Functional Landscape of PCGF Proteins Reveals Both RING1A/B-Dependent-and RING1A/B-Independent-Specific Activities.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes depending on the assembled PCGF protein (PCGF1-PCGF6); however, it is yet unclear whether these subcomplexes have also specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, while other PCGF proteins have high levels of specificity for distinct target genes. PCGF2 associates with transcription repression, whereas PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assemble and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.

The Cytidine N-Acetyltransferase NAT10 Participates in Peripheral Nerve Injury-Induced Neuropathic Pain by Stabilizing SYT9 Expression in Primary Sensory Neurons.

RNA N4-acetylcytidine (ac4C) modification is increasingly recognized as an important layer of gene regulation; however, the involvement of ac4C in pain regulation has not been studied. Here, we report that N-acetyltransferase 10 protein (NAT10; the only known ac4C "writer") contributes to the induction and development of neuropathic pain in an ac4C-dependent manner. Peripheral nerve injury increases the levels of NAT10 expression and overall ac4C in injured dorsal root ganglia (DRGs). This upregulation is triggered by the activation of upstream transcription factor 1 (USF1), a transcription factor that binds to the Nat10 promoter. Knock-down or genetic deletion of NAT10 in the DRG abolishes the gain of ac4C sites in Syt9 mRNA and the augmentation of SYT9 protein, resulting in a marked antinociceptive effect in nerve-injured male mice. Conversely, mimicking NAT10 upregulation in the absence of injury evokes the elevation of Syt9 ac4C and SYT9 protein and induces the genesis of neuropathic-pain-like behaviors. These findings demonstrate that USF1-governed NAT10 regulates neuropathic pain by targeting Syt9 ac4C in peripheral nociceptive sensory neurons. Our findings establish NAT10 as a critical endogenous initiator of nociceptive behavior and a promising new target for treating neuropathic pain.SIGNIFICANCE STATEMENT The cytidine N4-acetylcytidine (ac4C), a new epigenetic RNA modification, is crucial for the translation and stability of mRNA, but its role for chronic pain remains unclear. Here, we demonstrate that N-acetyltransferase 10 (NAT10) acts as ac4C N-acetyltransferase and plays an important role in the development and maintenance of neuropathic pain. NAT10 was upregulated via the activation of the transcription factor upstream transcription factor 1 (USF1) in the injured dorsal root ganglion (DRG) after peripheral nerve injury. Since pharmacological or genetic deleting NAT10 in the DRG attenuated the nerve injury-induced nociceptive hypersensitivities partially through suppressing Syt9 mRNA ac4C and stabilizing SYT9 protein level, NAT10 may serve as an effective and novel therapeutic target for neuropathic pain.

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

BACKGROUND: Endothelial-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. METHODS: The 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. RESULTS: USF1 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. CONCLUSION: Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

USF1 regulated circPRDM4 modulates tumorigenesis and immune escape in chemoresistant cervical cancer.

Cervical cancer (CC) represents a major global health concern, characterized by chemoresistance and immune evasion mechanisms. Circular RNAs (circRNAs), which play a crucial role in cancer pathogenesis, particularly in the case of CC, have gained significant attention. The primary objective of this study was to investigate the functional significance of circRNAs in chemoresistant CC. A significant upregulation of circPRDM4 expression in chemoresistant CC cells. To investigate the functional consequences, we conducted circPRDM4 knockdown experiments, which resulted in the effective blockade of immune escape mechanisms employed by chemoresistant CC cells. Furthermore, circPRDM4 knockdown demonstrated a significant suppression of tumorigenesis in CC cells, highlighting its contribution to the oncogenic potential of CC. Investigating the regulatory mechanisms involved, we found that the transcriptional factor upstream stimulatory factor 1 (USF1) acts as an inducer of circPRDM4 expression. Remarkably, USF1 was found to effectively modulate CC cell immune escape via its interaction with circPRDM4. Moreover, our results revealed that USF1 is intricately involved in CC cell tumorigenesis through the regulation of circPRDM4. Collectively, our study elucidates the significant roles of circPRDM4 and its upstream regulator USF1 in chemoresistant CC cells. These findings underscore the importance of circRNAs in CC pathogenesis and provide valuable insights into the mechanisms underlying immune escape and tumorigenesis.

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.

SEZ6L2, regulated by USF1, accelerates the growth and metastasis of breast cancer.

Breast cancer (BC) is the second cause of cancer-related mortality in women. Seizure related 6 homolog like 2 (SEZ6L2), a protein presented on cell surface, is involved in tumor development. It was found to be highly expressed in BC, however, its role in BC remains unclear. Herein, we aimed to explore the role of SEZ6L2 in BC. Firstly, the correlationship between SEZ6L2 expression and the clinic pathological characteristics of patients diagnosed with BC was analyzed. Subsequently, the role of SEZ6L2 was further explored using MTT, transwell invasion, flow cytometry, colony formation and wound healing assays. The result showed that the level of SEZ6L2 was remarkably correlated with the TNM stage, HER-2 status and lymph node metastasis of BC. Knockdown of SEZ6L2 significantly suppressed the proliferation of BC cells and induced cell cycle arrest at G1 phase. In addition, SEZ6L2 knockdown repressed their migration and invasion. On the contrary, SEZ6L2 overexpression performed the opposite effects. Furthermore, SEZ6L2 also accelerated the in vivo tumorigenesis of BC cells. Additionally, according to bioinformatics resources, we identified upstream transcription factor 1 (USF1) as a transcriptional factor which bound to the promoter of SEZ6L2 and positively regulated its transcription. In conclusion, this study demonstrated that SEZ6L2 was transcriptionally regulated by USF1 and was involved in the growth and metastasis of BC cells. Revealing the role of SEZ6L2 in BC provides additional knowledge for the pathogenesis of BC, which may benefit to BC therapy.

HCG15 is a hypoxia-responsive lncRNA and facilitates hepatocellular carcinoma cell proliferation and invasion by enhancing ZNF641 transcription.

Hypoxia, one of the key features of the hepatocellular carcinoma (HCC) microenvironment, transcriptionally regulates the expression of mRNAs and non-coding RNAs via hypoxia-inducible factors (HIFs), thereby promoting tumor progression. However, hypoxia-responsive long non-coding RNAs (lncRNAs) in HCC required further investigation. We found that HLA complex group 15 (HCG15) was a new hypoxia-related lncRNA in HCC cells. Both hypoxia and HIF prolyl-hydroxylase inhibitor significantly increased HCG15 expression in HCC cells. At the same time, HIF-1α knockdown blocked hypoxia-induced the upregulation of HCG15. TCGA data revealed that HCG15 was markedly overexpressed and closely associated with the poor prognosis of HCC. HCG15 knockdown prominently suppressed the migration, invasion and proliferation of Hep3B and Huh7 cells. HCG15 overexpression markedly enhanced the proliferation and mobility of Huh7 cells. Zinc finger protein 641 (ZNF641) mRNA was frequently overexpressed and positively associated with HCG15 level in HCC samples from the TCGA database. Either HCG15 or upstream transcription factor 1 (USF1) silencing markedly reduced the ZNF641 level in HCC cells. RNA immunoprecipitation assay confirmed the interaction between HCG15 and USF1. Either HCG15 or USF1 knockdown prominently reduced the luciferase activity of reporter plasmid containing ZNF642 promoter. HCG15 knockdown remarkably abolished USF1-activated ZNF641 transcription in Hep3B cells. Finally, we confirmed that restoring ZNF641 expression remarkably abolished the effects of HCG15 knockdown on HCC cell migration, invasion and proliferation. Collectively, our study demonstrated that HCG15 was a new HIF-1 target gene and played a tumor-promoting role in HCC cells by enhancing USF1-mediated ZNF641 transcription.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis.

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

LncRNA LINC00152 promotes oral squamous cell carcinoma growth via enhancing Upstream Transcription Factor 1 mediated Mitochondrial Ribosomal Protein L52 transcription.

BACKGROUND: LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. METHODS: Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152 subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in vivo tumor cell growth models were used for functional assays. RESULTS: NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152 knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52 knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152-mediated growth-promoting effects. CONCLUSION: This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Polymorphism rs3737787 of Upstream Stimulatory Factor 1 gene is associated with serum lipid phenotype in Nigerian population.

Serum lipid profile which is determined by genotype-phenotype relationship plays a significant role in the development of cardiovascular disease. Upstream stimulatory factor 1 (USF1), has been reported to be associated with serum lipid levels in different population, hence, this study investigated the association of variants in USF1 with serum lipid profile in adults in Lagos state, Nigeria. We genotyped rs3737787 (11235C > T) and rs550376620 (10488G > A) with PCR-RFLP in 384 participants and we used logistic regression to assess the association of these variants with serum lipid levels. The minor allele frequency observed in 10488G > A in both case and control groups was 5% while the minor allele of 11235C > T was observed to be more frequent in the control when compared to the dyslipidemic subjects (24% vs 12%; p = 1.84e-05). Levels of total cholesterol, triglycerides, and LDL-c in dyslipidemic subjects with CC genotype of 11235C > T were significantly higher compared to CT and TT genotypes (p < 0.001; p < 0.0001 and p < 0.0001 respectively). Logistic regression with adjustment for age, gender and BMI, showed that the minor allele carriers of 11235C > T have a reduced risk of dyslipidemia (Odds ratio: 0. 0.043, 95% confidence interval (CI): (0.006-0.331, p = 0.002). Our findings revealed that rs3737787 is associated with lipid phenotype in Nigerian population.

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.

[USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells].

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.

USF1-induced overexpression of long noncoding RNA WDFY3-AS2 promotes lung adenocarcinoma progression via targeting miR-491-5p/ZNF703 axis.

Lung adenocarcinoma (LUAD) is one of the most common diagnosed pathological categories of lung cancer. Long noncoding RNAs (lncRNAs) have been manifested to be key regulators in modulating multiple cancers. Nevertheless, the pathologic role of lncRNA WDFY3-AS2 in LUAD remains elusive. The relative messenger RNA and protein levels were assessed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses, respectively. Colony formation, carboxyfluorescein succinimidyl ester, terminal deoxynucleotidyl transferase dUTP nick-end labeling, wound-healing, and transwell invasion assays were performed to study the underlying role of WDFY3-AS2 in LUAD. Luciferase reporter assay, chromatin immunoprecipitation, RNA pull down, and RNA immunoprecipitation assays were conducted to probe into the interactions between relevant genes. WDFY3-AS2 expression was elevated in LUAD and WDFY3-AS2 transcription was activated by transcription factor USF1. Silencing WDFY3-AS2 could suppress cell proliferation, migration, and invasion, whereas accelerate cell apoptosis in LUAD. Molecular mechanism assays revealed that WDFY3-AS2 could bind to miR-491-5p and miR-491-5p inhibition could reverse the inhibitory effect of WDFY3-AS2 silence on LUAD progression. Besides, zinc finger protein 703 (ZNF703) was identified as a downstream target of miR-491-5p and its expression could be upregulated by WDFY3-AS2. Further, rescue assays uncovered that ZNF703 overexpression could restore the suppressive influence of silenced WDFY3-AS2 on LUAD development. USF1-acitvated WDFY3-AS2 promotes LUAD progression via targeting miR-491-5p/ZNF703 axis, suggesting the potential value of WDFY3-AS2 as a novel target for LUAD treatment.

Up-regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR-296-5p.

LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR-296-5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT-PCR. The targeting relationship between miR-296-5p and ZFAS1 or USF1 was validated by dual-luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK-8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR-296-5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up-regulated in tumour tissues and cells while miR-296-5p was significantly down-regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR-296-5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR-296-5p.

Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1).

Interleukin-like EMT inducer (ILEI, FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell-biological process that confers metastatic properties to a tumor cell. However, very little is known about how ILEI is regulated. Here we demonstrate that ILEI is an in vivo regulator of melanoma invasiveness and is transcriptionally up-regulated by the upstream stimulatory factor-1 (USF-1), an E-box-binding, basic-helix-loop-helix family transcription factor. shRNA-mediated knockdown of ILEI in melanoma cell lines attenuated lung colonization but not primary tumor formation. We also identified the mechanism underlying ILEI transcriptional regulation, which was through a direct interaction of USF-1 with the ILEI promoter. Of note, stimulation of endogenous USF-1 by UV-mediated activation increased ILEI expression, whereas shRNA-mediated USF-1 knockdown decreased ILEI gene transcription. Finally, we report that knocking down USF-1 decreases tumor cell migration. In summary, our work reveals that ILEI contributes to melanoma cell invasiveness in vivo without affecting primary tumor growth and is transcriptionally up-regulated by USF-1.

The rs2516839 variation of USF1 gene is associated with 4-year mortality of nonagenarian women: The Vitality 90+ study.

Upstream transcription factor 1 (USF1) regulates the transcription of many genes related to cell and organism survival processes such as stress and immune response, regulation of cellular senesce, and carcinogenesis. In this study, our aim was to investigate the effect of USF1 single nucleotide variations (SNVs) on longevity in the Vitality 90+ study, a population-based study of nonagenarians (90 ±1 years of age) living in the area of Tampere municipality, Finland. Altogether 509 voluntary nonagenarians (115 males, 394 females) were genotyped using the 5'-nuclease assay for rs2774279G > A, rs2516839T > C, and rs2073658C > T SNVs. During the 4 years of follow-up, the total mortality rate was 64.2%. In the study, we found that the frequency of C-allele of rs2516839 among nonsurviving nonagenarians (52.5%) was higher than those who survived (41.2%; P = 0.0006, odds ratio = 1.575, 95% confidence interval [CI]: 1.215-2.041). Furthermore, carriage of this variation and its haplotypes had a significant gender by genotype interaction (P < 0.05) on mortality. Kaplan-Meier log-rank test during 4-years of follow-up showed significantly higher mortality rate in the case of CC genotype carriage than other genotype carriages in nonagenarian women (P < 0.0001). In addition, after adjusting for age in Cox regression analysis, cardiovascular disease, diabetes, infectious disease, dementia, and living place (nursing home or home), CC genotype of rs2516839T > C was found to be associated with shorter life expectancy in nonagenarian women (hazard ratio = 2.27; 95% CI, 1.34-3.85 P = 0.002). In conclusion, rs2516839 variation and related haplotypes of the USF1 gene are strongly related to all-cause mortality in Finnish nonagenarians, especially among women.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

HDAC5-LSD1 axis regulates antineoplastic effect of natural HDAC inhibitor sulforaphane in human breast cancer cells.

Our recent studies have shown that cross-talk between histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) facilitates breast cancer progression. In this work, we demonstrated that regulatory activity at -356 to -100 bp promoter element plays a critical role in governing HDAC5 transcription. By using DNA affinity precipitation and mass spectrometry, we identified a group of factors that bind to this element. Among these factors, Upstream Transcription Factor 1 (USF1) was shown to play a critical role in controlling HDAC5 transcription. Through screening a panel of epigenetic modifying drugs, we showed that a natural bioactive HDAC inhibitor, sulforaphane, downregulated HDAC5 transcription by blocking USF1 activity. Sulforaphane facilitated LSD1 ubiquitination and degradation in an HDAC5-dependent manner. A comparative microarray analysis demonstrated a genome wide cooperative effect of HDAC5 and LSD1 on cancer-related gene expression. shRNA knockdown and sulforaphane inhibition of HDAC5/LSD1 exhibited similar effects on expression of HDAC5/LSD1 target genes. We also showed that coordinated cross-talk of HDAC5 and LSD1 is essential for the antitumor efficacy of sulforaphane. Combination treatment with sulforaphane and a potent LSD1 inhibitor resulted in synergistic growth inhibition in breast cancer cells, but not in normal breast epithelial cells. Furthermore, combined therapy with sulforaphane and LSD1 inhibitor exhibited superior inhibitory effect on MDA-MB-231 xenograft tumor growth. Taken together, our work demonstrates that HDAC5-LSD1 axis is an effective drug target for breast cancer. Inhibition of HDAC5-LSD1 axis with sulforaphane blocks breast cancer growth and combined treatment with LSD1 inhibitor improves the therapeutic efficacy of sulforaphane.

USF1 deficiency alleviates inflammation, enhances cholesterol efflux and prevents cholesterol accumulation in macrophages.

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.

USF1 gene polymorphisms may associate with the efficacy and safety of chemotherapy based on paclitaxel and prognosis in the treatment of ovarian cancer.

This study was supposed to investigate the correlation between the functional single nucleotide polymorphisms (SNPs) (rs2516839 and rs3737787) in USF1 gene and the efficacy and safety of paclitaxel-based chemotherapy and prognosis in the treatment of ovarian cancer (OC). In total 100 OC patients were selected and divided into the sensitive group and the resistantgroup according to the tumor response to paclitaxel-based chemotherapy after surgery, and the incidence of observed and recorded toxic reaction. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied to test the polymorphisms of rs2516839 and rs3737787 in USF1 gene after extraction of DNA. The correlation between USF1 gene polymorphisms and paclitaxel-based chemotherapy resistance was analyzed using Logistic regression analysis. Stratified analysis was used to test the incidence of toxic reaction in OC patients. Cox proportional hazard model was adapted to make a multiple-factor survival analysis. Significant differences exhibited in the genotype and the allele frequencies of rs2516839 between the sensitive and resistant groups, which showed no obvious difference in the genotype and allele frequencies of rs3737787. OC patients carrying the GA+AA genotype had higher incidence of serious toxic reaction than those carrying the GG genotype. Physical status score, tumor type, maximum tumor diameter and rs2516839 were the independent risk factors for the prognosis of OC patients. Taken together, our results suggest that the rs2516839 polymorphism in USF1 gene may associate with the efficacy and safety of paclitaxel-based chemotherapy and prognosis in the treatment of OC.