Reciprocal stabilization of transcription factor binding integrates two signaling pathways to regulate fission yeast fbp1 transcription.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Driving transcriptional regulators in melanoma metastasis.

The progression of melanoma toward the metastatic phenotype occurs in a defined stepwise manner. While many molecular changes take place early in melanoma development, progression toward the malignant phenotype, most notably during the transition from the radial growth phase (RGP) to the vertical growth phase (VGP) involves deregulated expression of several transcription factors. For example, the switch from RGP to VGP is associated with the loss of the transcription factor AP2α and gain of transcriptional activity of cAMP-responsive element binding protein. Together with the upregulation of microphthalmia-associated transcription factor, activating transcription factor 2, nuclear factor kappa B, and other transcription factors, these changes lead to dysregulated expression or function of important cellular adhesion molecules, matrix degrading enzymes, survival factors, as well as other factors leading to metastatic melanoma. Additionally, recent evidence suggests that microRNAs and RNA editing machinery influence the expression of transcription factors or are regulated themselves by transcription factors. Many of the downstream signaling molecules regulated by transcription factors, such as protease activated receptor-1, interleukin-8, and MCAM/MUC18 represent new treatment prospects.

NLRC5 cooperates with the RFX transcription factor complex to induce MHC class I gene expression.

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.

[Cell surface protein Ecm33 is involved in negative feedback regulation of MAP kinase signalling and development of the in vivo real-time monitoring of MAP kinase signalling].

The mitogen-activated protein kinase (MAPK) pathways are signal transduction mechanisms that regulate many cellular processes in eukaryotic organisms, from yeasts to mammals. Multiple MAPKs regulate eukaryotic gene expression in response to various extracellular stimuli through phosphorylation of transcription factors. We have been studying the Pmk1 MAPK, a homologue of the mammalian ERK/MAPK in fission yeast. The Pmk1 MAPK regulates cell integrity and cell morphology. We have previously demonstrated that Atf1, a transcription factor downstream of the stress-activated MAPK pathway, serves also as a target of the Pmk1 MAPK signaling in fission yeast. Here, we identified ecm33⁺ gene, encoding a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. The gene expression of ecm33⁺ is regulated by two transcription factors Atf1 and Mbx1. We also developed an in vivo real-time monitoring system of Atf1 or Mbx1 transcriptional activity, which enables to monitor the activation of the Pmk1 MAPK pathway by various stimuli. Finally, we demonstrated that Ecm33 is involved in the negative regulation of the Pmk1 MAPK signaling through the control of Ca²âº homeostasis. The ecm33 deleted cells displayed Ca²âº sensitivity and increased phosphorylation levels of Pmk1 MAPK. In addition, the Ecm33 overproducing cells displayed phenotypes closely similar to those of the pmk1 knockout cell. Collectively, Ecm33 plays a role in the negative feedback regulation of Pmk1 cell integrity signaling.

Molecular mechanisms underlying PGF2alpha-induced hypertrophy of vascular smooth muscle cells.

The present review focuses primarily on the studies we made in recent years to improve the understanding of the molecular mechanisms of PGF2alpha-induced hypertrophy of Vascular Smooth Muscle Cells (VSMC). In this review, we will summarize the recent findings in the context of the PGF2alpha signaling pathway in three parts: PGF2alpha binding to its receptor, transactivation of EGF receptor, two independent signaling transduction pathways increasing the expression of NOX1 gene.

ATF-1 is a hypoxia-responsive transcriptional activator of skeletal muscle mitochondrial-uncoupling protein 3.

Hypoxia induces oxidative damage in skeletal muscle. Uncoupling protein 3 (UCP3) is the skeletal muscle enriched uncoupling protein and has previously been shown to confer resistance against oxidative stress. We show that hypoxia robustly up-regulates skeletal muscle UCP3 and that the absence of UCP3 in primary skeletal myocytes exacerbates hypoxia-induced reactive oxygen species generation. In this context, we reasoned that the investigation of the regulation of UCP3 may identify novel hypoxia-responsive regulatory pathways that modulate intrinsic anti-oxidant defenses. By screening a transcription factor array of 704 full-length cDNAs in murine C2C12 myoblasts following cotransfection of a murine UCP3 promoter-luciferase construct and myoD we identified numerous candidate regulatory factors that up-regulate UCP3. Active transcription factor-1 (ATF-1) was identified, and as this transcription factor is a known component of a multiprotein hypoxia-induced regulatory complex, we explored its role in hypoxia-mediated UCP3 up-regulation. Site-directed mutagenesis and chromatin immunoprecipitation assays identify a 10-bp region required for ATF-1 induction of UCP3 promoter activity. Hypoxia promotes the phosphorylation of ATF-1, and the knockdown of ATF-1 by shRNA prevents hypoxia-mediated up-regulation of UCP3. Pharmacologic inhibition of p38 MAP kinase prevents both hypoxia-mediated ATF-1 phosphorylation and UCP3 up-regulation. PKA signaling does not modulate hypoxia-induced UCP3 up-regulation and neither does HIF-1alpha activation by cobalt chloride. In conclusion, ATF-1, via p38 MAP kinase activation, functions as a novel regulatory pathway driving UCP3 expression. These data reinforce the role of ATF-1 as a hypoxia-responsive trans-activator and identifies a novel regulatory program that may modulate cellular responses to oxygen-deficit.

PTH regulation of the human cytomegalovirus immediate-early gene promoter.

Secondary hyperparathyroidism and human cytomegalovirus (hCMV) seropositivity are highly prevalent in patients undergoing renal transplantation, and both are linked to the development of chronic allograft nephropathy (CAN). We investigated the hypothesis that parathyroid hormone (PTH) 1-84 regulates hCMV immediate-early gene (IEG) promoter activation in proximal renal tubular cells. PTH 1-84 enhanced hCMV IEG promoter (-548 to +92) activity in opossum kidney cells. Deletion analysis from the 5' end of the promoter localized the PTH 1-84 associated activity to the DNA sequence between -123 and -45. Mutation of an imperfect ATF/AP-1 DNA element within this region abrogated the PTH 1-84 effect and also strongly attenuated basal gene expression. Mobility shift analyses using this DNA element revealed that a member of the ATF-1 family was in the binding complex. In summary, we present evidence for a novel pathogenic role of PTH 1-84 in promoting hCMV immediate-early gene transcription.

Depletion of cAMP-response element-binding protein/ATF1 inhibits adipogenic conversion of 3T3-L1 cells ectopically expressing CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBP beta, or PPAR gamma 2.

The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the "master adipogenic regulators," CCAAT/enhancer-binding protein (C/EBP) beta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBP alpha. In addition, activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion and prevent apoptosis of mature adipocytes. In this report we used small interfering RNA to deplete CREB and the closely related factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARgamma, C/EBP alpha, and adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically expressing C/EBP alpha, C/EBP beta, or PPARgamma2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing both PPARgamma2 and C/EBP alpha, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively active CREB overcame the blockade of adipogenesis due to depletion of C/EBP beta but not due to loss of PPARgamma2 or C/EBP alpha. Depletion of CREB/ATF1 did not suppress the expression of C/EBP beta as we had previously observed using dominant negative forms of CREB. Finally results are presented showing that CREB promotes PPARgamma2 gene transcription. The results indicate that CREB and ATF1 play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also at later stages.