Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

Hepatitis C Virus Induces the Ubiquitin-Editing Enzyme A20 via Depletion of the Transcription Factor Upstream Stimulatory Factor 1 To Support Its Replication.

Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20, is a ubiquitin-editing enzyme capable of ubiquitination or deubiquitination of its target proteins. In this study, we show that hepatitis C virus (HCV) infection could induce the expression of A20 via the activation of the A20 promoter. The induction of A20 by HCV coincided with the loss of upstream stimulatory factor 1 (USF-1), a transcription factor known to suppress the A20 promoter. The role of USF-1 in the regulation of the A20 promoter in HCV-infected cells was confirmed by the chromatin immunoprecipitation (ChIP) assay, and its depletion was apparently mediated by proteasomes, as USF-1 could be stabilized by the proteasome inhibitor MG132 to suppress the A20 expression. As the overexpression of A20 enhanced the replication of HCV and the silencing of A20 had the opposite effect, A20 is a positive regulator of HCV replication. Our further studies indicated that A20 enhanced the activity of the HCV internal ribosome entry site (IRES). In conclusion, our results demonstrated that HCV could induce the expression of A20 via the depletion of USF-1 to enhance its replication. Our study provided important information for further understanding the interaction between HCV and its host cells.IMPORTANCE Hepatitis C virus establishes chronic infection in approximately 85% of the patients whom it infects. However, the mechanism of how HCV evades host immunity to establish persistence is unclear. In this report, we demonstrate that HCV could induce the expression of the ubiquitin-editing enzyme A20, an important negative regulator of the tumor necrosis factor alpha (TNF-α) and NF-κB signaling pathways. This induction of A20 enhanced HCV replication as it could stimulate the HCV IRES activity to enhance the translation of HCV proteins. The induction of A20 was mediated by the depletion of USF-1, a suppressor of the A20 promoter. Our study thus provides important information for further understanding the interaction between HCV and its host cells.

Genome-wide analyses in neuronal cells reveal that upstream transcription factors regulate lysosomal gene expression.

UNLABELLED: The upstream transcription factors (USFs) USF1 and USF2 are ubiquitously expressed transcription factors that are characterized by a conserved basic helix-loop-helix/leucine zipper DNA-binding domain. They form homo- or heterodimers, and recognize E-box motifs to modulate gene expression. They are known to regulate diverse cellular functions, including the cell cycle, immune responses and glucose/lipid metabolism, but their roles in neuronal cells remain to be clarified. Here, we performed chromatin immunoprecipitation of USF1 from mouse brain cortex. Subsequent promoter array analysis (ChIP-chip) indicated that USF1 exclusively bound to the CACGTG E-box motifs in the proximal promoter regions. Importantly, functional annotation of the USF1-binding targets revealed an enrichment of genes related to lysosomal functions. Gene expression array analysis using a neuronal cell line subsequently revealed that knockdown of USFs de-regulated lysosomal gene expression. Altered expression was validated by quantitative RT-PCR, supporting the conclusion that USFs regulate lysosomal gene expression. Furthermore, USF knockdown slightly increased LysoTracker Red staining, implying a role for USFs in modulating lysosomal homeostasis. Together, our comprehensive genome-scale analyses identified lysosomal genes as targets of USFs in neuronal cells, suggesting a potential additional pathway of lysosomal regulation. DATABASE: The data for the gene expression array and ChIP-chip have been submitted to the Gene Expression Omnibus (GEO) under accession numbers GSE76615 and GSE76616, respectively.

(E)-4-(3,4-Dimethoxyphenyl)but-3-en-1-ol Enhances Melanogenesis through Increasing Upstream Stimulating Factor-1-Mediated Tyrosinase Expression.

We investigated the potential melanogenic effect of compounds from Zingiber cassumunar Roxb. Our data revealed that chloroform-soluble extract of Z. cassumunar enhanced melanin synthesis in B16F10 melanoma cells. Among the components of the chloroform extract, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol (DMPB) increased melanogenesis in both B16F10 cells and human primary melanocytes. In B16F10 cells, DMPB enhanced the activation of ERK and p38, and the level of tyrosinase. Although the level of microphthalmia-associated transcription factor was unchanged in DMPB-treated B16F10 cells, DMPB increased levels and nuclear localization of upstream stimulating factor-1 (USF1). Consistently, DMPB-mediated melanin synthesis was diminished in USF1-knockdown cells. Furthermore, DMPB induced hyperpigmentation in brown guinea pigs in vivo. Together, these data suggest that DMPB may promote melanin synthesis via USF1 dependent fashion and could be used as a clinical therapeutic agent against hypopigmentation-associated diseases.

Suppression of adipogenesis by valproic acid through repression of USF1-activated fatty acid synthesis in adipocytes.

VPA (valproic acid), a short-chain fatty acid that is a HDAC (histone deacetylase) inhibitor, is known to suppress adipogenesis. In the present study, we identified the molecular mechanism of VPA-mediated suppression of adipogenesis in adipocytes. VPA suppressed the accumulation of intracellular triacylglycerol. The expression levels of PPARγ (peroxisome-proliferator-activated receptor γ) and C/EBPα (CCAAT/enhancer-binding protein α), which are key regulators of adipogenesis, as well as the expression of SCD (stearoyl-CoA desaturase), were decreased by the treatment with VPA. Moreover, glycerol release was decreased in the VPA-treated cells, even though the transcription levels of ATGL (adipose triacylglycerol lipase), HSL (hormone-sensitive lipase) and MGL (monoacylglycerol lipase), all of which are involved in lipolysis, were elevated by the treatment with VPA. It is noteworthy that the expression level of FAS (fatty acid synthase) was significantly suppressed when the cells were cultured in medium containing VPA. Furthermore, VPA-mediated suppression of the accumulation of the intracellular triacylglycerols was prevented by the treatment with palmitic acid, a major product of FAS. The results of promoter-luciferase and chromatin immunoprecipitation assays demonstrated that USF1(upstream stimulating factor 1) bound to the E-box of the promoter region of the FAS gene. In addition, the expression of USF1 was decreased by the treatment with VPA. siRNA-mediated knockdown of the expression of the USF1 gene repressed adipogenesis along with the decreased expression of the FAS gene. The overexpression of USF1 enhanced both adipogenesis and the expression of FAS in VPA-treated cells. These results indicate that VPA suppressed adipogenesis through the down-regulation of USF1-activated fatty acid synthesis in adipocytes.

Upstream stimulatory factor 2 and hypoxia-inducible factor 2α (HIF2α) cooperatively activate HIF2 target genes during hypoxia.

While the functions of hypoxia-inducible factor 1α (HIF1α)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2α/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2α protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2α, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2α knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.

DNA double-strand breaks relieve USF-mediated repression of Dß2 germline transcription in developing thymocytes.

Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRß locus (Tcrb) D segments, Dß1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJß2 segment cluster is initially restricted to the J segments and only redirected upstream of Dß2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJß2 recombination is less efficient than that of DJß1. Because inefficient DJß2 assembly offers the potential for V-to-DJß2 recombination to rescue frameshifted V-to-DJß1 joints, we wished to determine how Dß2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dß2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dß2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dß2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dß2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dß2 recombinational accessibility during thymocyte development.

The IFN-gamma-induced transcriptional program of the CIITA gene is inhibited by statins.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-gamma induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins; however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-gamma-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-gamma inducible expression of CIITA, and statins inhibit IFN-gamma-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-gamma induction of the CIITA gene is regulated by the transcription factors STAT-1alpha, interferon regulatory factor (IRF)-1 and upstream stimulatory factor (USF)-1. We previously reported that statins inhibit constitutive STAT-1alpha expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1alpha and IRF-1 to the endogenous CIITA promoter IV (pIV). The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA polymerase II (Pol II), p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene.