DREAM regulates insulin promoter activity through newly identified DRE element

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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MafA Is a Glucose-regulated and Pancreatic β-Cell-specific Transcriptional Activator for the Insulin Gene

Journal of Biological Chemistry ◽

10.1074/jbc.m206796200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49903-49910 ◽

Cited By ~ 190

Author(s):

Kohsuke Kataoka ◽

Song-iee Han ◽

Setsuko Shioda ◽

Momoki Hirai ◽

Makoto Nishizawa ◽

...

Keyword(s):

Sequence Similarity ◽

Regulatory Element ◽

Transcriptional Activator ◽

Dominant Negative ◽

Insulin Gene ◽

Pcr Analysis ◽

Β Cells ◽

Β Cell ◽

Basic Leucine Zipper ◽

Insulin Promoter

The insulin gene is specifically expressed in β-cells of the Langerhans islets of the pancreas, and its transcription is regulated by the circulating glucose level. Previous reports have shown that an unidentified β-cell-specific nuclear factor binds to a conservedcis-regulatory element called RIPE3b and is critical for its glucose-regulated expression. Based on the sequence similarity of the RIPE3b element and the consensus binding sequence of the Maf family of basic leucine zipper transcription factors, we here identified mammalianhomologueof avian MafA/L-Maf, an eye-specific member of the Maf family, as the RIPE3b-binding transcriptional activator. Reverse transcription-PCR analysis showed thatmafAmRNA is detected only in the eyes and in pancreatic β-cells and not in α-cells. MafA protein as well as its mRNA is up-regulated by glucose, consistent with the glucose-regulated binding of MafA to the RIPE3b element in β-cell nuclear extracts. In transient luciferase assays, we also showed that expression of MafA greatly enhanced insulin promoter activity and that a dominant-negative form of MafA inhibited it. Therefore, MafA is a β-cell-specific and glucose-regulated transcriptional activator for insulin gene expression and thus may be involved in the function and development of β-cells as well as in the pathogenesis of diabetes.

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Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression

Journal of Molecular Endocrinology ◽

10.1530/jme-11-0016 ◽

2011 ◽

Vol 47 (3) ◽

pp. 273-283 ◽

Cited By ~ 7

Author(s):

Julie Amyot ◽

Isma Benterki ◽

Ghislaine Fontés ◽

Derek K Hagman ◽

Mourad Ferdaoussi ◽

...

Keyword(s):

Transcription Factor ◽

Gene Promoter ◽

Insulin Gene ◽

Mcf7 Cells ◽

Rat Islets ◽

Insulin Promoter ◽

Isolated Rat Islets ◽

Direct Binding ◽

Activating Transcription Factor ◽

Isolated Rat

Pancreatic β-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter–reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.

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Hedgehog Signaling Regulation of Homeodomain Protein Islet Duodenum Homeobox-1 Expression in Pancreatic β-Cells*

Endocrinology ◽

10.1210/endo.142.3.8007 ◽

2001 ◽

Vol 142 (3) ◽

pp. 1033-1040 ◽

Cited By ~ 26

Author(s):

Melissa K. Thomas ◽

Jee H. Lee ◽

Naina Rastalsky ◽

Joel F. Habener

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Gene Promoter ◽

Insulin Gene ◽

Reporter Construct ◽

Β Cells ◽

Pancreatic Β Cells ◽

Insulin Promoter ◽

Insulin Gene Expression ◽

Hh Signaling

Abstract Insulin gene expression in pancreatic β-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5′-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 β-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a− 4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic β-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic β-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.

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Elevated Glucose Attenuates Human Insulin Gene Promoter Activity in INS-1 Pancreatic β-Cells via Reduced Nuclear Factor Binding to the A5/Core and Z Element

Molecular Endocrinology ◽

10.1210/me.2003-0493 ◽

2005 ◽

Vol 19 (5) ◽

pp. 1343-1360 ◽

Cited By ~ 15

Author(s):

Maria F. Pino ◽

Diana Z. Ye ◽

Katrina D. Linning ◽

Christopher D. Green ◽

Barton Wicksteed ◽

...

Keyword(s):

Human Insulin ◽

Promoter Activity ◽

Gene Promoter ◽

Insulin Gene ◽

Nuclear Factor ◽

Β Cells ◽

Pancreatic Β Cells ◽

Factor Binding ◽

Human Insulin Gene ◽

Elevated Glucose

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Identification of an INSM1-binding site in the insulin promoter: negative regulation of the insulin gene transcription

Journal of Endocrinology ◽

10.1677/joe-08-0001 ◽

2008 ◽

Vol 198 (1) ◽

pp. 29-39 ◽

Cited By ~ 15

Author(s):

Hong-Wei Wang ◽

Michelle Muguira ◽

Wei-Dong Liu ◽

Tao Zhang ◽

Chiachen Chen ◽

...

Keyword(s):

Cyclin D1 ◽

Binding Site ◽

Gene Transcription ◽

Promoter Activity ◽

Promoter Sequence ◽

Inhibitory Effect ◽

Human Islets ◽

Insulin Gene ◽

Insulin Promoter ◽

Insulin Gene Transcription

In this study, an insulinoma-associated antigen-1 (INSM1)-binding site in the proximal promoter sequence of the insulin gene was identified. The co-transfection of INSM1 with rat insulin I/II promoter-driven reporter genes exhibited a 40–50% inhibitory effect on the reporter activity. Mutational experiments were performed by introducing a substitution, GG to AT, into the INSM1 core binding site of the rat insulin I/II promoters. The mutated insulin promoter exhibited a three- to 20-fold increase in the promoter activity over the wild-type promoter in several insulinoma cell lines. Moreover, INSM1 overexpression exhibited no inhibitory effect on the mutated insulin promoter. Chromatin immunoprecipitation assays using βTC-1, mouse fetal pancreas, and Ad-INSM1-transduced human islets demonstrated that INSM1 occupies the endogenous insulin promoter sequence containing the INSM1-binding site in vivo. The binding of the INSM1 to the insulin promoter could suppress ∼50% of insulin message in human islets. The mechanism for transcriptional repression of the insulin gene by INSM1 is mediated through the recruitment of cyclin D1 and histone deacetylase-3 to the insulin promoter. Anti-INSM1 or anti-cyclin D1 morpholino treatment of fetal mouse pancreas enhances the insulin promoter activity. These data strongly support the view that INSM1 is a new zinc-finger transcription factor that modulates insulin gene transcription during early pancreas development.

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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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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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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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Glucose enhances insulin promoter activity in MIN6 β-cells independently of changes in intracellular Ca2+ concentration and insulin secretion

Biochemical Journal ◽

10.1042/bj3420275 ◽

1999 ◽

Vol 342 (2) ◽

pp. 275-280 ◽

Cited By ~ 9

Author(s):

Helen J. KENNEDY ◽

Imran RAFIQ ◽

Aristea E. POULI ◽

Guy A. RUTTER

Keyword(s):

Promoter Activity ◽

Photon Counting ◽

Firefly Luciferase ◽

Receptor Activation ◽

Luciferase Reporter ◽

Β Cells ◽

Viral Promoter ◽

Luciferase Reporter Construct ◽

Insulin Promoter ◽

Firefly Luciferase Reporter

Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca2+ concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 β-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca2+ channel inhibitor, verapamil (100 μM). Increases in intracellular [Ca2+] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet β-cells, via a signalling pathway which does not require increases in intracellular [Ca2+] nor insulin release and insulin receptor activation.

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