Octamer-binding Transcription Factor-1 Research Articles

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

HSV-1 selectively packs the transcription factor Oct-1 into EVs to facilitate its infection.

HSV-1 hijacks the cellular vesicular secretion system and promotes the secretion of extracellular vesicles (EVs) from infected cells. This is believed to facilitate the maturation, secretion, intracellular transportation and immune evasion of the virus. Intriguingly, previous studies have shown that noninfectious EVs from HSV-1-infected cells exert antiviral effects on HSV-1 and have identified host restrictive factors, such as STING, CD63, and Sp100 packed in these lipid bilayer-enclosed vesicles. Octamer-binding transcription factor-1 (Oct-1) is shown here to be a pro-viral cargo in non-virion-containing EVs during HSV-1 infection and serves to facilitate virus dissemination. Specifically, during HSV-1 infection, the nuclear localized transcription factor Oct-1 displayed punctate cytosolic staining that frequently colocalized with VP16 and was increasingly secreted into the extracellular space. HSV-1 grown in cells bereft of Oct-1 (Oct-1 KO) was significantly less efficient at transcribing viral genes during the next round of infection. In fact, HSV-1 promoted increased exportation of Oct-1 in non-virion-containing EVs, but not the other VP16-induced complex (VIC) component HCF-1, and EV-associated Oct-1 was promptly imported into the nucleus of recipient cells to facilitate the next round of HSV-1 infection. Interestingly, we also found that EVs from HSV-1-infected cells primed cells for infection by another RNA virus, vesicular stomatitis virus. In summary, this investigation reports one of the first pro-viral host proteins packed into EVs during HSV-1 infection and underlines the heterogenetic nature and complexity of these noninfectious double-lipid particles.

1887-P: Intermittent Hypoxia Increased the Expressions of Interleukin (IL)-8, Osteonectin, and Myonectin via OCT1 and Nrf2 Binding to the Promoters in Muscle Cells

Sleep apnea syndrome (SAS) is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human RD and mouse C2C12 muscle cells either to 70 cycles/24 h of IH (5 min hypoxia [1% O2]/10 min normoxia [21% O2]), mimicking SAS patients, or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction, and found that IH significantly increased the mRNA levels of interleukin-8 (IL-8), osteonectin (ON), and myonectin (MN). IL-8, ON, and MN in IH-treated RD cell medium were also increased (P<0.0001, P<0.0001, and P=0.0079, respectively). We further analyzed the IH-induced expression mechanisms of IL-8, ON, and MN genes in RD muscle cells. Deletion analyses revealed that -152 to -151 region in IL-8, -105∼-99 region in ON, and -3,741∼-3,738 region in MN promoter(s) were responsible for the activation by IH. The promoters contain consensus transcription factor binding sequences for octamer binding transcription factor 1 (OCT1) in IL-8 and MN, and for nuclear factor erythroid 2-related factor 2 (NRF2) in ON, respectively. The introduction of siRNA for OCT1, and NRF2 abolished the increase of mRNAs for IL-8 and MN, and ON, respectively. As IL-8, ON, and MN are reported to be myokines and associated with glucose intolerance, insulin resistance, and diabetes, the results could well explain a mechanism of glucose intolerance, insulin resistance, and diabetes in SAS patients. Disclosure S. Takasawa: None. R. Shobatake: None. A. Itaya-Hironaka: None. M. Makino: None. S. Sakuramoto-Tsuchida: None. T. Uchiyama: None. H. Ota: None. A. Yamauchi: None.

Metabolic and Biomolecular Changes Induced by Incremental Long-Term Training in Young Thoroughbred Racehorses during First Workout Season

Training has a huge effect on physiological homeostasis. The Thoroughbred racehorse is a valid animal model to investigate such changes for training schedule fine-tuning. As happens in human athletes, it is hypothesized that biochemical and immune response changes and related biomolecular variations could be induced by training programs. The aim of this study was to investigate, for the first time, the long-term metabolic and biomolecular modifications in young untrained Thoroughbred racehorses in the first 4-month timeframe training period. Twenty-nine clinically healthy, untrained, two-year-old Thoroughbred racehorses were followed during their incremental 4-month sprint exercise schedule. Blood collection was performed once a month, five times (T-30, T0, T30, T60, and T90). For each sample, lactate concentration, plasma cell volume (PCV), and hematobiochemical parameters (glucose, urea, creatinine, aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), total bilirubin (Tbil), lactate dehydrogenase (LDH), creatine kinase (CK), cholesterol, triglycerides, albumin (Alb), total proteins (TPs), phosphorus (P), calcium (Ca2+), magnesium (Mg), sodium (Na+), potassium (K-), and chloride (Cl)) were determined. At T-30 and T90, serum protein electrophoresis (SPE), serum amyloid A (SAA), and real-time qPCR were performed on all samples to evaluate the expression of key genes and cytokines related to inflammatory and Th2 immunity responses: Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Interleukin-1β (IL-1β), Octamer-Binding Transcription Factor 1 (OCT1), B-cell lymphoma/leukemia 11A (BCL11A). Statistical analysis was performed (ANOVA and t test, p < 0.05). Significant modifications were identified compared with T-30 for PCV, glucose, triglycerides, cholesterol, lactate, urea, creatinine, Tbil, ALP, LDH, Na+, K-, Ca2+, SAA, TPs, SPE, IL-6, IL-4, Oct-1, and BCL11A. In conclusion, the first long-term training period was found to induce fundamental systemic changes in untrained Thoroughbreds.

Abnormality of hepatic triglyceride metabolism in ApcMin/+ mice with colon cancer cachexia

AimsColorectal cancer syndrome has been one of the greatest concerns in the world. Although several epidemiological studies have shown that hepatic low lipoprotein lipase (LPL) mRNA expression may be associated with dyslipidemia and tumor progression, it is still not known whether the liver plays an essential role in hyperlipidemia of ApcMin/+ mice. Main methodsWe measured the expression of metabolic enzymes that involved fatty acid uptake, de novo lipogenesis (DNL), β-oxidation and investigated hepatic triglyceride production in the liver of wild-type and ApcMin/+ mice. Key findingsWe found that hepatic fatty acid uptake and DNL decreased, but there was no significant difference in fatty acid β-oxidation. Interestingly, the production of hepatic very low-density lipoprotein-triglyceride (VLDL-TG) decreased at 20 weeks of age, but marked steatosis was observed in the livers of the ApcMin/+ mouse. To further explore hypertriglyceridemia, we assessed the function of hepatic glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) for the first time. GPIHBP1 is governed by the transcription factor octamer-binding transcription factor-1 (Oct-1) which are involved in the nuclear factor-κB (NF-κB) signaling pathway in the liver of ApcMin/+ mice. Importantly, it was also confirmed that sn50 (100 μg/mL, an inhibitor of the NF-κB) reversed the tumor necrosis factor α (TNFα)-induced Oct-1 and GPIHBP1 reduction in HepG2 cells. SignificanceAltogether, these findings highlighted a novel role of GPIHBP1 that might be responsible for hypertriglyceridemia in ApcMin/+ mice. Hypertriglyceridemia in these mice may be associated with their hepatic lipid metabolism development.

Characterization of Hepatic Triglyceride Metabolism in the ApcMin/＋ Mouse

The ApcMin/＋ mouse exhibits an intestinal cachexia accompanied by hypertriglyceridemia (HTG). Because the liver governs systemic energy demands through lipid metabolism regulation, the liver is a likely pathological target of HTG in the ApcMin/＋ mouse. Methods: On the basal diet, at 8, 14 and 20 weeks of age, the mice were fasted for 4 h prior to undergoing a hepatic triglyceride production test. Several rate-limiting enzymes and transcription factors participate in hepatic lipid metabolism were determined by RT-qPCR and western blot analysis. Findings: Hepatic de novo lipogenesis (DNL) and β-oxidation were found to be regulated by the AMPK pathway in the ApcMin/＋ mouse at 20 weeks of age. Although hepatic triglyceride production was significantly decreased at 20 weeks of age (p<0.001), hepatic steatosis remained in the ApcMin/＋ mouse. Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is governed by the transcription factor octamer-binding transcription factor-1 (Oct-1), which is involved in the nuclear factor-κB (NF-κB) signaling pathway in the liver of the ApcMin/＋ mouse. Additionally, in HepG2 cells, sn50 (an inhibitor of the NF-κB signaling pathway) reversed the tumor necrosis factor α (TNFα)-induced Oct-1 and GPIHBP1 reduction. Interpretation: Our findings indicate that the liver might be responsible for the metabolic complications of HTG in individuals with APC defects and in the ApcMin/＋ mouse. Funding Statement: This work was financially supported by the National Natural Science Foundation of China (Grant No. 31472053 and 31572345) and Graduate Innovation Fund of Jilin University (Grant No. 2017094). The Program for JLU Science and Technology Innovative Research Team (JLUSTIRT, No. 2017TD497 28), the Fundamental Research Funds for the Central Universities. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal welfare and experimental procedures were performed strictly according to the guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978). Additionally, the procedures were approved by the Institutional Animal Care and Use Committee of Jilin University under the approved protocol number 201707025.

Glucose dependent miR-451a expression contributes to parathyroid hormone mediated osteoblast differentiation

Parathyroid hormone (PTH; amino acid 1-34, known as teriparatide) has reported promoting differentiation and glucose uptake in osteoblasts. However, how PTH regulates glucose metabolism to facilitate osteoblast differentiation is not understood. Here, we report that PTH promotes glucose dependent miR-451a expression which stimulates osteoblast differentiation. In addition to glucose uptake, PTH suppresses AMPK phosphorylation via PI3K-mTOR-AKT axis thereby preventing phosphorylation and inactivation of octamer-binding transcription factor 1 (OCT-1) which has been reported to act on the promoter region of miR-451a. Modulation of AMPK activity controls miR-451a levels in differentiating osteoblasts. Moreover, pharmacological inhibition of PI3K-mTOR-AKT axis suppressed miR-451a via increased AMPK activity. We report that this glucose regulated miRNA is an anabolic target and transfection of miR-451a mimic induces osteoblast differentiation and mineralization in vitro. These actions were mediated through the suppression of Odd-skipped related 1 (Osr1) and activation of Runx2 transcription. When injected in vivo, the miR-451a mimic significantly increased osteoblastogenesis, mineralization, reversed ovariectomy induced bone loss and improved bone strength. Together, these findings suggest that enhanced osteoblast differentiation associated with bone formation in case of PTH therapy is also a consequence of elevated miR-451a levels via glucose regulation. Consequently, this miRNA has the potential to be a therapeutic target for conditions of bone loss.

Collaborative interaction of Oct-2 with Oct-1 in transactivation of lactogenic hormones-induced β-casein gene expression in mammary epithelial cells

Octamer-binding transcription factor-1 (Oct-1) is found to mediate lactogenic hormones (prolactin and glucocorticoids, HP)-induced β-casein gene expression in mammary alveolar secretory epithelial cells (MECs). The mammary gland also expresses Oct-2 isoform. In this study, we show that Oct-2 is also involved in HP-induced β-casein expression. Oct-2 endogenously binds to the β-casein promoter in MECs, and HP induce Oct-2 binding activity via mechanisms other than increasing Oct-2 expression or inducing Oct-2 translocation to the nucleus. Oct-2 transactivates HP-induced β-casein gene expression and this function is exchangeable with Oct-1. In MECs, Oct-2 is found to physically interact with Oct-1 regardless of HP treatment. However, HP induce physical interactions of Oct-2 with both signal transducer and activator of transcription 5 (STAT5) and glucocorticoid receptor (GR). These results provided biochemical evidence that Oct-2 may form a heteromer with Oct-1 in induction of β-casein gene expression by HP in MECs.

A Novel Transcription Mechanism Activated by Ethanol: INDUCTION OF Slc7a11 GENE EXPRESSION VIA INHIBITION OF THE DNA-BINDING ACTIVITY OF TRANSCRIPTIONAL REPRESSOR OCTAMER-BINDING TRANSCRIPTION FACTOR 1 (OCT-1)

Solute carrier family 7, member 11 (Slc7a11) is a plasma membrane cystine/glutamate exchanger that provides intracellular cystine to produce glutathione, a major cellular antioxidant. Oxidative and endoplasmic reticulum stresses up-regulate Slc7a11 expression by activation of nuclear factor erythroid 2-related factor 2 and transcription factor 4. This study examined the effect of ethanol on Slc7a11 expression and the underlying mechanism involved. Treatment of mouse hepatic stellate cells with ethanol significantly increased Slc7a11 mRNA and protein levels. Deletion of a 20-bp DNA sequence between -2044 to -2024 upstream of the transcription start site significantly increased basal activity and completely abolished the ethanol-induced activity of the Slc7a11 promoter. This deletion did not affect Slc7a11 promoter activity induced by oxidative or endoplasmic reticulum stress. DNA sequence analysis revealed a binding motif for octamer-binding transcription factor 1 (OCT-1) in the deleted fragment. Mutation of this OCT-1 binding motif resulted in a similar effect as the deletion experiment, i.e. it increased the basal promoter activity and abolished the response to ethanol. Ethanol exposure significantly inhibited OCT-1 binding to the Slc7a11 promoter region, although it did not alter OCT-1 mRNA and protein levels. OCT-1 reportedly functions as either a transcriptional enhancer or repressor, depending on the target genes. Results from this study suggest that OCT-1 functions as a repressor on the Slc7a11 promoter and that ethanol inhibits OCT-1 binding to the Slc7a11 promoter, thereby increasing Slc7a11 expression. Taken together, inhibition of the DNA binding activity of transcriptional repressor OCT-1 is a mechanism by which ethanol up-regulates Slc711 expression.

Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells

Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1.

A novel transcription mechanism activated by ethanol: induction of Slc7a11 expression via inhibiting the DNA‐binding activity of repressor OCT1 in mouse hepatic stellate cells

The objective of this study is to determine the mechanism underlying ethanol‐induced expression of solute carrier family 7 member 11 (Slc7a11). Quantitative real‐time RT‐PCR assay, western blot analysis, promoter activity assays and chromatin immunoprecipitation analysis were performed in this study. Our data demonstrated that ethanol significantly increased Slc7a11 mRNA and protein expression in mouse hepatic stellate cells. Deletion of 20‐bp DNA sequence between −2044 to −2024 upstream of the transcription start site significantly increased the basal activity, and completely abolished the ethanol‐induced activity of the Slc7a11 promoter. DNA sequence analysis reveals a biding motif for octamer‐binding transcription factor 1 (OCT‐1) in this deleted fragment. Similarly, mutation of this OCT‐1 binding motif significantly increased the basal promoter activity and abolished the response to ethanol. In addition, Ethanol exposure significantly inhibited OCT‐1 binding to the Slc7a11 promoter region, while the OCT‐1 mRNA and protein levels were comparable in cells treated with or without ethanol. Results from the present study suggest that OCT‐1 functions as a repressor on the Slc7a11 promoter, and that ethanol inhibits OCT‐1 binding to the Slc7a11 promoter, and therefore increases Slc7a11 expression. Grant support: R01HL089382 (ZhongMao Guo) and SC1HL101431 (Hong Yang).

Androgen receptor repression of GnRH gene transcription.

Alterations in androgen levels lead to reproductive defects in both males and females, including hypogonadotropic hypogonadism, anovulation, and infertility. Androgens have been shown to down-regulate GnRH mRNA levels through an androgen receptor (AR)-dependent mechanism. Here, we investigate how androgen regulates expression from the GnRH regulatory region in the GT1-7 cell line, a model of GnRH neurons. A synthetic androgen, R1881, repressed transcription from the GnRH promoter (GnRH-P) in an AR-dependent manner, and liganded AR associated with the chromatin at the GnRH-P in live GT1-7 cells. The three known octamer-binding transcription factor-1 (Oct-1) binding sites in GnRH-P were required for AR-mediated repression, although other sequences were also involved. Although a multimer of the consensus Oct-1 binding site was not repressed, a multimer of the cluster of Oct-1, Pre-B cell leukemia transcription factor (Pbx)/Prep, and NK2 homeobox 1 (Nkx2.1) binding sites, found at -106/-91 in GnRH-P, was sufficient for repression. In fact, overexpression of any of these factors disrupted the androgen response, indicating that a balance of factors in this tripartite complex is required for AR repression. AR bound to this region in EMSA, indicating a direct interaction of AR with DNA or with other transcription factors bound to GnRH-P at this sequence. Collectively, our data demonstrate that GnRH transcription is repressed by AR via multiple sequences in GnRH-P, including three Oct-1 binding sites, and that this repression requires the complex interaction of several transcription factors.

Association of the POU class 2 homeobox 1 gene (POU2F1) with susceptibility to Type 2 diabetes in Chinese populations

POU class 2 homeobox 1 (POU2F1), also known as octamer-binding transcription factor-1 (OCT-1), is a ubiquitous transcription factor that plays a key role in the regulation of genes related to inflammation and cell cycles. POU2F1 is located on chromosome 1q24, a region with linkage for Type 2 diabetes in Chinese and other populations. We examined the association of POU2F1 genetic variants with Type 2 diabetes in Hong Kong Chinese using two independent cohorts. We genotyped five haplotype-tagging single nucleotide polymorphisms at POU2F1 in 1378 clinic-based patients with Type 2 diabetes and 601 control subjects, as well as 707 members from 179 families with diabetes. We found significant associations of rs4657652, rs7532692, rs10918682 and rs3767434 (OR = 1.26-1.59, 0.0003 < P(unadjusted) < 0.035) with Type 2 diabetes in the clinic-based case-control cohorts. Rs3767434 was also associated with Type 2 diabetes (OR = 1.55, P(unadjusted) = 0.013) in the family-based cohort. Meta-analysis revealed similar associations. In addition, the risk G allele of rs10918682 showed increased usage of insulin treatment during a mean follow-up period of 7 years [hazard ratio = 1.50 (1.05-2.14), P = 0.025]. Using separate cohorts, we observed consistent results showing the contribution of multiple variants at POU2F1 to the risk of Type 2 diabetes.

Functional characterization of a promoter polymorphism inAPE1/Ref‐1that contributes to reduced lung cancer susceptibility

Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein that possesses both DNA-repair and redox regulatory activities. Although it was originally identified as a DNA-repair enzyme, accumulating evidence supports a role of APE1/Ref-1 in tumor development. To investigate association between APE1/Ref-1 polymorphisms and lung cancer risk in Chinese populations, we first genotyped three variants of APE1/Ref-1 and found a -141 T-to-G variant (rs1760944) in the promoter associated with decreased risk of lung cancer [odds ratio (OR) = 0.62 for GG; P=0.043]. Similar results were obtained in a follow-up replication study. Combined data from the two studies comprising a total of 1072 lung cancer patients and 1064 cancer-free control participants generated a more significant association (P=0.002). We observed lower APE1/Ref-1 mRNA levels in the presence of the protective G allele in human peripheral blood mononuclear cells and normal lung tissues. The -141G-allele-promoter construct exhibited decreased luciferase reporter gene expression. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that the -141G allele impaired the binding affinity of some transcription factor, accounting for lower APE1/Ref-1-promoter activity. Supershift assays further revealed that the protein of interest was octamer-binding transcription factor-1 (Oct-1). Chromatin immunoprecipitation reconfirmed binding of Oct-1 to the APE1/Ref-1 -141-promoter region. We also found that Oct-1 conferred attenuated transactivation capacity toward the -141G variant by exogenously introducing Oct-1. These data indicate that genetic variations in APE1/Ref-1 may modify susceptibility to lung cancer and provide new insights into an unexpected effect of APE1/Ref-1 on lung carcinogenesis.

Transcriptional regulation of the fucosyltransferase VI gene in hepatocellular carcinoma cells

The alpha1,3-fucosyltransferase VI (FUT VI) protein is a key enzyme for synthesis of sialyl Lewis X and Lewis X in epithelial cells. Despite its importance, how FUT VI expression is regulated has not previously been elucidated. In this work, we examined transcriptional regulation of the FUT VI gene in hepatocellular carcinoma HepG2 cells. 5'-Rapid amplification of cDNA ends analysis revealed transcription start sites of FUT VI in HepG2 cells at +65 and +278 nucleotides (nt) downstream of the position registered in the Data Base of Human Transcription Start Sites. We determined promoter regions for FUT VI in HepG2 cells using a luciferase reporter gene assay. The promoter activities of constructs located 5'-upstream of the transcription start site decreased when the -186 to -156 and -56 to -19 nt regions were deleted. Site-directed mutagenesis of these regions revealed that two hepatocyte nuclear factor-4 alpha (HNF-4 alpha) and one octamer binding transcription factor-1 (Oct-1) binding sites are essential for FUT VI transcription. Furthermore, transient over-expression of HNF-4 alpha but not Oct-1 enhanced both FUT VI promoter activities and FUT VI mRNA levels in HuH-7 cells. These results suggest that two defined regions in the 5'-flanking region of the FUT VI transcription start site are critical for FUT VI transcription in HepG2 cells.

Octamer-Binding Transcription Factor-1 Gene Is Upregulated in Primary Varicose Veins

While the pathogenesis of varicose veins is assumed to be multifactorial, including the aging process, the etiology, especially in the very young, is strongly linked to genetics and is believed to be associated with improper development and regulation of venous tissue maturation. The aim of this study was to identify the genes whose expression is different in primary varicose veins compared to normal veins in the legs. To test this hypothesis, the differentially expressed gene technique was performed on a large-scale screen of mRNA from varicose and normal veins. Transcriptional products corresponding to cDNA were compared between the two vein types, and one gene showed the greatest differential expression between the samples in all sets of experiments, confirmed by reverse-transcriptase polymerase chain reaction. Octamer-binding transcription factor-1 gene (Oct-1) was upregulated in primary varicose veins. Therefore, we suggest that Oct-1 may play an important role in the development of primary lower extremity varicose veins.

Phylogenetic footprinting reveals evolutionarily conserved regions of the gonadotropin-releasing hormone gene that enhance cell-specific expression.

Reproductive function is controlled by the hypothalamic neuropeptide, GnRH, which serves as the central regulator of the hypothalamic-pituitary-gonadal axis. GnRH expression is limited to a small population of neurons in the hypothalamus. Targeting this minute population of neurons (as few as 800 in the mouse) requires regulatory elements upstream of the GnRH gene that remain to be fully characterized. Previously, we have identified an evolutionarily conserved promoter region (-173 to +1) and an enhancer (-1863 to -1571) in the rat gene that targets a subset of the GnRH neurons in vivo. In the present study, we used phylogenetic sequence comparison between human and rodents and analysis of the transcription factor clusters within conserved regions in an attempt to identify additional upstream regulatory elements. This approach led to the characterization of a new upstream enhancer that regulates expression of GnRH in a cell-specific manner. Within this upstream enhancer are nine binding sites for Octamer-binding transcription factor 1 (OCT1), known to be an important transcriptional regulator of GnRH gene expression. In addition, we have identified nuclear factor I (NF1) binding to multiple elements in the GnRH-regulatory regions, each in close proximity to OCT1. We show that OCT1 and NF1 physically and functionally interact. Moreover, the OCT1 and NF1 binding sites in the regulatory regions appear to be essential for appropriate GnRH gene expression. These findings indicate a role for this upstream enhancer and novel OCT1/NF1 complexes in neuron-restricted expression of the GnRH gene.

Functional Interaction between Oct-1 and Retinoid X Receptor

The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily and heterodimerizes with a variety of other family members such as the thyroid hormone receptor (TR),1 retinoic acid receptor, vitamin D receptor, and peroxisome proliferator-activated receptor. Therefore, RXR is supposed to play a key role in a ligand-dependent regulation of gene transcription by nuclear receptors. In this study, we have identified the octamer-binding transcription factor-1 (Oct-1) as a novel interaction factor of RXR. In vitro pull-down assays using RXR deletion mutants showed that the interaction surfaces were located in the region encompassing the DNA binding domain (C domain) and the hinge domain (D domain) of RXR. We also showed that RXR interacted with the POU homeodomain but not with the POU-specific domain of Oct-1. Gel shift analysis revealed that Oct-1 reduced the binding of TR/RXR heterodimers to the thyroid hormone response element (TRE). In transient transfection assays using COS1 cells, Oct-1 repressed the T3-dependent transcriptional activity of TR/RXR heterodimers, consistent with in vitro DNA binding data; however, transcriptional activation by Gal4-TR(LBD) (LBD, ligand binding domain), which lacks its own DNA binding domain but retains responsiveness to T3, was not influenced by Oct-1. These results suggest that Oct-1 functionally interacts with RXR and negatively regulates the nuclear receptor signaling pathway by altering the DNA binding ability of the receptors.

Activation of Octamer-Containing Promoters by Either Octamer-Binding Transcription Factor 1 (OTF-1) or OTF-2 and Requirement of an Additional B-Cell-Specific Component for Optimal Transcription of Immunoglobulin Promoters

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters.

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.