Transcription Factor Sp1 Research Articles

Long Non-Coding RNA Small Nucleolar RNA Host Gene 4 Induced by Transcription Factor SP1 Promoted the Progression of Nasopharyngeal Carcinoma Through Modulating microRNA-510-5p/Centromere Protein F Axis.

Long non-coding RNAs (LncRNAs) are implicated with tumorigenesis and the development of nasopharyngeal carcinoma (NPC). Previous studies suggested that long non-coding RNA small nucleolar RNA host gene 4 (SNHG4) exerted oncogenic roles in various cancers. However, the function and molecular mechanism of SNHG4 in NPC have not been investigated. In our study, it was confirmed that the SNHG4 level was enriched in NPC tissues and cells. Functional assays indicated that SNHG4 depletion inhibited the proliferation and metastasis but promoted apoptosis of NPC cells. Furthermore, we identified miR-510-5p as a downstream gene of SNHG4 in NPC cells and SNHG4 upregulated CENPF expression by binding to miR-510-5p. Moreover, there was a positive (or negative) association between CENPF and SNHG4 (or miR-510-5p) expression in NPC. In addition, rescue experiments verified that CENPF overexpression or miR-510-5p silencing abrogated inhibitory effects on NPC tumorigenesis caused by SNHG4 deficiency. The study demonstrated that SNHG4 promoted NPC progression via miR-510-5p/CENPF axis, providing a novel potential therapeutic target for NPC treatments.

Specificity Proteins (Sp) and Cancer.

The specificity protein (Sp) transcription factors (TFs) Sp1, Sp2, Sp3 and Sp4 exhibit structural and functional similarities in cancer cells and extensive studies of Sp1 show that it is a negative prognostic factor for patients with multiple tumor types. In this review, the role of Sp1, Sp3 and Sp4 in the development of cancer and their regulation of pro-oncogenic factors and pathways is reviewed. In addition, interactions with non-coding RNAs and the development of agents that target Sp transcription factors are also discussed. Studies on normal cell transformation into cancer cell lines show that this transformation process is accompanied by increased levels of Sp1 in most cell models, and in the transformation of muscle cells into rhabdomyosarcoma, both Sp1 and Sp3, but not Sp4, are increased. The pro-oncogenic functions of Sp1, Sp3 and Sp4 in cancer cell lines were studied in knockdown studies where silencing of each individual Sp TF decreased cancer growth, invasion and induced apoptosis. Silencing of an individual Sp TF was not compensated for by the other two and it was concluded that Sp1, Sp3 and Sp4 are examples of non-oncogene addicted genes. This conclusion was strengthened by the results of Sp TF interactions with non-coding microRNAs and long non-coding RNAs where Sp1 contributed to pro-oncogenic functions of Sp/non-coding RNAs. There are now many examples of anticancer agents and pharmaceuticals that induce downregulation/degradation of Sp1, Sp3 and Sp4, yet clinical applications of drugs specifically targeting Sp TFs are not being used. The application of agents targeting Sp TFs in combination therapies should be considered for their potential to enhance treatment efficacy and decrease toxic side effects.

Tra2β Enhances Cell Proliferation by Inducing the Expression of Transcription Factor SP1 in Cervical Cancer

ObjectiveIn this study, the role and potential mechanism of transformer 2β (Tra2β) in cervical cancer were explored. MethodsThe transcriptional data of Tra2β in patients with cervical cancer from Gene Expression Profiling Interactive Analysis (GEPIA) and cBioPortal databases were investigated. The functions of Tra2β were evaluated by using Western blot, MTT, colony formation, Transwell assays, and nude mouse tumor formation experiments. Target genes regulated by Tra2β were studied by RNA-seq. Subsequently, representative genes were selected for RT-qPCR, confocal immunofluorescence, Western blot, and rescue experiments to verify their regulatory relationship. ResultsThe dysregulation of Tra2β in cervical cancer samples was observed. Tra2β overexpression in Siha and Hela cells enhanced cell viability and proliferation, whereas Tra2β knockdown showed the opposite effect. Alteration of Tra2β expression did not affect cell migration and invasion. Furthermore, tumor xenograft models verified that Tra2β promoted cervical cancer growth. Mechanically, Tra2β positively regulated the mRNA and protein level of SP1, which was critical for the proliferative capability of Tra2β. ConclusionThis study demonstrated the important role of the Tra2β/SP1 axis in the progression of cervical cancer in vitro and in vivo, which provides a comprehensive understanding of the pathogenesis of cervical cancer.

The UDPase ENTPD5 regulates ER stress-associated renal injury by mediating protein N-glycosylation

Impaired protein N-glycosylation leads to the endoplasmic reticulum (ER) stress, which triggers adaptive survival or maladaptive apoptosis in renal tubules in diabetic kidney disease (DKD). Therapeutic strategies targeting ER stress are promising for the treatment of DKD. Here, we report a previously unappreciated role played by ENTPD5 in alleviating renal injury by mediating ER stress. We found that ENTPD5 was highly expressed in normal renal tubules; however, ENTPD5 was dynamically expressed in the kidney and closely related to pathological DKD progression in both human patients and mouse models. Overexpression of ENTPD5 relieved ER stress in renal tubular cells, leading to compensatory cell proliferation that resulted in hypertrophy, while ENTPD5 knockdown aggravated ER stress to induce cell apoptosis, leading to renal tubular atrophy and interstitial fibrosis. Mechanistically, ENTPD5-regulated N-glycosylation of proteins in the ER to promote cell proliferation in the early stage of DKD, and continuous hyperglycemia activated the hexosamine biosynthesis pathway (HBP) to increase the level of UDP-GlcNAc, which driving a feedback mechanism that inhibited transcription factor SP1 activity to downregulate ENTPD5 expression in the late stage of DKD. This study was the first to demonstrate that ENTPD5 regulated renal tubule cell numbers through adaptive proliferation or apoptosis in the kidney by modulating the protein N-glycosylation rate in the ER, suggesting that ENTPD5 drives cell fate in response to metabolic stress and is a potential therapeutic target for renal diseases.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90β expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

Downregulated mitochondrial transcription factor A enhances mycoplasma infection to promote the metastasis of hepatocellular carcinoma.

Mycoplasma is widespread in various hosts and may cause various diseases in animals. Interestingly, the occurrence of mycoplasma infection was observed in many tumor types. However, the mechanism regulating its infection is far from clear. We unexpectedly found that the knockdown of mitochondrial transcription factor A (TFAM) remarkably enhanced mycoplasma infection in hepatocellular carcinoma (HCC) cells. More importantly, we found that mycoplasma infection facilitated by TFAM knockdown significantly promoted HCC cell metastasis. Mycoplasma infection was further found to be positively correlated with poor prognosis in patients with HCC. Mechanistically, the decreased TFAM expression upregulated the transcription factor Sp1 to increase the expression level of Annexin A2 (ANXA2), which was reported to interact with membrane protein of mycoplasma. Moreover, we found that mycoplasma infection enhanced by the TFAM downregulation promoted HCC migration and invasion by activating the nuclear factor-κB signaling pathway. The downregulation of TFAM enhanced mycoplasma infection in HCC cells and promoted HCC cell metastasis. Our study contributes to the understanding of the pathological role of mycoplasma infection and provides supporting evidence that targeting TFAM could be a potential strategy for the treatment of HCC with mycoplasma infection.

Transcriptional activation of CSTB gene expression by transcription factor Sp3

CSTB has been reported to be associated with the pathogenesis of many malignant tumors, especially hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated is largely unknown. We initially cloned and analyzed the promoter region of the CSTB gene by luciferase assay and the Sp3 binding site (CCCCGCCCCGCG) was found in it. The results of electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments verified that the transcription factor, Sp3 could bind to the " CCCCGCCCCGCG ″ site of the CSTB gene promoter. We showed that the overexpression of Sp3 significantly increased the endogenous mRNA and protein expression levels of CSTB, whereas knockdown of Sp3 decreased the mRNA and protein expression levels according to quantitative real-time PCR (qRT‒PCR) and western blotting. In conclusion, CSTB gene expression is closely regulated by transcription factor Sp3, which may be a potential mechanism for the dysregulation of CSTB expression in HCC.

N-Acetyltransferase 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Proliferation by N-Terminal Acetylation of the Structural Protein GP5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the global swine industry. As a typical immunosuppressive virus, PRRSV has developed a variety of complex mechanisms to escape the host innate immunity. In this study, we uncovered a novel immune escape mechanism of PRRSV infection. Here, we demonstrate for the first time that the endoplasmic reticulum (ER)-resident N-acetyltransferase Nat9 is an important host restriction factor for PRRSV infection. Nat9 inhibited PRRSV proliferation in an acetyltransferase activity-dependent manner. Mechanistically, glycoprotein 5 (GP5) of PRRSV was identified as interacting with Nat9 and being N-terminally acetylated by it, which generates a GP5 degradation signal, promoting the K27-linked-ubiquitination degradation of GP5 to decrease virion assembly. Meanwhile, the expression of Nat9 was inhibited during PRRSV infection. In detail, two transcription factors, ETV5 and SP1, were screened out as the key transcription factors binding to the core promoter region of Nat9, and the PRRSV nonstructural protein 1β (Nsp1β), Nsp4, Nsp9, and nucleocapsid (N) proteins were found to interfere significantly with the expression of ETV5 and SP1, thereby regulating the transcription activity of Nat9 and inhibiting the expression of Nat9. The findings suggest that PRRSV decreases the N-terminal acetylation of GP5 to support virion assembly by inhibiting the expression of Nat9. Taken together, our findings showed that PRRSV has developed complex mechanisms to inhibit Nat9 expression and trigger virion assembly. IMPORTANCE To ensure efficient replication, a virus must hijack or regulate multiple host factors for its own benefit. Understanding virus-host interactions and the molecular mechanisms of host resistance to PRRSV infection is necessary to develop effective strategies to control PRRSV. The N-acetyltransferase Nat9 plays important roles during virus infection. Here, we demonstrate that Nat9 exhibits an antiviral effect on PRRSV proliferation. The GP5 protein of PRRSV is targeted specifically by Nat9, which mediates GP5 N-terminal acetylation and degradation via a ubiquitination-dependent proteasomal pathway. However, PRRSV manipulates the transcription factors ETV5 and SP1 to inhibit the expression of Nat9 and promote virion assembly. Thus, we report a novel function of Nat9 in PRRSV infection and elucidate a new mechanism by which PRRSV can escape the host innate immunity, which may provide novel insights for the development of antiviral drugs.

GPX4 utilization by selenium is required to alleviate cadmium-induced ferroptosis and pyroptosis in sheep kidney.

Cadmium (Cd), a persistent and harmful heavy metal in the environment, can accumulate in the kidneys and cause nephrotoxicity. Selenium (Se) is a beneficial natural element that alleviates the toxicity of Cd. To ascertain the relationship between the protective mechanism of Se against Cd nephrotoxicity and ferroptosis and pyroptosis, we randomly divided 48 sheep into four groups and treated them with Cd chloride and/or sodium selenite for 50 days. The data confirmed that Cd apparently resulted in impaired kidney histology and function, depletion of GSH and nicotinamide adenine dinucleotide phosphate contents and CAT and SOD activities, elevation of MDA level, as well as the reduction in selenoprotein mRNA (GPX1, GPX4, TXNRD1, SELP) levels and GPX4 protein level and immunofluorescence intensity. Meanwhile, Cd induced ferroptosis by causing iron overload, up-regulating PTGS2, NCOA4, TFR1, and LC3B mRNA levels and PTGS2 and LC3B-II/LC3B-I protein levels, reducing SLC7A11 and FTH1 mRNA and protein levels, and enhancing the immunofluorescence co-localization of FTH1/LC3B. Moreover, it was also found that Cd triggered pyroptosis, which was evidenced by the increase of NLRP3 immunohistochemical positive signal, GSDMD-N immunofluorescence intensity, IL-1β and IL-18 release and the levels of pyroptosis-related mRNA (NLRP3, ASC, Caspase-1, GSDMD, IL-1β and IL-18) and proteins (NLRP3, Caspase-1p20, GSDMD-N, IL-1β and IL-18). Notably, Se increased the expression level of GPX4 and the transcription factors TFAP2c and SP1, and ameliorated Cd-induced changes in aforementioned factors. In conclusion, GPX4 utilization by Se might be required to alleviate Cd-induced ferroptosis and pyroptosis in sheep kidney.

Midline 1 interacting protein 1 promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in HCC.

Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis. Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities. Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression.

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1)in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.

G protein inhibitory α subunit 2 is a molecular oncotarget of human glioma.

Identification of novel therapeutic oncotargets for human glioma is extremely important. Here we tested expression, potential functions and underlying mechanisms of G protein inhibitory α subunit 2 (Gαi2) in glioma. Bioinformatics analyses revealed that Gαi2 expression is significantly elevated in human glioma, correlating with poor patients' survival, higher tumor grade and wild-type IDH status. Moreover, increased Gαi2 expression was also in local glioma tissues and different glioma cells. In primary and immortalized (A172) glioma cells, Gαi2 shRNA or knockout (KO, by Cas9-sgRNA) potently suppressed viability, proliferation, and mobility, and induced apoptosis. Ectopic Gαi2 overexpression, using a lentiviral construct, further augmented malignant behaviors in glioma cells. p65 phosphorylation, NFκB activity and expression of NFκB pathway genes were decreased in Gαi2-depleted primary glioma cells, but increased following Gαi2 overexpression. There was an increased binding between Gαi2 promoter and Sp1 (specificity protein 1) transcription factor in glioma tissues and different glioma cells. In primary glioma cells Gαi2 expression was significantly reduced following Sp1 silencing, KO or inhibition. In vivo studies revealed that Gαi2 shRNA-expressing AAV intratumoral injection hindered growth of subcutaneous glioma xenografts in nude mice. Moreover, Gαi2 KO inhibited intracranial glioma xenograft in nude mice. Gαi2 depletion, NFκB inhibition and apoptosis induction were observed in subcutaneous and intracranial glioma xenografts with Gαi2 depletion. Together, overexpressed Gαi2 is important for glioma cell growth possibly by promoting NFκB cascade activation.

The Cancer/Testis Antigen CT45A1 Promotes Transcription of Oncogenic Sulfatase-2 Gene in Breast Cancer Cells and Is Sensible Targets for Cancer Therapy.

Invasive breast carcinomas (BRCAs) are highly lethal. The molecular mechanisms underlying progression of invasive BRCAs are unclear, and effective therapies are highly desired. The cancer-testis antigen CT45A1 promotes overexpression of pro-metastatic sulfatase-2 (SULF2) and breast cancer metastasis to the lungs, but its mechanisms are largely unknown. In this study, we aimed to elucidate the mechanism of CT45A1-induced SULF2 overexpression and provide evidence for targeting CT45A1 and SULF2 for breast cancer therapy. The effect of CT45A1 on SULF2 expression was assessed using reverse transcription polymerase chain reaction and western blot. The mechanism of CT45A1-induced SULF2 gene transcription was studied using protein-DNA binding assay and a luciferase activity reporter system. The interaction between CT45A1 and SP1 proteins was assessed using immunoprecipitation and western blot. Additionally, the suppression of breast cancer cell motility by SP1 and SULF2 inhibitors was measured using cell migration and invasion assays. CT45A1 and SULF2 are aberrantly overexpressed in patients with BRCA; importantly, overexpression of CT45A1 is closely associated with poor prognosis. Mechanistically, gene promoter demethylation results in overexpression of both CT45A1 and SULF2. CT45A1 binds directly to the core sequence GCCCCC in the promoter region of SULF2 gene and activates the promoter. Additionally, CT45A1 interacts with the oncogenic master transcription factor SP1 to drive SULF2 gene transcription. Interestingly, SP1 and SULF2 inhibitors suppress breast cancer cell migration, invasion, and tumorigenicity. Overexpression of CT45A1 is associated with poor prognosis in patients with BRCA. CT45A1 promotes SULF2 overexpression by activating the promoter and interacting with SP1. Additionally, SP1 and SULF2 inhibitors suppress breast cancer cell migration, invasion, and tumorigenesis. Our findings provide new insight into the mechanisms of breast cancer metastasis and highlight CT45A1 and SULF2 as sensible targets for developing novel therapeutics against metastatic breast cancer.

Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

Specificity Protein 1-Mediated Promotion of CXCL12 Advances Endothelial Cell Metabolism and Proliferation in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a rare yet devastating and incurable disease with few treatment options. The underlying mechanisms of PAH appear to involve substantial cellular proliferation and vascular remodeling, causing right ventricular overload and eventual heart failure. Recent evidence suggests a significant seminal role of the pulmonary endothelium in the initiation and promotion of PAH. Our previous work identified elevated reactive oxygen species (ROS)-producing enzyme NADPH oxidase 1 (NOX1) in human pulmonary artery endothelial cells (HPAECs) of PAH patients promoting endothelial cell proliferation in vitro. In this study, we interrogated chemokine CXCL12's (aka SDF-1) role in EC proliferation under the control of NOX1 and specificity protein 1 (Sp1). We report here that NOX1 can drive hypoxia-induced endothelial CXCL12 expression via the transcription factor Sp1 leading to HPAEC proliferation and migration. Indeed, NOX1 drove hypoxia-induced Sp1 activation, along with an increased capacity of Sp1 to bind cognate promoter regions in the CXCL12 promoter. Sp1 activation induced elevated expression of CXCL12 in hypoxic HPAECs, supporting downstream induction of expression at the CXCL12 promoter via NOX1 activity. Pathological levels of CXCL12 mimicking those reported in human PAH patient serum restored EC proliferation impeded by specific NOX1 inhibitor. The translational relevance of our findings is highlighted by elevated NOX1 activity, Sp1 activation, and CXCL12 expression in explanted lung samples from PAH patients compared to non-PAH controls. Analysis of phosphofructokinase, glucose-6-phosphate dehydrogenase, and glutaminase activity revealed that CXCL12 induces glutamine and glucose metabolism, which are foundational to EC cell proliferation. Indeed, in explanted human PAH lungs, demonstrably higher glutaminase activity was detected compared to healthy controls. Finally, infusion of recombinant CXCL12 into healthy mice amplified pulmonary arterial pressure, right ventricle remodeling, and elevated glucose and glutamine metabolism. Together these data suggest a central role for a novel NOX1-Sp1-CXCL12 pathway in mediating PAH phenotype in the lung endothelium.

NF-κB1 p50 stabilizes HIF-1α protein through suppression of ATG7-dependent autophagy

The function and underlying mechanisms of p50 in the regulation of protein expression is much less studied because of its lacking of transactivation domain. In this study, we discovered a novel function of p50 in its stabilization of hypoxia-inducible factor 1α (HIF-1α) protein under the condition of cells exposed to arsenic exposure. In p50-deficient (p50−/−) cells, the HIF-1α protein expression was impaired upon arsenic exposure, and such defect could be rescued by reconstitutional expression of p50. Mechanistic study revealed that the inhibition of autophagy-related gene 7 (ATG7)-dependent autophagy was in charge of p50-mediated HIF-1α protein stabilization following arsenic exposure. Moreover, p50 deletion promoted nucleolin (NCL) protein translation to enhance ATG7 mRNA transcription via directly binding transcription factor Sp1 mRNA and increase its stability. We further discovered that p50-mediated miR-494 upregulation gave rise to the inhibition of p50-mediated NCL translation by interacting with its 3’-UTR. These novel findings provide a great insight into the understanding of biomedical significance of p50 protein in arsenite-associated disease development and therapy.

Identification of 5’ upstream sequence involved in HSPBP1 gene transcription and its downregulation during HIV-1 infection

The Human Immunodeficiency Virus-1 (HIV-1) is known to modulate the host environment for successful replication and propagation like other viruses. The virus utilises its proteins to interact with or modulate host factors and host signalling pathways that may otherwise restrict the virus. A previous study from our lab has shown that the host heat shock protein 70 (HSP70) binding protein (HSPBP1) is a co-chaperone that inhibits viral replication. We have also shown that the virus downregulates HSPBP1 during infection. However, the mechanism of downregulation remains to be elucidated. In the present study, we hypothesized that the HSPBP1 promoter may be repressed during infection leading to its downmodulation at the RNA and protein levels. The 5’ upstream region of the HSPBP1 gene was first mapped and it was identified that a fragment comprising of a ∼600 bp upstream region of the transcription start site show the highest promoter-like activity. Further, the Sp1 transcription factor was shown to be essential for normal promoter activation. Our results further demonstrate that HIV-1 downregulates the activity of the identified promoter. It was seen that the viral transactivator protein, Tat, was responsible for the downmodulation of the HSPBP1 promoter. HIV-1 Tat is known to bind and regulate several cellular promoters during infection, thereby making the environment conducive for establishment of the virus. Our results further show that Tat is recruited to the HSPBP1 promoter and in the presence of Tat, recruitment of Sp1 on HSPBP1 promoter was decreased, which explains the suppression of HSPBP1 during HIV-1 infection. Therefore, this study further adds to the list of cellular promoters that are modulated by Tat during HIV-1 infection either directly or indirectly.

Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma

Gliomas are highly aggressive intracranial tumors that are difficult to resect and have high lethality and recurrence rates. According to WHO grading criteria, glioblastoma with wild-type IDH1 has a poorer prognosis than WHO grade 4 IDH-mutant astrocytomas. To date, no effective therapeutic strategies have been developed to treat glioblastoma. Clinical trials have shown that herpes simplex virus (HSV)-1 is the safest and most efficacious oncolytic virus against glioblastoma, but the molecular antitumor mechanism of action of HSV-1 has not yet been determined. Deletion of the γ34.5 and ICP47 genes from a strain of HSV-1 yielded the oncolytic virus, oHSV-1, which reduced glioma cell viability, migration, and invasive capacity, as well as the growth of microvilli. Infected cell polypeptide 4 (ICP4) expressed by oHSV-1 was found to suppress the expression of the transcription factor Sp1, reducing the expression of host invasion-related genes. In vivo, oHSV-1 showed significant antitumor effects by suppressing the expression of Sp1 and invasion-associated genes, highly expressed in high-grade glioblastoma tissue specimens. These findings indicate that Sp1 may be a molecular marker predicting the antitumor effects of oHSV-1 in the treatment of glioma and that oHSV-1 suppresses host cell invasion through the ICP4-mediated downregulation of Sp1.

Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells.

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

Deep learning for MYC binding site recognition.

Motivation: The definition of the genome distribution of the Myc transcription factor is extremely important since it may help predict its transcriptional activity particularly in the context of cancer. Myc is among the most powerful oncogenes involved in the occurrence and development of more than 80% of different types of pediatric and adult cancers. Myc regulates thousands of genes which can be in part different, depending on the type of tissues and tumours. Myc distribution along the genome has been determined experimentally through chromatin immunoprecipitation This approach, although powerful, is very time consuming and cannot be routinely applied to tumours of individual patients. Thus, it becomes of paramount importance to develop in silico tools that can effectively and rapidly predict its distribution on a given cell genome. New advanced computational tools (DeeperBind) can then be successfully employed to determine the function of Myc in a specific tumour, and may help to devise new directions and approaches to experiments first and personalized and more effective therapeutic treatments for a single patient later on. Results: The use of DeeperBind with DeepRAM on Colab platform (Google) can effectively predict the binding sites for the MYC factor with an accuracy above 0.96 AUC, when trained with multiple cell lines. The analysis of the filters in DeeperBind trained models shows, besides the consensus sequence CACGTG classically associated to the MYC factor, also the other consensus sequences G/C box or TGGGA, respectively bound by the SP1 and MIZ-1 transcription factors, which are known to mediate the MYC repressive response. Overall, our findings suggest a stronger synergy between the machine learning tools as DeeperBind and biological experiments, which may reduce the time consuming experiments by providing a direction to guide them.