Upstream Stimulating Factor 1 Research Articles

USF1 transcriptionally activates USP14 to drive atherosclerosis by promoting EndMT through NLRC5/Smad2/3 axis

BackgroundEndothelial-to-Mesenchymal Transformation (EndMT) plays key roles in endothelial dysfunction during the pathological progression of atherosclerosis; however, its detailed mechanism remains unclear. Herein, we explored the biological function and mechanisms of upstream stimulating factor 1 (USF1) in EndMT during atherosclerosis.MethodsThe in vivo and in vitro atherosclerotic models were established in high fat diet-fed ApoE−/− mice and ox-LDL-exposed human umbilical vein endothelial cells (HUVECs). The plaque formation, collagen and lipid deposition, and morphological changes in the aortic tissues were evaluated by hematoxylin and eosin (HE), Masson, Oil red O and Verhoeff-Van Gieson (EVG) staining, respectively. EndMT was determined by expression levels of EndMT-related proteins. Target molecule expression was detected by RT-qPCR and Western blotting. The release of pro-inflammatory cytokines was measured by ELISA. Migration of HUVECs was detected by transwell and scratch assays. Molecular mechanism was investigated by dual-luciferase reporter assay, ChIP, and Co-IP assays.ResultsUSF1 was up-regulated in atherosclerosis patients. USF1 knockdown inhibited EndMT by up-regulating CD31 and VE-Cadherin, while down-regulating α-SMA and vimentin, thereby repressing inflammation, and migration in ox-LDL-exposed HUVECs. In addition, USF1 transcriptionally activated ubiquitin-specific protease 14 (USP14), which promoted de-ubiquitination and up-regulation of NLR Family CARD Domain Containing 5 (NLRC5) and subsequent Smad2/3 pathway activation. The inhibitory effect of sh-USF1 or sh-USP14 on EndMT was partly reversed by USP14 or NLRC5 overexpression. Finally, USF1 knockdown delayed atherosclerosis progression via inhibiting EndMT in mice.ConclusionOur findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

USF1-mediated ALKBH5 stabilizes FLII mRNA in an m6A-YTHDF2-dependent manner to repress glycolytic activity in prostate adenocarcinoma.

Upstream-stimulating factor 1 (USF1) is a ubiquitously expressed transcription factor implicated in multiple cellular processes, including metabolism and proliferation. This study focused on the function of USF1 in glycolysis and the malignant development of prostate adenocarcinoma (PRAD). Bioinformatics predictions suggested that USF1 is poorly expressed in PRAD. The clinical PRAD samples revealed a low level of USF1, which was correlated with an unfavorable prognosis. Artificial upregulation of USF1 significantly repressed glycolytic activity in PRAD cells and reduced cell growth and metastasis in vitro and in vivo. Potential downstream genes of USF1 were probed by integrated bioinformatics analyses. The chromatin immunoprecipitation and luciferase assays indicated that USF1 bound to the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) promoter for transcription activation. Flightless I (FLII) was identified as the gene showing the highest degree of correlation with ALKBH5. As an m6A demethylase, ALKBH5 enhanced FLII mRNA stability by inducing m6A demethylation in an m6A-YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2)-dependent manner. Either silencing of ALKBH5 or FLII blocked the role of USF1 in PARD cells and restored glycolysis, cell proliferation, and invasion. This study demonstrates that USF1 activates ALKBH5 to stabilize FLII mRNA in an m6A-YTHDF2-dependent manner, thereby repressing glycolysis processes and the progression of PRAD.

Silencing RPL8 inhibits the progression of hepatocellular carcinoma by down-regulating the mTORC1 signalling pathway

This study aimed to explore the role of ribosomal protein L8 (RPL8) in controlling hepatocellular carcinoma (LIHC) development. We measured RPL8 expression, apoptosis, cell viability, proliferation, migration, invasion, glucose uptake, lactate production, and the ATP/ADP ratio of LIHC cells to investigate the effect of RPL8 on LIHC. Bioinformatic analysis was employed to analyse RPL8 expression and its potential mechanism in LIHC. RPL8 was upregulated in LIHC tissues and cells. RPL8 silencing accelerated apoptosis and suppressed viability, growth, and movement of LIHC cells. Additionally, RPL8 silencing inhibited glycolysis in LIHC cells. Bioinformatic analysis revealed that RPL8 is regulated by the upstream transcription factor upstream stimulating factor 1 (USF1) and activates the mTORC1 signalling pathway. USF1 overexpression eliminated the inhibitory effect of RPL8 silencing in LIHC cells. RPL8 overexpression increased cell growth, movement, and glycolysis in LIHC. However, inhibition of the mTORC1 signalling pathway eliminated the effect of RPL8 overexpression on LIHC cells. In conclusion, RPL8 may affect LIHC progression by regulating the mTORC1 signalling pathway.

USF1-CHCHD4 axis promotes lung adenocarcinoma progression partially via activating the MYC pathway

This study aimed to identify genes related to lung adenocarcinoma (LUAD) and investigate the effects and molecular mechanisms of coiled-coil-helix-coiled-coil-helix domain containing 4 (CHCHD4) in the progression of LUAD. The GEPIA database was used to evaluate the differential expression of CHCHD4 and the survival data of LUAD patients compared to controls. TCGA-LUAD database, JASPAR website, and GSEA were used to analyse the relationship between CHCHD4 and the upstream stimulating factor 1 (USF1) or MYC pathways. The proliferation, apoptosis, migration, and invasion of LUAD cells were evaluated using cell counting kit-8, 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, wound healing, and transwell assays. qRT-PCR, western blotting, and immunohistochemistry were used to detect the mRNA and protein expression, respectively. Furthermore, xenograft tumours from nude mice were used to verify the effect of CHCHD4 on LUAD in vivo. CHCHD4 overexpression was found in LUAD tumor tissues and cells, and high CHCHD4 was associated with a poor prognosis. Interestingly, CHCHD4 knockdown suppressed the malignant phenotype of the LUAD cells. Moreover, we found that USF1 upregulated CHCHD4 and promoted LUAD progression. CHCHD4 knockdown also inhibited the progression of LUAD. In addition, CHCHD4 knockdown suppressed xenograft tumour growth. USF1-CHCHD4 axis can promote LUAD progress, which may be through activating MYC pathway.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

USF1 promotes the development of knee osteoarthritis by activating the NF-κB signaling pathway.

The current study mainly aims to evaluate the expression pattern and underlying mechanism of upstream stimulating factor 1 (USF1) in the muscle tissues of knee osteoarthritis (KOA) patients. In accordance with previous findings, our data showed that muscle strength was significantly decreased in KOA patients compared with controls. Furthermore, several inflammatory factors, including tumor necrosis factor α (TNFα), IL-8, IL-6 and MCP-1, were associated with reduced muscle strength in KOA patients. Not surprisingly, NF-κB signaling was significantly activated in the muscle tissues of KOA patients compared with control individuals. Furthermore, we showed that USF1 was increased in the muscles of KOA patients compared with controls. More importantly, overexpression of USF1 in primary human skeletal muscle cells significantly increased the activation of NF-κB signaling as well as the levels of pro-inflammatory factors. In summary, we showed novel data that the upregulation of USF1 promoted NF-κB activation-induced inflammatory responses in muscle tissues of KOA patients.

Upstream stimulating factor 1 suppresses autophagy and hepatic lipid droplet catabolism by activating mTOR.

Previous studies indicate that the transcription factor upstream stimulating factor 1 (USF1) is involved in the regulation of lipid and glucose metabolism. However, the role of USF1 in lipid‐induced autophagy remains unknown. Interestingly, we found that USF1 overexpression suppresses autophagy‐related gene expression in HepG2 cells. Further assays confirmed that USF1 could transcriptionally activate mTOR expression, thereby suppressing rapamycin‐induced autophagy in HepG2 cells. Moreover, pharmacological activation of autophagy with rapamycin decreases the numbers and sizes of lipid droplets (LDs) in HepG2 cells exposed to an oleate/palmitate mixture. Of note, USF1 upregulation decreases colocalization of LDs and autophagosomes. In conclusion, our data provide evidence that USF1 contributes to abnormal lipid accumulation in the liver by suppressing autophagy via regulation of mTOR transcription.

Upstream Stimulating Factor 1 (USF-1) Gene Polymorphisms and the Risk, Symptoms, and Outcome of Pediatric Ischemic Stroke

Pediatric ischemic stroke is an important cause of morbidity and mortality. As previous studies of children after stroke showed, dyslipidemias were very common in Polish and other European populations. Thus, looking for genetic factors predisposing to pediatric stroke, its symptoms, and outcome, we have analyzed 2 polymorphisms of the upstream stimulating factor 1 (USF-1) gene. The study group consisted of 82 children with stroke, 156 parents, and 146 controls. We used 2 alternative methods: the case-control model and the analysis of families using the transmission disequilibrium test. The 2 polymorphisms, rs2516839 and rs3737787, were genotyped using the TaqMan Pre-Designed SNP Genotyping Assay. The Statistica 10.0 software was used in all statistical analyses. We did not observe any statistical differences in genotype and allele frequencies between patients and controls. There were also no significant differences in the transmission of alleles from the parents to the affected children. However, we have observed that the TT genotype of the rs2516839 polymorphism was more common in patients with epilepsy and dysarthria, whereas the TT genotype of the rs3737787 polymorphism was more frequent in the group of patients with a decrease in intellectual functioning. Our study did not show any associations between the 2 analyzed polymorphisms of the USF-1 gene and pediatric ischemic stroke. However, we have observed an influence of specific genotypes on the outcome of stroke, including epilepsy, dysarthria, and a decrease in intellectual functioning.

Cortical cells reveal APP as a new player in the regulation of GABAergic neurotransmission

The amyloid precursor protein (APP) modulates synaptic activity, resulting from the fine tuning of excitatory and inhibitory neurotransmission. GABAergic inhibitory neurotransmission is affected by modifications in intracellular chloride concentrations regulated by Na+-K+-2Cl− cotransporter 1 (NKCC1) and neuronal K+-Cl− cotransporter 2 (KCC2), allowing entrance and efflux of chloride, respectively. Modifications in NKCC1 and KCC2 expression during maturation of cortical cells induce a shift in GABAergic signaling. Here, we demonstrated that APP affects this GABA shift. Expression of APP in cortical cells decreased the expression of KCC2, without modifying NKCC1, eliciting a less inhibitory GABA response. Downregulation of KCC2 expression by APP was independent of the APP intracellular domain, but correlated with decreased expression of upstream stimulating factor 1 (USF1), a potent regulator of Slc12a5 gene expression (encoding KCC2). KCC2 was also downregulated in vivo following APP expression in neonatal mouse brain. These results argue for a key role of APP in the regulation of GABAergic neurotransmission.

(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.

Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development.

Upstream stimulating factor 1 (USF1) is a basic helix-loop-helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8-16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.

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.

Transcriptional Repression of C4 Complement by Hepatitis C Virus Proteins

The fourth component of human complement (C4) plays an important role in innate immune function. C4 activity has been observed to be significantly lower in patients with chronic hepatitis C virus (HCV) infections, although the mechanism remains unknown. In this study, we have examined the mechanisms of C4 regulation by HCV. Liver biopsy specimens from patients with chronic HCV infections displayed significantly lower C4 mRNA levels than liver tissue samples from patients with unrelated liver disease. Further, C4 mRNA levels of the two isoforms (C4A and C4B) were significantly reduced in hepatocytes transfected with RNA from HCV genotype 1a or 2a. Subsequently, a significant C4 regulatory role of HCV core or NS5A upon C4 promoter activity was observed. HCV core or NS5A transgenic mice displayed a reduction in C4 mRNA. Gamma interferon (IFN-γ)-induced C4 promoter activation was also impaired in the presence of HCV proteins. We further demonstrated that HCV core reduced the expression of upstream stimulating factor 1 (USF-1), a transcription factor important for basal C4 expression. On the other hand, the expression of interferon regulatory factor 1 (IRF-1), which is important for IFN-γ-induced C4 expression, was inhibited by hepatocytes expressing HCV NS5A. These results underscore the roles of HCV proteins in innate immune regulation in establishing a chronic infection.

Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice

Glycerol-3-phosphate acyltransferase 1 (GPAT1) is a rate limiting enzyme in de novo glycerophospholipid synthesis. The murine GPAT1 promoter sequence (the “classical” sequence) was reported previously. However, the organization of this DNA sequence does not fully match the mouse genome sequences on NCBI/GenBank. Here we have identified net cis-acting promoter sequences for the mouse GPAT1 gene: promoter 1a which includes part of the classical sequence and the downstream promoter 1b. Promoter 1a facilitates transcription of two alternative GPAT1 transcript variants, GPAT1-V1 and V2, while promoter 1b produces a third transcript variant, GPAT1-V3. Upstream stimulating factor-1 (USF-1) controlled both promoters whereas sterol regulatory element-binding protein-1 (SREBP-1) exclusively regulated promoter 1a activity in vitro. Feeding increased GPAT1-V1 and V2, but not V3 mRNA levels in mouse liver. The obese condition of db/ db mice did not alter the hepatic expression levels of any of the three GPAT1 variants. Feeding enhanced hepatic mRNA levels, intranuclear protein levels and promoter 1a-binding levels of SREBP-1, but not of USF-1. Thus, promoter 1a was exclusively activated by routine feeding in vivo. Our results indicate differential roles of the two promoters in the regulation of hepatic GPAT1 gene expression in mice.

SOX9 and the tanning response: something new under the sun

SOX9 and the tanning response: something new under the sun

Upstream stimulating factor-1 mediates the E-box-dependent transcriptional repression of the plasminogen activator inhibitor-1 gene in human mast cells

Plasminogen activator inhibitor (PAI)-1 promotes development of asthma. PAI-1 mRNA and protein are markedly induced in activated mast cells (MCs), a major effector cell type in asthma. However, regulatory mechanisms of PAI-1 transcription in MCs are unknown. We present first evidence that PAI-1 is transcriptionally regulated in human MCs (hMCs). In addition to three enhancer regions, we demonstrated that the E-box at −566 bp to −561 bp is the negative regulatory element, and the specific and constitutive binding of the upstream stimulating factor-1 to this E-box is the key mechanism of the negative regulation of PAI-1 expression in hMCs.

UV-induced Expression of Key Component of the Tanning Process, the POMC and MC1R Genes, Is Dependent on the p-38-activated Upstream Stimulating Factor-1 (USF-1)

Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the alpha-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.

Human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer activity is inhibited by transcriptional factors, from the upstream stimulating factors family, via E-box and newly identified initiator element (Inr) in FSH-R non-expressing cells

To localize the regulatory elements in the human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer and to determine the role of upstream stimulating factors (USFs) in these elements, we transiently transfected constructs of FSH-R promoter/enhancer in pGL3 luciferase reporter plasmids into Chinese hamster ovary cells and the activities were determined by measuring luciferase luminescence of the cell lysates. The 5′-flanking regions of the human FSH-R gene from nt –1485 to –1 with respect to the gene translation start site were amplified by polymerase chain reaction (PCR) and subcloned in pGL3. Deletion mutants were created using PCR or restriction enzyme digestion. Mutation in the E-box sequence from nt –124 to –119 (E-box 3), in the construct from –224 to nt –1 or in the Inr element, which encompasses the transcriptional start site at nt –99, resulted in a substantial reduction in the human FSH-R promoter/enhancer activity. Overexpression of upstream stimulating factor-1 (USF1) suppresses the activity of the human FSH-R promoter/enhancer via Inr and E-box elements. Upstream stimulating factor-2 (USF2) decreases FSH-R promoter/enhancer activity by acting on E-box 3. The results indicate that E-box 3 and the Inr element are important elements of the human FSH-R promoter/enhancer. USF family members inhibit FSH-R gene activity by acting via these elements. USF1 and USF2 suppress human FSH-R promoter/enhancer activity by acting on E-box 3. USF1 also decreases activity by interacting with the Inr element.

Thrombopoietin stimulates Hoxb4 expression: an explanation for the favorable effects of TPO on hematopoietic stem cells

AbstractThrombopoietin (TPO), the primary regulator of platelet production, also plays an important role in hematopoietic stem cell (HSC) biology. In previous studies we demonstrated that the self-renewal and expansion of HSCs is 10 to 20 times less robust in tpo–/– mice than in controls. To explore the molecular basis of this effect, we postulated that Hoxb4 might mediate at least part of the TPO effect on these cells. We first analyzed the effects of TPO on Hoxb4 expression in primitive hematopoietic cell lines; TPO increased expression of the gene 2- to 3-fold in EML and UT-7/TPO cells. We also compared Hoxb4 levels in a candidate HSC population derived from tpo–/– and control mice; Hoxb4 expression was 2- to 5-fold lower in null HSCs. Of the numerous signal transduction molecules induced by TPO, we found that p38 mitogen-activated protein kinase (MAPK) was responsible for the TPO-induced Hoxb4 elevation. We also demonstrated that upstream stimulating factor 1 (USF-1), a transcription factor previously shown to regulate Hoxb4 expression, is also induced by TPO in a p38-dependent manner. Together, these data provide a molecular pathway by which a growth factor can modulate a transcription factor and thereby help direct a critical developmental process.

IFN-gamma regulation of class II transactivator promoter IV in macrophages and microglia: involvement of the suppressors of cytokine signaling-1 protein.

The discovery of the class II transactivator (CIITA) transcription factor, and its IFN-gamma-activated promoter (promoter IV), have provided new opportunities to understand the molecular mechanisms of IFN-gamma-induced class II MHC expression. Here, we investigated the molecular regulation of IFN-gamma-induced murine CIITA promoter IV activity in microglia/macrophages. In the macrophage cell line RAW264.7, IFN-gamma inducibility of CIITA promoter IV is dependent on an IFN-gamma activation sequence (GAS) element and adjacent E-Box, and an IFN response factor (IRF) element, all within 196 bp of the transcription start site. In both RAW cells and the microglia cell line EOC20, two IFN-gamma-activated transcription factors, STAT-1alpha and IRF-1, bind the GAS and IRF elements, respectively. The E-Box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Functionally, the GAS, E-Box, and IRF elements are each essential for IFN-gamma-induced CIITA promoter IV activity. The effects of the suppressors of cytokine signaling-1 (SOCS-1) protein on IFN-gamma-induced CIITA and class II MHC expression were examined. Ectopic expression of SOCS-1 inhibits IFN-gamma-induced activation of CIITA promoter IV and subsequent class II MHC protein expression. Interestingly, SOCS-1 inhibits the constitutive expression of STAT-1alpha and its IFN-gamma-induced tyrosine phosphorylation and binding to the GAS element in CIITA promoter IV. As well, IFN-gamma-induced expression of IRF-1 and its binding to the IRF element is inhibited. These results indicate that SOCS-1 may be responsible for attenuating IFN-gamma-induced CIITA and class II MHC expression in macrophages.