Publications

The emerging appreciation of plasticity among pancreatic lineages has created interest in harnessing cellular reprogramming for beta cell replacement therapy of diabetes. Current reprogramming methodologies are inefficient, largely because of a limited understanding of the underlying mechanisms, Using an in vitro reprogramming system, we reveal the transcriptional repressor RE-1 silencing transcription factor (REST) as a barrier for beta cell gene expression in the reprogramming of pancreatic exocrine cells. We observe that REST-bound loci lie adjacent to the binding sites of multiple key beta cell transcription factors, including PDX1. Accordingly, a loss of REST function combined with PDX1 expression results in the synergistic activation of endocrine genes. This is accompanied by increased histone acetylation and PDX1 binding at endocrine gene loci. Collectively, our data identify a mechanism for REST activity involving the prevention of PDX1-mediated activation of endocrine genes and uncover REST downregulation and the resulting chromatin alterations as key events in beta cell reprogramming.

Bleomycin is an anticancer antibiotic effective against a range of human malignancies. Yet its usefulness is limited by serious side effects. In this study, we converted bleomycin into a prodrug by covalently linking 2-sulfo, 9 fluorenylmethoxycarbonyl (FMS) to the primary amino side chain of bleomycin. FMS-bleomycin lost its efficacy to bind transition metal ions and therefore was converted into an inactive derivative. Upon incubation in vitro under physiological conditions, the FMS-moiety undergoes spontaneous hydrolysis, generating native bleomycin possessing full anti-bacterial potency. FMS hydrolysis and reactivation takes place with a t(1/2) value of 17 +/- 1 h. In silico simulation predicts a narrow therapeutic window in human patients of seven hours, starting 40 min after administration. In mice, close agreement was obtained between the experimental and the simulated pharmacokinetic profiles for FMS-bleomycin. FMS-bleomycin is thus shown to be a classical prodrug: it is inactive at the time of administration and the non-modified (active) bleomycin is released with a desirable pharmacokinetic profile following administration, suggesting it may have therapeutic value in the clinic.

Insulin secretion by pancreaticb-cells is primarily regulated by glucose; however, hormones and additional nutrients, such as long-chain fatty acids, also play an important role inadjusting insulinoutput to physiologicneeds.To examine the role of short-chain fatty acids (SCFAs) in β-cell function, we analyzed mouse models of gain and loss of function of GPR41 (FFAR3), a receptor for SCFAs, vs. wild-Type control mice. GPR41 gain of function [GPR41-overexpressing transgenic (41 Tg) model] and GPR41 loss of function [GPR41-knockout (KO 41) model] resulted in complementary changes in glucose tolerance, without significant effects on insulin sensitivity. KO 41 mice showed fasting hypoglycemia, which was consistent with increased basal and glucose-induced insulin secretion by islets in vitro.Mirroring this, 41 Tg islets showed impaired glucose responsiveness in vitro.Microarray analysis of islets from 41 Tg mice indicated significant alterations in gene expression patterns; several of the altered genes were chosen for further analysis and were also observed to change upon incubation of islets and cultured b-cells with SCFAs in a GPR41-dependent manner. Taken together, our results indicate that GPR41 and its ligands, SCFAs, may play an important role inthefine-Tuning of insulin secretion infedandfastingstates.

The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9^high/CD56⁺), glucagon-producing alpha cells (CD9^-/CD56⁺) and trypsin-producing acinar cells (CD9^-/CD56^-). This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

MicroRNAs are transcribed by RNA polymerase II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA box in microRNA and protein-coding genes is associated with increased sensitivity to slow RNA polymerase II. Promoters driven by TATA box or NF-κB elicit high re-initiation rates, but paradoxically lower microRNA levels. MicroRNA synthesis becomes more productive by decreasing the initiation rate, but less productive when the re-initiation rate increases. This phenomenon is associated with a delay in miR-146a induction by NF-κB. Finally, we demonstrate that microRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of microRNA synthesis directed by TATA box or NF-κB is a consequence of frequent transcription initiations that lead to RNA polymerase II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for microRNA synthesis, and have implications for TATA-box promoters in general.

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa-immune system crosstalk necessary for mounting protective T helper type 2 (T(H)2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1(Delta gut)), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELM beta, a key T(H)2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1(Delta gut) mice with helminths favored a futile T(H)1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a T(H)2-facilitating epithelial cytokine; this indicated a T(H)2-amplification loop. We found that miR-375 was required for RELM beta expression in vivo; miR-375-deficient mice had significantly less intestinal RELM beta, which possibly explains the greater susceptibility of Dicer1(Delta gut) mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.

microRNAs (miRNAs) are known to play an essential role in controlling a broad range of biological processes including animal development. Accordingly, many miRNAs are expressed preferentially in one or a small number of cell types. Yet the mechanisms responsible for this selectivity are not well understood. The aim of this study was to elucidate the molecular basis of cell-specific expression of the pri-miR-375 gene, which is selectively expressed in pancreatic islets, and has been implicated both in the development of islets, and the function of mature pancreatic beta cells. An evolutionarily conserved 768 bp region of DNA upstream of the pri-miR-375 gene was linked to GFP and luciferase reporter genes, and expression monitored in transgenic mice and transfected cultured cells. Deletion and targeted mutagenesis analysis was used to evaluate the functional significance of sequence blocks within the upstream fragment. 5′-RACE analysis was used for mapping the pri-miR-375 gene transcription start site. The conserved 768 bp region was able to direct preferential expression of a GFP reporter gene to pancreatic islets in transgenic mice. Deletion analysis using a luciferase reporter gene in transfected cultured cell lines confirmed the cell specificity of the putative promoter region, and identified several key cis-elements essential for optimal activity, including E-boxes and a TATA sequence. Consistent with this, 5′-RACE analysis identified a transcription start site within this DNA region, 24 bp downstream of the TATA sequence. These studies define the promoter of the pri-miR-375 gene, and show that islet-specific expression of the pri-miR-375 gene is controlled at the transcriptional level. Detailed analysis of the transcriptional mechanisms controlling expression of miRNA genes will be essential to permit a comprehensive understanding of the complex role of miRNAs such as miR-375 in developmental processes.

GPR40 is a G protein-coupled receptor expressed preferentially in pancreatic β cells. It is activated by long-chain fatty acids and has been implicated in mediating physiological and pathological effects of long-chain fatty acids on β cells. We mapped the GPR40 transcription start site to a location 1044 bp upstream of the translation start site. This permitted definition of the GPR40 core promoter and the organization of the gene, which comprises a 24-bp non-coding exon, a 698-bp intron and a 4402-bp second exon, containing the entire protein coding sequence. Sequence analysis of the GPR40 locus revealed three evolutionarily conserved regions upstream to the translation start site (HR1-HR3). DNase I-hypersensitive sites were present in the HR2 and HR3 regions in β cells but not in non-βcells. The 5′-flanking region of the GPR40 gene was capable of directing transcriptional activity selectively in β cells. An important component of this is attributable to the HR2 region, which showed strong β cell-specific enhancer activity. Systematic mutagenesis of HR2 revealed several important sub-regions. Mutagenesis of sub-regions 4-5, and 9 reduced transcriptional activity by ∼60 and 40%, respectively. These sub-regions can bind the β cell-specific transcription factors PDX1 and BETA2, respectively, both in vitro and in vivo. Thus, cell-specific expression of the GPR40 gene involves a characteristic chromatin organization of the locus and is controlled at the transcriptional level through HR2, a potent β cell-specific enhancer.

The insulin gene is efficiently expressed only in pancreatic beta cells. Using reverse transcriptase-polymerase chain reaction analysis, we show that insulin mRNA levels are at least 10⁵-fold higher in beta cells than non-beta cells. To examine the underlying mechanisms, we expressed beta cell transcription factors by transfection of non-beta cells. Separate expression of BETA2, E2A, or PDX1 led to modest (

A PCR-based subtractive cloning procedure was used to identify genes expressed at higher levels in the pancreatic beta cell line βTC1, as compared to the pancreatic alpha cell line αTC1. One of the clones isolated by this procedure corresponded to the regulatory subunit (RIα) of protein kinase A (PKA). Using antibodies directed against RIα, we now demonstrate both by immunoblot and immunofluorescence that RIα protein is present at higher levels in cultured beta cells as compared to alpha cells. In vitro PKA assays revealed high basal PKA activity in αTC1 extracts, which changed little on addition of exogenous cAMP. On the other hand, extracts from beta cells showed very low basal activity of PKA, which was elevated upon addition of cAMP. A similar trend was observed in vivo using transfected luciferase constructs bearing multiple copies of a CRE element: in αTC1 cells, no induction by forskolin was observed, whereas in βTC1 cells, forskolin produced a 9-fold increase in activity. Therefore, the results indicate that RIα of PKA is selectively expressed in pancreatic beta cells as compared to alpha cells: this selective expression is associated with major differences in the properties of the PKA signal transduction pathway. Differential expression of the regulatory subunit may play a role in determining the patterns of gene expression and signal transduction characteristic of alpha and beta cells.

The complex anatomy of the mammalian pancreas, in which the endocrine cells are grouped in islets dispersed among the predominant exocrine component, has hampered study of the molecular events governing the development of pancreatic cell lineages. To investigate whether fish may provide relevant, complementary models of pancreas development, we characterized the trout insulin (tINS) promoter and its molecular interactions with PDX1, a key transcriptional and developmental factor of the mammalian pancreas. Transfection of a luciferase reporter plasmid containing the 280 bp 5'-flanking region of the tINS gene resulted in strong activity in mammalian pancreatic β cells but not in CHO or pituitary cells. Footprinting assays and cotransfection experiments indicated that mammalian PDX1 binds to and activates the tINS promoter. By microinjecting plasmids to fertilized zebrafish eggs, we showed that the expression of mouse PDX1 is capable of activating the coinjected tINS promoter plasmid in most cell types of the 24-h zebrafish embryo. The conserved role of PDX1 in vertebrate insulin gene regulation opens the possibility to exploit fish models in the study of pancreas development.

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5′ flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5 - 10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine β cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis Indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5′ flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression.

Cell-specific expression of the insulin gene is controlled by cis-acting DNA sequences located within approximately equal to 350 base pairs of the 5' flanking DNA immediately upstream from the transcription start site. Using synthetic oligonucleotides, we have constructed a systematic series of block replacement mutants spanning this region. No single sequence appears to be absolutely required for expression. However, three of the mutants exhibit 5-10 times less activity and several others show 2-3 times less. Simultaneous mutation of two of the most mutationally sensitive regions leads to virtual abolition of activity. These two elements are structurally related and presumably represent key components of the machinery determining the cell-specific expression of the insulin gene.

Replacement of the early region of simian virus 40 results in virus that cannot replicate in a normal host, CV-1 cells, but can replicate in COS cells, a derivative of CV-1 cells that constitutively express simian virus 40 tumor antigen (T antigen). However, passage of such an early replacement simian virus 40 mutant in COS cells results in the emergence of virus that can propagate in CV-1 cells. Analysis of this virus revealed that the mutant rescued the integrated T-antigen gene from the COS cell genome. Comparison of the sequence of the recovered virus with that of the viral DNA resident in COS cells (strain 776) and the mutant used in our studies (derived from strain 777) proves that the mutant virus acquired the T-antigen gene from the COS cell chromosome via homologous recombination. Most probably this process was mediated by a direct genetic exchange.

Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pstl site of pBR322 plasmid and transformation of E.coli x1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

A system was established in which nuclear preparations from rat brains were capable of protein synthesis under cell free conditions. The electrophoretic pattern of the synthesized proteins was similar to that found in vivo provided that the reaction mixture contained pH 5 precipate factors derived from the high speed supernatant fraction of brain. In the absence of the pH 5 factors, using nuclear preparations from brains of 2 day old rats, approximately 1.5% and 2% of the newly synthesized proteins were identified as tubulin and actin, respectively. In the presence of pH 5 factors, protein synthesis was stimulated and the proportion of the newly synthesized tubulin and actin increased to 26% and 11%, respectively. In contrast to nuclear fractions from 2 day old rats, when nuclei from brains of 1 month old rats were tested in the presence of pH 5 factors, the proportion of tubulin and actin synthesized was lower and amounted to 10% and 4%, respectively. The age dependent change in the relative amount of the tubulin and actin synthesized is in good agreement with the translational pattern shown by brain polyribosomes in a brain cell free system as well as with the pattern obtained with brain mRNA translated in a wheat germ cell free system. Nuclei enriched for either neuronal or glial populations synthesized similar proportions of tubulin and actin in vitro. We conclude that the reduction in the synthesis of tubulin and actin during the postnatal development of the rat brain occurs in both neuronal and glial cells.

The increased rate of synthesis of the "estrogen-induced protein" (IP) first detected by Notides and Gorski is one of the earliest macromolecular responses to estrogen by the rat uterus. The IP detected by autoradiography of sodium dodecyl sulfate polyacrylamide gel electropherograms was shown to correspond to a stainable protein band in electropherograms of cytosol from uteri of both untreated and estrogen-treated rats. The uteri of 9-10-day-old rats showed a maximum rate of IP synthesis 1 h after estrogen-treatment, followed by a decrease to constitutive levels by 24 h. The rate of synthesis of IP was doubled by estrogen administration to 10-30-day-old rats. The improved sensitivity of the autoradiographic method permitted the detection of some stimulation of IP synthesis in uteri of 5-day-old rats, in contrast to our previous findings in Wistar rats using a double isotope labelling technique.