Characterization of the human DYRK1A promoter and its regulation by the transcription factor E2F1

<p>Abstract</p> <p>Background</p> <p>Overexpression of the human <it>DYRK1A </it>gene due to the presence of a third gene copy in trisomy 21 is thought to play a role in the pathogenesis of Down syndrome. The observation of gene dosage effects in transgenic mouse models implies that subtle changes in expression levels can affect the correct function of the <it>DYRK1A </it>gene product. We have therefore characterized the promoter of the human <it>DYRK1A </it>gene in order to study its transcriptional regulation.</p> <p>Results</p> <p>Transcription start sites of the human <it>DYRK1A </it>gene are distributed over 800 bp within a region previously identified as an unmethylated CpG island. We have identified a new alternative noncoding 5'-exon of the <it>DYRK1A </it>gene which is located 772 bp upstream of the previously described transcription start site. Transcription of the two splicing variants is controlled by non-overlapping promoter regions that can independently drive reporter gene expression. We found no evidence of cell- or tissue-specific promoter usage, but the two promoter regions differed in their activity and their regulation. The sequence upstream of exon 1A (promoter region A) induced about 10-fold higher reporter gene activity than the sequence upstream of exon 1B (promoter region B). Overexpression of the transcription factor E2F1 increased <it>DYRK1A </it>mRNA levels in Saos2 and Phoenix cells and enhanced the activity of promoter region B three- to fourfold.</p> <p>Conclusion</p> <p>The identification of two alternatively spliced transcripts whose transcription is initiated from differentially regulated promoters regions indicates that the expression of the <it>DYRK1A </it>gene is subject to complex control mechanisms. The regulatory effect of E2F1 suggests that DYRK1A may play a role in cell cycle regulation or apoptosis.</p

Mechanism of attenuation of leptin signaling under chronic ligand stimulation

<p>Abstract</p> <p>Background</p> <p>Leptin is an adipocyte-derived hormone that acts via its hypothalamic receptor (LEPRb) to regulate energy balance. A downstream effect essential for the weight-regulatory action of leptin is the phosphorylation and activation of the latent transcription factor STAT3 by LEPRb-associated Janus kinases (JAKs). Obesity is typically associated with chronically elevated leptin levels and a decreased ability of LEPRb to activate intracellular signal transduction pathways (leptin resistance). Here we have studied the roles of the intracellular tyrosine residues in the negative feedback regulation of LEPRb-signaling under chronic leptin stimulation.</p> <p>Results</p> <p>Mutational analysis showed that the presence of either Tyr985 and Tyr1077 in the intracellular domain of LEPRb was sufficient for the attenuation of STAT3 phosphorylation, whereas mutation of both tyrosines rendered LEPRb resistant to feedback regulation. Overexpression and RNA interference-mediated downregulation of suppressor of cytokine signaling 3 (SOCS3) revealed that both Tyr985 and Tyr1077 were capable of supporting the negative modulatory effect of SOCS3 in reporter gene assays. In contrast, the inhibitory effect of SOCS1 was enhanced by the presence of Tyr985 but not Tyr1077. Finally, the reduction of the STAT-phosphorylating activity of the LEPRb complex after 2 h of leptin stimulation was not accompanied by the dephosphorylation or degradation of LEPRb or the receptor-associated JAK molecule, but depended on Tyr985 and/or Tyr1077.</p> <p>Conclusions</p> <p>Both Tyr985 and Tyr1077 contribute to the negative regulation of LEPRb signaling. The inhibitory effects of SOCS1 and SOCS3 differ in the dependence on the tyrosine residues in the intracellular domain of LEPRb.</p

Leptin induces inflammation-related genes in RINm5F insulinoma cells

<p>Abstract</p> <p>Background</p> <p>Leptin acts not only on hypothalamic centers to control food intake but has additional functions in peripheral tissues, <it>e.g</it>. inhibition of insulin secretion from pancreatic islets. The leptin receptor (LEPRb) is a class I cytokine receptor that mediates activation of STAT transcription factors. In this study, we characterise the regulation of inflammation-related genes by leptin in insulinoma cells and compare the effect of transcriptional regulation by leptin with that of other cytokines.</p> <p>Results</p> <p>We have used RINm5F insulinoma cells as a model system for a peripheral target cell of leptin. Six transcripts encoding inflammation-related proteins were found to be upregulated by activation of LEPRb, namely lipocalin-2, pancreatitis-associated protein, preprotachykinin-1, fibrinogen-β, tissue-type plasminogen activator (tPA) and manganese-dependent superoxide dismutase (MnSOD). Four of these transcripts (fibrinogen-β, lipocalin-2, tPA, MnSOD) were also induced by the proinflammatory cytokine interleukin-1β (IL-1β). Interferon-γ alone had no effect on the leptin-induced transcripts but enhanced the upregulation by IL-1β of lipocalin-2, tPA and MnSOD mRNA levels. Experiments with LEPRb point mutants revealed that the upregulation of the inflammation-related genes depended on the presence of tyrosine-1138 which mediates the activation of the transcription factors STAT1 and STAT3. Reporter gene assays showed that leptin induced the expression of preprotachykinin-1 and lipocalin-2 on the level of promoter regulation. Finally, leptin treatment increased caspase 3-like proteolytic activity in RINm5F cells.</p> <p>Conclusion</p> <p>The present data show that leptin induces a cytokine-like transcriptional response in RINm5F cells, consistent with the proposed function of leptin as a modulator of immune and inflammatory responses.</p

Экономические перспективы повышения уровня использования попутного нефтяного газа

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2

Differentielle Genexpression in der Insulinoma-Zelllinie RINm5F nach Stimulation mit Leptin

Leptin, a cytokine secreted by the white adipose tissue, decreases food intake via hypothalamic pathways and is a major regulator of energy homoestasis. Leptin has also important peripheral effects, such as the attenuation of insulin secre-tion from pancreatic islets. Leptin signals through the JAK/STAT pathway which is also used by other cytokines such as interleukin-6 (IL-6) or growth hormone (GH). Activation of this pathway leads to phosphorylation of STAT transcription factors which migrate into the nucleus and regulate gene expression. The aim of this study was to compare the leptin signalling pathway with that of IL-6 and GH and to identify leptin-regulated genes. I have used the pancreatic beta cell line RINm5F, which I transfected stably with the leptin receptor (LepRb) cDNA using a retroviral expression system. These cells were stimulated with leptin, IL-6 or GH, and STAT factor phosphorylation was analysed. Leptin stimulated tyrosine phosphorylation of a broad range of STAT factors (STAT1, STAT3, STAT5 and STAT6). In contrast, IL6 and GH only induced tyrosine phosphorylation of a smaller subset of STAT factors (STAT5 and STAT6 with GH; STAT1 and STAT3 with IL-6). Leptin treatment also led to the phosphorylation of the serine residue 727 in STAT3 and to the activation of the ERK pathway. In order to identify leptin-induced genes, a filter array containing 27,648 different cDNAs was screened by differential hybridization with a complex cDNA probe isolated from leptin-stimulated or non-stimulated RINm5F-LepRb cells. 35 transcripts were found to be upregulated after 16 h of leptin stimulation and 21 to be upregulated after 1 h of leptin stimulation. Northern blot analysis allowed to distinguish immediate early, delayed early and late target genes, which had their expression maximum at 1 h, 7 h and 16 h of leptin stimulation, respectively. The following genes were assigned to the first group: SOCS3 (suppressor of cytokine signalling 3), CISH (cytokine-inducible SH2-containing protein), fibrinogen-beta and RGS4 (regulator of Gprotein signalling 4). To the second group was assigned: PAP1 (pancreatitis associated protein 1), and to the third group tPA (tissue plasminogen activator), Ppap2a (phosphatidate phosphohydrolase type 2a), SOD2 (superoxide dismutase 2), lipocalin-2 and tachykinin. Several of those genes encoded proteins which are secreted during inflammatory processes (fibrinogen-beta, PAP1, lipocalin-2 and tachykinin), whereas others play roles in intracellular signalling pathways (SOCS3, CISH, RGS4 and Ppap2a). The effect of the intracellular leptin receptor tyrosine residues 985, 1077 and 1138 on the expression of RGS4 and PAP1 was also evaluated using point mutants of the leptin receptor stably transfected into RINm5F cells. This experiment revealed that the expression of RGS4 and PAP1 mainly depended on Tyr1138, the known STAT3 binding site

Characterization of the human promoter and its regulation by the transcription factor E2F1-6

Le E2F1 binding sites. B) The inducible E2F1-expressing Saos2 cells were transfected with the -742 promoter construct or the mutated versions thereof as indicated. Five hours after transfection, the medium was changed and cells were treated with doxycycline or were not treated. Data were normalized to β-galactosidase activity and are presented as the ratio relative to the activity of the unstimulated wild type construct. Luciferase activity of induced cells was significantly different from that in untreated cells (two-sided t test, p < 0.05, except for the double mutant) but there was no significant difference between wild type and mutants. Bars reflect the means +/- SD of 3 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Characterization of the human promoter and its regulation by the transcription factor E2F1"</p><p>http://www.biomedcentral.com/1471-2199/9/30</p><p>BMC Molecular Biology 2008;9():30-30.</p><p>Published online 26 Mar 2008</p><p>PMCID:PMC2292204.</p><p></p