMO-interacting motif 1 (SIM1) at the carboxyl-terminal region of PIASy was required to repress the synergistic transactivation of MafA, Pdx1, and Beta2, transcription factors playing central roles in β-cell differentiation and function. The PINIT and SP-RING domains in the middle region of PIASy were dispensable. These findings suggest that PIASy binds to MafA through the SAP domain and negatively regulates the insulin gene promoter through a novel SIM1-dependent mechanism.

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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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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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The NeuroD1/BETA2 Sequences Essential for Insulin Gene Transcription Colocalize with Those Necessary for Neurogenesis and p300/CREB Binding Protein Binding

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.704 ◽

1999 ◽

Vol 19 (1) ◽

pp. 704-713 ◽

Cited By ~ 56

Author(s):

Arun Sharma ◽

Melissa Moore ◽

Edoardo Marcora ◽

Jacqueline E. Lee ◽

Yi Qiu ◽

...

Keyword(s):

Gene Transcription ◽

Binding Protein ◽

Ectopic Expression ◽

Amino Acid Sequences ◽

Insulin Gene ◽

Creb Binding Protein ◽

Β Cells ◽

Helix Loop Helix ◽

Transactivation Domains ◽

Neuronal Precursors

ABSTRACT NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet β cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and convert ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. We have identified amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 that are necessary for insulin gene expression and ectopic neurogenesis. Our results indicate that evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1, aa 189 to 299; AD2, aa 300 to 355) were within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos was regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrated that only aa 252 to 355 contained activation domain function, although both aa 156 to 251 and 300 to 355 were found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet β 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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