MafA, NeuroD1, and HNF1β synergistically activate Slc2a2 (Glut2) gene in β-cells

2021 ◽  
Author(s):  
Yuka Ono ◽  
Kohsuke Kataoka
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Glucose Transporter ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Β Cells ◽  
Β Cell ◽  
Target Sites

Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet β-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to β-cell functionality in rodents, but the mechanism by which β-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 β-cell line. We also found that three β-cell-enriched transcription factors, MafA, NeuroD1, and HNF1β, synergistically activate transcription through this 3’ downstream distal enhancer (ECR3’) and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1β to its target sites within the promoter and ECR3’ of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3’ enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1β interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1β is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in β-cells.

scholarly journals Identification of a novel distal enhancer in human adiponectin gene

2008 ◽  
Vol 200 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Katsumori Segawa ◽  
Morihiro Matsuda ◽  
Atsunori Fukuhara ◽  
Kentaro Morita ◽  
Yosuke Okuno ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Luciferase Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Adiponectin Gene ◽  
Distal Enhancer

Adiponectin is exclusively expressed in adipose tissue and secreted from adipocytes, and shows anti-diabetic and anti-atherogenic properties. However, the precise transcriptional mechanism of adiponectin remains elusive. In this study, the 5′ flanking promoter region of human adiponectin gene was analyzed using UCSC genome browser, and a 10 390-bp fragment, containing an evolutionally conserved region among species, was investigated. The luciferase reporter assay using this fragment identified a novel distal enhancer of human adiponectin gene. Promoter constructs with the distal enhancer exhibited high promoter activities in 3T3-L1 mature adipocytes. However, no such activity was observed in other types of cell lines. The distal enhancer is highly conserved, and contains two completely conserved CCAAT boxes. In 3T3-L1 mature adipocytes, deletion or each point mutation of these CCAAT boxes markedly reduced luciferase activity driven by adiponectin promoter. Knockdown of CCAAT/enhancer-binding protein α (CEBPA; also known as C/EBPα) using small interfering RNA diminished adiponectin mRNA expression and luciferase activity driven by adiponectin promoter with the distal enhancer. However, adiponectin promoter with each mutation of two CCAAT boxes in the distal enhancer did not respond to knockdown of CEBPA expression. Furthermore, CEBPA bound to the distal enhancer both in vitro and in vivo. We also identified a proximal promoter region responsible for transcriptional activation by the distal enhancer in human adiponectin gene. Our results indicate that CEBPA plays a pivotal role in the transcription of human adiponectin gene via the distal enhancer and proximal region in its promoter.

Glucose regulation of insulin gene expression in pancreatic β-cells

2008 ◽  
Vol 415 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Sreenath S. Andrali ◽  
Megan L. Sampley ◽  
Nathan L. Vanderford ◽  
Sabire Özcan
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Blood Glucose ◽  
Insulin Gene ◽  
Β Cells ◽  
Β Cell ◽  
Insulin Synthesis ◽  
Glucose Levels ◽  
Blood Glucose Levels ◽  
Insulin Gene Expression

Production and secretion of insulin from the β-cells of the pancreas is very crucial in maintaining normoglycaemia. This is achieved by tight regulation of insulin synthesis and exocytosis from the β-cells in response to changes in blood glucose levels. The synthesis of insulin is regulated by blood glucose levels at the transcriptional and post-transcriptional levels. Although many transcription factors have been implicated in the regulation of insulin gene transcription, three β-cell-specific transcriptional regulators, Pdx-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation 1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A), have been demonstrated to play a crucial role in glucose induction of insulin gene transcription and pancreatic β-cell function. These three transcription factors activate insulin gene expression in a co-ordinated and synergistic manner in response to increasing glucose levels. It has been shown that changes in glucose concentrations modulate the function of these β-cell transcription factors at multiple levels. These include changes in expression levels, subcellular localization, DNA-binding activity, transactivation capability and interaction with other proteins. Furthermore, all three transcription factors are able to induce insulin gene expression when expressed in non-β-cells, including liver and intestinal cells. The present review summarizes the recent findings on how glucose modulates the function of the β-cell transcription factors Pdx-1, NeuroD1 and MafA, and thereby tightly regulates insulin synthesis in accordance with blood glucose levels.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Pericytes contribute to the islet basement membranes to promote beta-cell gene expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Sakhneny ◽  
Alona Epshtein ◽  
Limor Landsman
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
Basement Membranes ◽  
Cell Physiology ◽  
Β Cells ◽  
Β Cell ◽  
Cell Clustering ◽  
Β Cell Function ◽  
Cell Gene Expression ◽  
Glucose Stimulated Insulin Secretion

Abstractβ-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support β-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate β-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in β-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse β-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of β-cell genes, including Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper β-cell function.

scholarly journals Ontology of the apelinergic system in mouse pancreas during pregnancy and relationship with β-cell mass

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Brenda Strutt ◽  
Sandra Szlapinski ◽  
Thineesha Gnaneswaran ◽  
Sarah Donegan ◽  
Jessica Hill ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Insulin Secretion ◽  
Glucose Transporter ◽  
Cell Mass ◽  
Elevated Serum ◽  
Pancreatic Β Cell ◽  
Β Cells ◽  
Β Cell ◽  
Glucose Transporter 2 ◽  
Glucose Stimulated Insulin Secretion

AbstractThe apelin receptor (Aplnr) and its ligands, Apelin and Apela, contribute to metabolic control. The insulin resistance associated with pregnancy is accommodated by an expansion of pancreatic β-cell mass (BCM) and increased insulin secretion, involving the proliferation of insulin-expressing, glucose transporter 2-low (Ins+Glut2LO) progenitor cells. We examined changes in the apelinergic system during normal mouse pregnancy and in pregnancies complicated by glucose intolerance with reduced BCM. Expression of Aplnr, Apelin and Apela was quantified in Ins+Glut2LO cells isolated from mouse pancreata and found to be significantly higher than in mature β-cells by DNA microarray and qPCR. Apelin was localized to most β-cells by immunohistochemistry although Aplnr was predominantly associated with Ins+Glut2LO cells. Aplnr-staining cells increased three- to four-fold during pregnancy being maximal at gestational days (GD) 9–12 but were significantly reduced in glucose intolerant mice. Apelin-13 increased β-cell proliferation in isolated mouse islets and INS1E cells, but not glucose-stimulated insulin secretion. Glucose intolerant pregnant mice had significantly elevated serum Apelin levels at GD 9 associated with an increased presence of placental IL-6. Placental expression of the apelinergic axis remained unaltered, however. Results show that the apelinergic system is highly expressed in pancreatic β-cell progenitors and may contribute to β-cell proliferation in pregnancy.

Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation

2005 ◽  
Vol 288 (5) ◽  
pp. F899-F909 ◽  
Author(s):  
Zubaida Saifudeen ◽  
Susana Dipp ◽  
Hao Fan ◽  
Samir S. El-Dahr
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Spatial Organization ◽  
Kidney Development ◽  
Collecting Duct ◽  
Proximal Promoter ◽  
Bradykinin B2 Receptor ◽  
Nephron Differentiation ◽  
Terminal Nephron

Despite a wealth of knowledge regarding the early steps of epithelial differentiation, little is known about the mechanisms responsible for terminal nephron differentiation. The bradykinin B2 receptor (B2R) regulates renal function and integrity, and its expression is induced during terminal nephron differentiation. This study investigates the transcriptional regulation of the B2R during kidney development. The rat B2R 5′-flanking region has a highly conserved cis-acting enhancer in the proximal promoter consisting of contiguous binding sites for the transcription factors cAMP response element binding protein (CREB), p53, and Krüppel-like factor (KLF-4). The B2R enhancer drives reporter gene expression in inner medullary collecting duct-3 cells but is considerably weaker in other cell types. Site-directed mutagenesis and expression of dominant negative mutants demonstrated the requirement of CREB DNA binding and Ser-133 phosphorylation for optimal enhancer function. Moreover, helical phasing experiments showed that disruption of the spatial organization of the enhancer inhibits B2R promoter activity. Several lines of evidence indicate that cooperative interactions among the three transcription factors occur in vivo during terminal nephron differentiation: 1) CREB, p53, and KLF-4 are coexpressed in B2R-positive differentiating cells; 2) the maturational expression of B2R correlates with CREB/p53/KLF-4 DNA-binding activity; 3) assembly of CREB, p53, and KLF-4 on chromatin at the endogenous B2R promoter is developmentally regulated and is accompanied by CBP recruitment and histone hyperacetylation; and 4) CREB and p53 occupancy of the B2R enhancer is cooperative. These results demonstrate that combinatorial interactions among the transcription factors, CREB, p53, and KLF-4, and the coactivator CBP, may be critical for the regulation of B2R gene expression during terminal nephron differentiation.

scholarly journals Differentiation of Sendai Virus-Reprogrammed iPSC into β Cells, Compared with Human Pancreatic Islets and Immortalized β Cell Line

2018 ◽  
Vol 27 (10) ◽  
pp. 1548-1560 ◽  
Author(s):  
Silvia Pellegrini ◽  
Fabio Manenti ◽  
Raniero Chimienti ◽  
Rita Nano ◽  
Linda Ottoboni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Cell Line ◽  
Expression Profile ◽  
Sendai Virus ◽  
Secretory Function ◽  
Β Cells ◽  
Β Cell ◽  
Insulin Producing Cells ◽  
Gene And Protein Expression

Background: New sources of insulin-secreting cells are strongly in demand for treatment of diabetes. Induced pluripotent stem cells (iPSCs) have the potential to generate insulin-producing cells (iβ). However, the gene expression profile and secretory function of iβ still need to be validated in comparison with native β cells. Methods: Two clones of human iPSCs, reprogrammed from adult fibroblasts through integration-free Sendai virus, were differentiated into iβ and compared with donor pancreatic islets and EndoC-βH1, an immortalized human β cell line. Results: Both clones of iPSCs differentiated into insulin+ cells with high efficiency (up to 20%). iβ were negative for pluripotency markers (Oct4, Sox2, Ssea4) and positive for Pdx1, Nkx6.1, Chromogranin A, PC1/3, insulin, glucagon and somatostatin. iβ basally secreted C-peptide, glucagon and ghrelin and released insulin in response either to increasing concentration of glucose or a depolarizing stimulus. The comparison revealed that iβ are remarkably similar to donor derived islets in terms of gene and protein expression profile and similar level of heterogeneity. The ability of iβ to respond to glucose instead was more related to that of EndoC-βH1. Discussion: We demonstrated that insulin-producing cells generated from iPSCs recapitulate fundamental gene expression profiles and secretory function of native human β cells.

221 EPIGENETIC DYNAMICS OF PLURIPOTENCY GENES IN THE CONTEXT OF DIFFERENTIATION AND NUCLEAR REPROGRAMMING DETERMINED BY Q2ChIP, A QUICK AND QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION TECHNIQUE APPLICABLE TO SMALL CELL SAMPLES

2007 ◽  
Vol 19 (1) ◽  
pp. 227 ◽  
Author(s):  
J. A. Dahl ◽  
C. K. Taranger ◽  
P. Collas
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Regulation Of Gene Expression ◽  
Cell Extract ◽  
Proximal Promoter ◽  
Pcr Analysis ◽  
Cross Linking ◽  
Nuclear Reprogramming ◽  
Distal Enhancer ◽  
Developmentally Regulated

Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) is a key technique for mapping histone modifications and transcription factor binding on DNA and thereby unraveling the role of epigenetics in the regulation of gene expression. Current ChIP protocols require extensive sample handling and large numbers of cells (5-10 million). primarily owing to ample loss of material during the procedure. We altered critical steps of conventional ChIP to develop a quick and quantitative (Q2) ChIP assay suitable for cell numbers 100- to 1000-fold lower than those required for conventional ChIP. Key modifications of the ChIP procedure include (i) formaldehyde DNA–protein cross-linking in suspended cells, (ii) cross-linking in the presence of 20 mM sodium butyrate to enhance specificity of precipitation of acetylated histones, (iii) transfer of washed precipitated immune complexes to a clean tube ('tube shift') to increase ChIP specificity by virtually eliminating nonspecifically bound chromatin, and (iv) combination of cross-link reversal, protein digestion, and DNA elution into a single 2-h step. We used Q2ChIP to monitor changes in 6 histone H3 modifications on the human developmentally regulated genes OCT4 (POU5F1), NANOG, and LMNA (lamin A) in the context of retinoic acid (RA)-mediated differentiation of embryonal carcinoma cells and upon reprogramming of kidney epithelial 293T cells to pluripotency in carcinoma cell extract (Taranger et al. 2005 Mol. Biol. Cell 16, 5719–5735). Real-time PCR analysis of precipitated DNA unravels an unexpected two-step heterochromatin assembly elicited by RA on the OCT4 proximal promoter, proximal enhancer, and distal enhancer, and on the NANOG promoter, whereby methylation of H3K9 and H3K27 is followed by H3K9 deacetylation. H3K4 di- and trimethylation remain relatively unaffected by RA treatment. In contrast, reprogramming of 293T cells in carcinoma extract promotes assembly of histone marks characteristic of transcriptional induction of OCT4 and NANOG, such as acetylation and demethylation of H3K9. The results argue toward ordered chromatin repackaging at developmentally regulated promoters upon differentiation or, conversely, nuclear reprogramming to pluripotency.

scholarly journals Gene activation precedes DNA demethylation in response to infection in human dendritic cells

2019 ◽  
Vol 116 (14) ◽  
pp. 6938-6943 ◽  
Author(s):  
Alain Pacis ◽  
Florence Mailhot-Léonard ◽  
Ludovic Tailleux ◽  
Haley E. Randolph ◽  
Vania Yotova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dendritic Cells ◽  
Transcriptional Activation ◽  
Time Course ◽  
Gene Activation ◽  
Dna Demethylation ◽  
Epigenetic Mark ◽  
Distal Enhancer ◽  
Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

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