Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1.

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo.

Background: The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.Methods: IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis.Results: In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL-6 and IL-17 mRNA expression and to a lesser extent with IL-22 mRNA expression in IBD. To further explore the role of IRF4 under in vivo conditions, we studied IRF4-deficient and wildtype mice in experimental colitis. In contrast to DSS colitis, IRF4 deficiency was protective in T-cell-dependent transfer colitis associated with reduced ROR alpha/gamma t levels and impaired IL-6, IL-17a, and IL-22 production, suggesting that IRF4 acts as a master regulator of mucosal Th17 cell differentiation. Subsequent mechanistic studies using database analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays identified a novel IRF4 binding site in the IL-17 gene promoter. Overexpression of IRF4 using retroviral infection induced IL-17 production and IL-17 together with IL-6 induced ROR gamma t expression.Conclusions: IRF4 can directly bind to the IL-17 promotor and induces mucosal ROR gamma t levels and IL-17 gene expression thereby controlling Th17-dependent colitis. Targeting of this molecular mechanism may lead to novel therapeutic approaches in human IBD.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

The eukaryotic promoter database (EPD).

The Eukaryotic Promoter Database (EPD) is an annotated non-redundant collection of eukaryotic POL II promoters for which the transcription start site has been determined experimentally. Access to promoter sequences is provided by pointers to positions in nucleotide sequence entries. The annotation part of an entry includes a description of the initiation site mapping data, exhaustive cross-references to the EMBL nucleotide sequence database, SWISS-PROT, TRANSFAC and other databases, as well as bibliographic references. EPD is structured in a way that facilitates dynamic extraction of biologically meaningful promoter subsets for comparative sequence analysis. WWW-based interfaces have been developed that enable the user to view EPD entries in different formats, to select and extract promoter sequences according to a variety of criteria, and to navigate to related databases exploiting different cross-references. The EPD web site also features yearly updated base frequency matrices for major eukaryotic promoter elements. EPD can be accessed at http://www.epd.isb-sib.ch

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in pregnant C57BL/6 mice infected with L. major at different gestational periods.

BACKGROUND AND OBJECTIVE: To assess if gestational factors affect the resistance of C57BL/6 mice to L major infection, this study determined the levels of IL-4 and IFN-gamma in popliteal lymph node cells of pregnant C57BL/6 mice infected with L. major at 16 hours, 5 days-, 10 days- and 15 days- post plug by PCR, ELISA and BIOASSAY. DESIGN/SETTING: Experimental. RESULTS: Infected pregnant C57BL/6 mice developed larger cutaneous footpad lesions compared with non-pregnant C57BL/6 mice (that showed signs of resolution 7-10 weeks after infection). But, the lesions in infected pregnant C57BL/6 mice and infected non-pregnant C57BL/6 mice were not as large as in susceptible BALB/c mice. The mean litter weight was also reduced in pregnant infected C57BL/6 mice particularly in the groups infected at later stages of pregnancy (day 10- and day 15-post plug). The levels of both IL-4 and IFN-gamma increased with gestation in pregnant infected C57BL/6 mice compared with pregnant non-infected group, while only IL-4 was raised in pregnant infected mice compared with infected non pregnant mice. CONCLUSIONS: It may be concluded that increased IL-4 in pregnant infected C57BL/6 mice caused the transient susceptibility to L major infection while reduced litter weight was associated with increased IFN-gamma. These effects were pronounced in C57BI/6 mice infected with L major in late pregnancy.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Mating system and avpr1a promoter variation in primates.

It has been suggested that primate mating and social behaviours may be influenced by variation in promoter region repetitive DNA of the vasopressin receptor 1a gene (avpr1a). We show that male mating behaviour does not covary in a simple way with promoter repetitive DNA in 12 Old World primates. We found that one microsatellite (-553 bp upstream) was present in all species, irrespective of their behaviour. By contrast, two microsatellites (-3956 and -3625 bp upstream) were present only in some species, yet this variation did not correlate with behaviour. These findings agree with a recent comparative analysis of voles and show that the variation in repetitive DNA in the avpr1a promoter region does not generally explain variation in male mating behaviour. Phylogenetic analysis revealed a GAGTA motif that has been independently deleted three times and involved in another larger deletion. Importantly, the presence/absence of this GAGTA motif leads to changes in predicted transcription factor-binding sites. Given the repeated loss of this motif, we speculate that it might be of functional relevance. We suggest that such non-repetitive variation, either in indels or in sequence variation, are likely to be important in explaining interspecific variation in avpr1a expression.

Novel function for interleukin-7 in dendritic cell development.

Interleukin-7 (IL-7) is crucial for the development of T and B lymphocytes from common lymphoid progenitors (CLPs) and for the maintenance of mature T lymphocytes. Its in vivo role for dendritic cells (DCs) has been poorly defined. Here, we investigated whether IL-7 is important for the development or maintenance of different DC types. Bone marrow-derived DCs expressed the IL-7 receptor (IL-7R) and survived significantly longer in the presence of IL-7. Migratory DCs (migDCs) isolated from lymph nodes also expressed IL-7R. Surprisingly, IL-7R was not required for their maintenance but indirectly for their development. Conventional DCs (cDCs) and plasmacytoid DCs (pDCs) resident in lymph nodes and spleen were IL-7R(-). Using mixed bone marrow chimeras, we observed an intrinsic requirement for IL-7R signals in their development. As the number of CLPs but not myeloid progenitors was reduced in the absence of IL-7 signals, we propose that a large fraction of cDCs and pDCs derives from CLPs and shares not only the lymphoid origin but also the IL-7 requirement with lymphocyte precursors.

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved.