Molecular characterization of the SP3a gene, a negative regulator of viral infection in the orange-spotted grouper, Epinephelus coioides.

SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry.

Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters.

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

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.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Caspase-Mediated Cleavage of the Transcription Factor Sp3: Possible Relevance to Cancer and the Lytic Cycle of Kaposi's Sarcoma-Associated Herpesvirus.

The open reading frame 50 (ORF50) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the master regulator essential for initiating the viral lytic cycle. Previously, we have demonstrated that the ORF50 protein can cooperate with Sp3 to synergistically activate a set of viral and cellular gene promoters through highly conserved ORF50-responsive elements that harbor a Sp3-binding motif. Herein, we show that Sp3 undergoes proteolytic cleavage during the viral lytic cycle, and the cleavage of Sp3 is dependent on caspase activation. Since similar cleavage patterns of Sp3 could be detected in both KSHV-positive and KSHV-negative lymphoma cells undergoing apoptosis, the proteolytic cleavage of Sp3 could be a common event during apoptosis. Mutational analysis identifies 12 caspase cleavage sites in Sp3, which are situated at the aspartate (D) positions D17, D19, D180, D273, D275, D293, D304 (or D307), D326, D344, D530, D543, and D565. Importantly, we noticed that three stable Sp3 C-terminal fragments generated through cleavage at D530, D543, or D565 encompass an intact DNA-binding domain. Like the full-length Sp3, the C-terminal fragments of Sp3 could still retain the ability to cooperate with ORF50 protein to activate specific viral and cellular gene promoters synergistically. Collectively, our findings suggest that despite the proteolytic cleavage of Sp3 under apoptotic conditions, the resultant Sp3 fragments may retain biological activities important for the viral lytic cycle or for cellular apoptosis. IMPORTANCE The ORF50 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3. In this report, we show that Sp3 is proteolytically cleaved during the viral lytic cycle, and up to 12 caspase cleavage sites are identified in Sp3. Despite the proteolytic cleavage of Sp3, several resulting C-terminal fragments that have intact zinc-finger DNA-binding domains still retain substantial influence in the synergy with ORF50 to activate specific gene promoters. Overall, our studies elucidate the caspase-mediated cleavage of Sp3 and uncover how ORF50 utilizes the cleavage fragments of Sp3 to transactivate specific viral and cellular gene promoters.

Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Editing of DNA methylation using CRISPR/Cas9 and a ssDNA template in human cells.

Programmable DNA methylation is required for understanding of transcriptional regulation and elucidating gene functions. We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells. ssDNA-mediated knock-in has recently been reported to completely reduce random integrations. We speculated that by changing MMEJ-to ssDNA-based knock-in, targeted DNA methylation may be achieved through a hemimethylation-symmetric methylation pathway. We herein successfully developed a new system that enables the replacement of an unmethylated promoter with a methylated ssDNA promoter through ssDNA-based knock-in. A DNA methylation ratio of approximately 100% was achieved at the cancer-associated gene SP3 in HEK293 cells. The present results provide a promising framework for artificial epigenetic modifications.

miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche.

Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular vesicles (sEVs) play key roles in intercellular communication. Specific sEV-miRNAs from several types of cancer were found to induce a premetastatic niche in target organs before tumor cell arrive. However, whether the primary GC affects hepatic microenvironment or the role of sEV-miRNAs in GC-LM is yet unclear. We report that GC-derived sEVs are primarily absorbed by Kupffer cells (KCs). sEV-miR-151a-3p is highly expressed in GC-LM patients' plasma and presents poor prognosis. Treating mice with sEVs-enriched in miR-151a-3p promotes GC-LM, whereas has no influence on the proliferation of GC cells in situ. Mechanistically, sEV-miR-151a-3p inhibits SP3 in KCs. Simultaneously, sEV-miR-151a-3p targets YTHDF3 to decrease the transcriptional inhibitory activity of SP3 by reducing SUMO1 translation in a N6-methyladenosine-dependent manner. These factors contribute to TGF-ß1 transactivation in KCs, subsequently activating the SMAD2/3 pathway and enhancing the stem cell-like properties of incoming GC cells. Furthermore, sEV-miR-151a-3p induces miR-151a-3p transcription in KCs to form a positive feedback loop. In summary, our results reveal a previously unidentified regulatory axis initiated by sEV-miR-151a-3p that establishes a hepatic stemness-permissive niche to support GC-LM. sEV-miR-151a-3p could be a promising diagnostic biomarker and preventive treatment candidate for GC-LM.

Amsp3 may act upstream of Amdnmt3 in female caste differentiation in the honeybee (Apis mellifera).

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

lncRNA UCA1 induced by SP1 and SP3 forms a positive feedback loop to facilitate malignant phenotypes of colorectal cancer via targeting miR-495.

AIMS: Long noncoding RNA (LncRNA) urothelial cancer associated 1 (UCA1) was dysregulated in colorectal cancers (CRC) and promoted tumor progression of CRC. The aims of this study are to further investigate the underlying mechanism. MAIN METHODS: Short hairpin RNAs (shRNAs) were applied for gene knockdown. microRNA mimic and pcDNA-UCA1 plasmids were transfected for miR-495 and UCA1 overexpression, respectively. MTT was applied to determine cell viability and sensitivity of 5-fluorouracil (FU). Transwell assays were performed to evaluate cell migration/invasion. Angiogenesis was evaluated by tube formation. Western blotting and quantitative PCR were utilized for protein and mRNA detection, respectively. The interaction of UCA1, miR-495 and SP1/SP3 were explored by dual-luciferase assay. RNA pulldown was adopted to determine the UCA1/miR-495 interaction. KEY FINDINGS: UCA1 was significantly upregulated in CRC tissues. UCA1 enhanced cell proliferation, migration/invasion, angiogenesis, epithelial-mesenchymal transition, and resistance to 5-FU in CRC cell lines. MiR-495 was inversely correlated to the expression of UCA1. The results indicated that UCA1 sponged miR-495, leading to the disinhibition of SP1/SP3 expression. SP1/SP3 induced the expression of DNA methyltransferases and, in turn, contributed to UCA1 mediated tumor-promoting actions. Reduction of SP1/SP3 exerted anti-cancer effects, which can be reversed by forced expression of UCA1. SIGNIFICANCE: UCA1-miR-495-SP1/SP3 axis is dysregulated in CRC and contributed to malignant phenotypes of CRC. UCA1-SP1/SP3 may form a positive feedback loop in CRC.

Phosphorylation of Specificity Protein 3 Is Critical for Activation of ß4-Galactosyltransferase 3 Gene Promoter in SH-SY5Y Human Neuroblastoma Cell Line.

Elevated expression of ß4-galactosyltransferase (ß4GalT) 3 is correlated with poor clinical outcome of neuroblastoma patients. Our recent study has revealed that the transcription of the ß4GalT3 gene is activated by Specificity protein (Sp) 3 in SH-SY5Y human neuroblastoma cell line. Here we report the biological significance of the Sp3 phosphorylation in the transcriptional activation of the ß4GalT3 gene. The treatment of SH-SY5Y cells with 10% fetal bovine serum (FBS) increased the mitogen-activated protein kinase (MAPK) signaling and the promoter activity of the ß4GalT3 gene. Meanwhile, the treatment with U0126, an inhibitor for MAPK kinase, decreased the MAPK signaling and the promoter activity. These findings indicate that the transcriptional activation of the ß4GalT3 gene is mediated by the MAPK signaling. In SH-SY5Y cells cultured in the medium containing 10% FBS, the serine (Ser) residues in Sp3 were phosphorylated. Human Sp3 contains four Ser residues, Ser73, Ser563, Ser566, and Ser646, as the putative phosphorylation sites. Sp3 mutant with the mutation of Ser73 did not decrease the promoter activation of the ß4GalT3 gene, indicating that Ser73 is uninvolved in the promoter activation of the ß4GalT3 gene by Sp3. In contrast, Sp3 mutants with the mutations of Ser563, Ser566, and Ser646 significantly reduced the promoter activation by Sp3. The results suggest that the phosphorylation of these Ser residues is implicated in the promoter activation by Sp3. This study demonstrates that the phosphorylation of Sp3 plays important roles in the transcriptional activation of the ß4GalT3 gene in human neuroblastoma.

The specificity protein 3 (SP3) gene in ducks (Anas platyrhynchos): cloning, characterization and expression during viral infection.

Specificity Protein 3 (SP3) is a newly identified regulator of tumor growth and invasiveness in humans. In this study, we identified and characterized the function of duck SP3 (duSP3). The full-length cDNA sequence of the duSP3 gene was cloned via rapid amplification of cDNA ends. It contained 2468 nucleotides, including a 111 base pair (bp) 5'-untranslated region (UTR), 215 bp 3'-UTR, and 2142 bp open reading frame (ORF), which encoded a 713 amino acid (AA) strongly conserved with Avian SP3. Tissue specificity analysis demonstrated that duSP3 was constitutively expressed in the eight tissues tested: liver, spleen, lung, heart, kidney, thymus, breast, and leg; and low expression levels were observed in all tissues, except the spleen and thymus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that duSP3 expression rapidly increased in vitro after stimulation with both the hepatitis virus (DHV-1) and polyriboinosinic polyribocytidylic acid (poly(I:C)). However, the expression under these treatments varied in kidney and liver tissues; in the liver, duSP3 increased significantly at 36 h after the DHV-1 treatment and peaked at 72 h after poly(I:C) stimulation. These results suggested that SP3 may play a positive role in immune responses against viral infections in ducks.

SP3 is associated with migration, invasion, and Akt/PKB signalling in MDA-MB-231 breast cancer cells.

Specificity proteins (SPs) have pro-oncogenic functions in cancer cells, ranging from cancer cell proliferation, migration, invasion, and angiogenesis. There is strong evidence that several antineoplastic drugs target depletion of SP proteins via different pathways. However, the mode of action of SP3 and the underlying consequences of its depletion are not well understood. Here, we demonstrate that SP3 is overexpressed in invasive breast cancer cells vs normal counterparts. The gene expression analysis from The Cancer Genome Atlas datasets indicated that SP3 is strongly correlated with Akt signalling-related proteins, G protein subunit alpha 13, and RAB33B (RAB33B, member RAS oncogene family). RNA interference of SP3 decreased active phosphorylation of Akt at serine and threonine sites. These findings indicate that SP3 exhibits a pro-oncogenic function, which clearly fits the description of an nononcogene addiction gene. Future analyses are prompted to uncover the SP3 gene regulation function and to reveal downstream targets of SP3 in breast cancer.

Estrogen-regulated expression of SK3 channel in rat colonic smooth muscle contraction.

AIMS: Estrogen can induce inhibition of colonic smooth muscle contraction in male and female mice, which may lead to constipation; however, the mechanisms of inhibition are poorly understood. Hence, this study investigated the effect of estrogen on rat colonic smooth muscle contraction and role of small-conductance Ca2+-activated K+ 3 (SK3) and transcription factors (Sp1 and Sp3) in the underlying mechanisms. MAIN METHODS: The experiment included 24 female Sprague-Dawley (SD) rats divided into 4 groups. The rats were oophorectomized surgically, and a silicone tube containing blank solvent, 0.3 mg/mL estrogen (E2), equal-concentration of estrogen and estrogen receptor antagonist (EI), and bovine serum albumin-E2 (BSA-E2) was implanted. The rats were sacrificed on day 14. The molecular insights were confirmed using real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analyses to determine the effect of estrogenic stimulation on gene and protein expression analyses, respectively. KEY FINDINGS: The E2 group showed significantly greater SK3 expression (P < .005) compared with other groups and significantly lowers smooth muscle cell (SMC) contractility (P < .005). Estrogen stimulation and SK3 overexpression resulted in a significant decrease (P < .05) in Ca2+ mobilization in the E2 group versus the control group. Further, the E2 group showed significantly higher Sp1 mRNA (P < .05) but lower Sp3 mRNA expression (P < .05) and protein expression (P < .001) compared with other groups. SIGNIFICANCE: E2 may promote SK3 expression by its genomic effect and inhibit colonic contraction by affecting SK3 expression via an interaction between Sp1 and Sp3.

Inhibition of Specificity Protein 1 Is Involved in Phloretin-Induced Suppression of Prostate Cancer.

Phloretin is a flavonoid existed in various plants and has been reported to possess anticarcinogenic activity. However, the anticancer mechanism of phloretin in prostate cancer (PCa) remains unclear. Here, our in vitro and in vivo experimental data demonstrate that phloretin inhibits the phosphorylation and the activation of EGFR and then inhibits its downstream PI3K/AKT and MEK/ERK1/2 pathways in PCa cells. Inhibition of these two pathways further decreases expression of Sp1 by inhibiting Sp1 gene transcription, induces degradation of Sp1 protein by inhibiting GSK3ß phosphorylation, suppresses nucleolin-enhanced translation of Sp1 mRNA by inhibiting nucleolin phosphorylation, and directly inactivates transcription activity of Sp1. Inhibition of Sp1 subsequently decreases the expression of Sp3/4, VEGF, and Survivin and then upregulates apoptosis-related proteins and downregulates cell cycle-related proteins in PCa cells. Finally, phloretin treatment in PCa cells induces cell growth inhibition and apoptosis, suggesting that phloretin may be an effective therapy compound in the treatment of prostate cancer.

Sp3 Transcription Factor Cooperates with the Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein To Synergistically Activate Specific Viral and Cellular Gene Promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.

ZEB1 Mediates Fibrosis in Corneal Endothelial Mesenchymal Transition Through SP1 and SP3.

Purpose: ZEB1 is induced during endothelial-mesenchymal transition (EnMT) in the cornea. Induction of SP1 and SP3 by ZEB1 along with identification of putative SP1 and SP3 binding sites in promoters of EnMT-associated gene lead us to investigate their roles in retrocorneal membrane formation in the corneal endothelium. Methods: Expressions of SP1, SP3, and EnMT associated genes were analyzed by immunoblotting and semiquantitative reverse transcription polymerase chain reaction. Accell SMARTpool siRNAs targeting ZEB1, SP1, and SP3 were used for gene knockdown. SP1 and SP3 binding to promoters of EnMT associated genes was investigated by chromatin immunoprecipitation assay. Corneal endothelium in mice was surgically injured in vivo under direct visualization. Results: Transient Fibroblast Growth Factor 2 stimulation increased the expression of both SP1 and SP3 in the human corneal endothelium ex vivo. ZEB1 siRNA knockdown inhibited FGF2-induced SP1 mRNA and protein but not the expression of SP3. FGF2-induced expression of EnMT-related genes, such as fibronectin, vimentin, and type I collagen, was reduced by both SP1 and SP3 siRNA knockdown, with inhibition of SP1 having a greater inhibitory effect than SP3. Additionally, although SP1 and SP3 proteins were found to bind together, SP1 and SP3 could bind to the same promoter binding sites of EnMT-related genes in the absence of the other. Moreover, siRNA knockdown of Zeb1 inhibited injury-dependent RCM formation in mouse corneal endothelium in vivo. Conclusions: Zeb1, through SP1 and SP3, plays a central role in mesenchymal transition induced fibrosis in the corneal endothelium and suggests that Zeb1 could be targeted to inhibit anterior segment fibrosis.

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin.

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

The transcription factor SP3 drives TNF-α expression in response to Smac mimetics.

The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.