Raghavendra G. Mirmira

Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding

The transcription factor Pdx1 is expressed in the pancreatic β-cell, where it is believed to regulate several β-cell-specific genes. Whereas binding by Pdx1 to elements of β-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which β-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from β-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using real-time PCR. We compared these in vivofindings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in β-cells. In contrast, in in vitroimmunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in β-cells and non-β-cells.

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.

Covalent histone modifications underlie the developmental regulation of insulin gene transcription in pancreatic β cells

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3–K4). To determine whether covalent histone modifications play a role in the β cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from β cell lines, non-β cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in β cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in β cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of β cells is hypermethylated at H3–K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of β cells and non-β cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the β cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.

Transcription factors direct the development and function of pancreatic β cells

Transcription factors orchestrate intricate pathways of cellular growth and differentiation by regulating the rate of transcription of an array of genes. Genetic and biochemical studies have begun to unravel the complex cascade of factors that controls the proliferation and differentiation of cells in the developing pancreas. The specific pathway leading to the development of the insulin-secreting b cell has been a focus of many of these studies because an understanding of the transcription factors governing this pathway will be crucial to the engineering of new b cells to cure diabetes. In recent years, the number of transcription factors that has been implicated in b-cell differentiation and function has grown considerably. Here, we outline the known role of transcription factors in b-cell development, and describe how these factors form a network of gene activation signals that mediates insulin transcription.

Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

Stimulation of Human and Rat Islet β-Cell Proliferation with Retention of Function by the Homeodomain Transcription Factor Nkx6.1

ABSTRACT The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet β-cell development, but its effects on adult β-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2′-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-βGAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are β cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating β-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of β-cell mass in treatment of both major forms of diabetes.

Transcriptional and Translational Regulation of β-Cell Differentiation Factor Nkx6.1

In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to β-cells. Nkx6.1 also is expressed in developing β-cells and plays an essential role in their differentiation. Among cell lines, both β- and α-cell lines express nkx6.1 mRNA; but no protein can be detected in the α-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to β-cells. To investigate the regulator of Nkx6.1 expression, we outlined the structure of the mouse nkx6.1 gene, and we identified regions that direct cell type-specific expression. Thenkx6.1 gene has a long 5′-untranslated region (5′-UTR) downstream of a cluster of transcription start sites.nkx6.1 gene sequences from −5.6 to +1.0 kilobase pairs have specific promoter activity in β-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about −800 base pairs and on the 5′-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at −800 base pairs. In addition, dicistronic assays establish that the 5′-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development.

Cyclical and Alternating Infusions of Glucose and Intralipid in Rats Inhibit Insulin Gene Expression and Pdx-1 Binding in Islets.

OBJECTIVE—Prolonged exposure of isolated islets of Langerhans to elevated levels of fatty acids, in the presence of high glucose, impairs insulin gene expression via a transcriptional mechanism involving nuclear exclusion of pancreas-duodenum homeobox-1 (Pdx-1) and loss of MafA expression. Whether such a phenomenon also occurs in vivo is unknown. Our objective was therefore to ascertain whether chronic nutrient oversupply inhibits insulin gene expression in vivo. RESEARCH DESIGN AND METHODS—Wistar rats received alternating 4-h infusions of glucose and Intralipid for a total of 72 h. Control groups received alternating infusions of glucose and saline, saline and Intralipid, or saline only. Insulin and C-peptide secretion were measured under hyperglycemic clamps. Insulin secretion and gene expression were assessed in isolated islets, and β-cell mass was quantified by morphometric analysis. RESULTS—Neither C-peptide secretion nor insulin sensitivity was different among infusion regimens. Insulin content and insulin mRNA levels were lower in islets isolated from rats infused with glucose plus Intralipid. This was associated with reduced Pdx-1 binding to the endogenous insulin promoter, and an increased proportion of Pdx-1 localized in the cytoplasm versus the nucleus. In contrast, MafA mRNA and protein levels and β-cell mass and proliferation were unchanged. CONCLUSIONS—Cyclical and alternating infusions of glucose and Intralipid in normal rats inhibit insulin gene expression without affecting insulin secretion or β-cell mass. We conclude that fatty acid inhibition of insulin gene expression, in the presence of high glucose, is an early functional defect that may contribute to β-cell failure in type 2 diabetes.

Glucose Regulation of Insulin Gene Transcription and Pre-mRNA Processing in Human Islets

Glucose is the primary regulator of insulin granule release from pancreatic islets. In rodent islets, the role of glucose in the acute regulation of insulin gene transcription has remained unclear, primarily because the abundance and long half-life of insulin mRNA confounds analysis of transcription by traditional methods that measure steady-state mRNA levels. To investigate the nature of glucose-regulated insulin gene transcription in human islets, we first quantitated the abundance and half-lives of insulin mRNA and pre-mRNAs after addition of actinomycin D (to stop transcription). Our results indicated that intron 1–and intron 2–containing pre-mRNAs were ∼150- and 2,000-fold less abundant, respectively, than mature mRNA. 5′ intron 2–containing pre-mRNAs displayed half-lives of only ∼60 min, whereas all other transcripts displayed more extended lifetimes. In response to elevated glucose, pre-mRNA species increased within 60 min, whereas increases in mature mRNA did not occur until 48 h, suggesting that measurement of mature mRNA species does not accurately reflect the acute transcriptional response of the insulin gene to glucose. The acute increase in pre-mRNA species was preceded by a sixfold increase in histone H4 acetylation and a twofold increase in RNA polymerase II recruitment at the insulin promoter. Taken together, our data suggest that pre-mRNA species may be a more reliable reflection of acute changes to human insulin gene transcriptional rates and that glucose acutely enhances insulin transcription by a mechanism that enhances chromatin accessibility and leads to recruitment of basal transcriptional machinery.

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase

BACKGROUNDnNon-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4-phosphopantetheine.nnnRESULTSnA truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, <1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP.nnnCONCLUSIONSnOur finding indicates the presence of an almost equimolar amount of 4-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.