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Transcription factors bind specific sequences upstream of the 5′ end of their target genes to ensure proper spatiotemporal expression of the target gene. This study aims to demonstrate that the transcription factor SP2 regulates expression of the Ski gene, which has specific binding sites for SP2, and thus enables Ski to regulate astrocyte proliferation. The upstream regulation mechanism of astrocyte proliferation was explored to further regulate the formation of glial scar in specific time and space after spinal cord injury. JASPAR and UCSC databases were used to predict transcription factor binding and the threshold was gradually reduced to screen transcription factors upstream of Ski, leading to the identification of SP2. Next, we analyzed the correlation between the expression of SP2 and Ski in normal astrocytes and reactive astrocytes, as well as the changes in astrocyte proliferation. To confirm that SP2 regulates Ski during astrocyte proliferation, astrocytes were transfected siRNA targeting SP2 and then astrocyte proliferation were analyzed. Finally, a dual luciferase reporter assay and Chromatin immunoprecipitation (ChIP) assay confirmed that the promoter region of Ski contained a specific SP2 binding site. This is the first that SP2 has been identified and confirmed to play an important role in astrocyte proliferation by regulating Ski expression. These results may help identify novel targets for the treatment of spinal cord injury.

Studies have found that RNA-binding proteins (RBPs) are dysfunctional and play a significant regulatory role in the development of glioma. Based on The Cancer Genome Atlas database and the previous studies, we selected heterogeneous nuclear ribonucleoprotein (HNRNPD) as the research candidate and sought its downstream targeted genes. In the present study, HNRNPD, linc00707, and specific protein 2 (SP2) were highly expressed, while zinc fingers and homeboxes 2 (ZHX2) and miR-651-3p were remarkedly downregulated in glioma tissues and cells. HNRNPD, linc00707, and SP2 knockdown or ZHX2 and miR-651-3p overexpression suppressed glioma cells proliferation, migration, and invasion and vasculogenic mimicry (VM) formation. Knockdown of HNRNPD increased the stability of ZHX2 mRNA. ZHX2 bound to the promoter region of linc00707 and negatively regulate its expression. Linc00707 could bind with miR-651-3p, while miR-651-3p bound to the 3′ untranslated region (3′UTR) of SP2 mRNA to negatively regulate its expression. The transcription factor SP2 directly bound to the promoter regions of the VM formation-related proteins MMP2, MMP9, and VE-cadherin, playing a role in promoting transcription in order to regulate the VM formation ability of glioma cells.

Objective To explore the biological function and molecular mechanism of Sp2 in hepatocellular carcinoma (HCC). Methods Tissue microarray immunohistochemistry and western blot were used to study the expression of Sp2 in hepatocellular tissue and adjacent non‐neoplastic tissues (ANT). In HCC cell lines, the role of Sp2 was determined by in vitro experiments such as CCK8, clone formation test, Transwell assay, wound‐healing assay, and flow cytometry apoptotic analysis, and its possible mechanism was analyzed. Results Compared with ANT, Sp2 expression in HCC tissues was significantly up‐regulated, which was strongly associated with stage of tumor and poor prognosis of patients. TCGA database were further confirmed these results. Besides, functional studies had shown that Sp2 knockdown not only leads to a decrease in cell proliferation and an increase in cell apoptosis but also inhibits the cells' abilities of migration and invasion. Sp2 silencing could inhibit the expression of TRIB3 protein and down‐regulate the endoplasmic reticulum stress (ERS) level of HCC. Conclusion Sp2 may play a part in promoting cancer by regulating TRIB3 protein, which may be a factor of prognostic and a potential new therapeutic target for HCC., We found that Sp2 expression was significantly up‐regulated in HCC tissues, which was closely related to tumor staging and poor prognosis of patients. Functional studies had shown that Sp2 knockdown not only leads to a decrease in cell proliferation and an increase in cell apoptosis, but also inhibits migration and invasion of hepatoma cells. Sp2 may play a role in promoting cancer by regulating TRIB3 protein, which may be a prognostic factor and a potential new therapeutic target for HCC.

Somatic mutations and changes in expression of RAD21 are common in many types of cancer. Moreover, sub-optimal levels of RAD21 expression in early development can result in cohesinopathies. Altered RAD21 levels can result directly from mutations in the RAD21 gene. However, whether DNA variants outside of the RAD21 gene could control its expression and thereby contribute to cancer and developmental disease is unknown. In this study, we searched for genomic variants that modify RAD21expression to determine their potential to contribute to development or cancer by RAD21 dysregulation. We searched 42,953,834 genomic variants for a spatial-eQTL association with the transcription of RAD21. We identified 123 significant associations (FDR 0.05), which are local (cis) or long-distance (trans) regulators of RAD21 expression. The 123 variants co-regulate a further seven genes (AARD, AKAP11, GRID1, KCNIP4, RCN1, TRIOBP, and USP32), enriched for having Sp2 transcription factor binding sites in their promoter regions. The Sp2 transcription factor and six of the seven genes had previously been associated with cancer onset, progression, and metastasis. Our results suggest that genome-wide variation in non-coding regions impacts on RAD21 transcript levels in addition to other genes, which then could impact on oncogenesis and the process of ubiquitination. This identification of distant co-regulation of oncogenes represents a strategy for discovery of novel genetic regions influencing cancer onset and a potential for diagnostics.

Mutations and changes in expression in RAD21 are common across cancers types and outside of cancer can result in cohesinopathy. As such, exploration of variants that modify RAD21 enhancer activity, across the genome, may also provide insights into mechanisms by which distinct variants impact healthy human development and disease. We searched 42,953,834 genomic variants for a spatial-eQTL association with the transcription of RAD21. We identified 123 significant associations (FDR < 0.05), which are local (cis) or long-distance (trans) regulators of RAD21 expression. The 123 variants co-regulate a further seven genes, enriched for having Sp2 transcription factor binding sites in their promoter regions. The Sp2 transcription factor and six of the seven genes had previously been associated with cancer onset, progression, and metastasis. Our results suggest that genome-wide variation in non-coding regions impacts on RAD21 transcript levels in addition to other genes, which then could impact on oncogenesis and the process of ubiquitination. This identification of distant co-regulation of oncogenes represents a strategy for discovery of novel genetic regions which impact cancer onset and a potential for diagnostics.

Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195-5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-β pathway. However, the transcriptional regulation of miR-195-5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195-5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195-5p. Cotransfection of SP2 shRNA and a miR-195-5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195-5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195-5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.

Background: Mitochondria-nuclear cross-talk and mitochondrial retrograde regulation are involved in the genesis and development of breast cancer (BC). Therefore, mitochondria can be regarded as a promising target for BC therapeutic strategies. The present study aimed to construct regulatory network and seek the potential biomarkers of BC diagnosis and prognosis as well as the molecular therapeutic targets from the perspective of mitochondrial dysfunction. Methods: The microarray data of mitochondria-related encoding genes in BC cell lines were downloaded from GEO including GSE128610 and GSE72319. GSE128610 was treated as test set and validation sets consisted of GSE72319 and TCGA tissue samples, intending to identify mitochondria-related differentially expressed genes (mrDEGs). We performed enrichment analysis, PPI network, hub mrDEGs and overall survival analysis and constructed transcription factor (TF)-miRNA-hub mrDEGs network. Results: A total of 23 up-regulated and 71 down-regulated mrDEGs were identified and validated in BC cell lines and tissues. Enrichment analyses indicated that mrDEGs were associated with several cancer-related biological processes. Moreover, 9 hub mrDEGs were identified and validated in BC cell lines and tissues. Finally, 5 hub coregulated mrDEGs, 21 miRNAs and 117 TFs were used to construct TF-miRNA-hub mrDEGs network. MYC associated zinc finger protein (MAZ), heparin binding growth factor (HDGF) and Sp2 transcription factor (SP2) regulated 3 hub mrDEGs. Hsa-mir-21-5p, hsa-mir-1-3p, hsa-mir-218-5p, hsa-mir-26a-5p and hsa-mir-335-5p regulated 2 hub mrDEGs. Overall survival analysis suggested that the up-regulation of fibronectin 1 (FN1), as well as the down-regulation of discoidin domain receptor tyrosine kinase 2 (DDR2) correlated with unfavorable prognosis in BC. Conclusion: TF-miRNA-hub mrDEGs had instruction significance for the exploration of BC etiology. The hub mrDEGs such as FN1 and DDR2 were likely to regulate mitochondrial function and be novel biomarkers for BC diagnosis and prognosis as well as the therapeutic targets.

The transcriptomic profile associated with osteo- and adipogenic differentiation in growth-arrested multipotent mesenchymal stromal cells (MSCs) from human adipose tissue was analyzed in vitro at 20% (standard laboratory) and 5% (tissue-related) O

Cellular and molecular mechanisms underlying the switch from self-amplification of cortical stem cells to neuronal and glial generation are incompletely understood, despite their importance for neural development. Here, we have investigated the role of the transcription factor specificity protein 2 (Sp2) in expansive and neurogenic divisions of the developing cerebral cortex by combining conditional genetic deletion with the mosaic analysis with double markers (MADM) system in mice. We find that loss of Sp2 in progenitors undergoing neurogenic divisions results in prolonged mitosis due to extension of early mitotic stages. This disruption is correlated with depletion of the populations of upper layer neurons in the cortex. In contrast, early cortical neural stem cells proliferate and expand normally in the absence of Sp2. These results indicate a stage-specific requirement for Sp2 in neural stem and progenitor cells, and reveal mechanistic differences between the early expansive and later neurogenic periods of cortical development. This article has an associated ‘The people behind the papers’ interview.

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

Oligodendrocytes and Schwann cells are the myelinating glia of the vertebrate nervous system and by generation of myelin sheaths allow rapid saltatory conduction. Previous in vitro work had pointed to a role of the zinc finger containing specificity proteins Sp1 and Sp3 as major regulators of glial differentiation and myelination. Here, we asked whether such a role is also evident in vivo using mice with specific deletions of Sp1 or Sp3 in myelinating glia. We also studied glia-specific conditional Sp2- and constitutive Sp4-deficient mice to include all related glutamine-rich Sp factors into our analysis. Surprisingly, we did not detect developmental Schwann cell abnormalities in any of the mutant mice. Oligodendrocyte development and differentiation was also not fundamentally affected as oligodendrocytes were present in all mouse mutants and retained their ability to differentiate and initiate myelin gene expression. The most severe defect we observed was a 50% reduction in Mbp- and proteolipid protein 1 (Plp1)-positive differentiating oligodendrocytes in Sp2 mutants at birth. Unexpectedly, glial development appeared undisturbed even in the joint absence of Sp1 and Sp3. We conclude that Sp2 has a minor effect on the differentiation of myelinating glia, and that glutamine-rich Sp proteins are not essential regulators of the process.

Purpose Expression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer. Methods Expression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR. Results mRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3–9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue. Conclusions In ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3–9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers.

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2–promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding–independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.

Background/Aims: Overexpression of cytosolic sulfotransferase 2B1b (SULT2B1b) has been commonly found in colorectal and hepatocellular carcinoma, suggesting that SULT2B1b might act as a potential oncogenic protein. However, its clinical significance and biological role in gastric cancer progression remain largely unknown. Methods: Expressions of SULT2B1b in clinical gastric cancer (GC) samples were examined using qRT-PCR and Western blot. Results: SULT2B1b was markedly overexpressed in human GC samples, and positively correlated with vessel density and associated with poor clinical features. We also demonstrated that overexpression of SULT2B1b resulted in increased tumor angiogenesis and tumor growth in mouse GC models. In addition, ablation of SULT2B1b in human GC cells lines BGC823 and MKN45 decreased the capability of the cells to recruit endothelial cells. Moreover, depletion of SULT2B1b in GC cells reduced VEGF-A expression by downregulating SP1 and AP2. Conclusion: Our results suggested that the SULT2B1b-mediated angiogenic pathway could serve as biomarkers for GC diagnosis and prognosis, and suppressing SULT2B1b-mediated angiogenic signaling might be a promising strategy for developing novel GC treatment.

The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1–4 bound to IE62 and amino acids 238–258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner.

Although the search for quantitative trait loci for behaviour remains a considerable challenge, the complicated genetic architecture of quantitative traits is beginning to be understood. The current project utilised heterogeneous stock (HS) male mice (n = 580) to investigate the genetic basis for brain weights, activity, anxiety and cognitive phenotypes. We identified 126 single nucleotide polymorphisms (SNPs) in genes involved in regulation of neurotransmitter systems, nerve growth/death and gene expression, and subsequently investigated their associations with changes in behaviour and/or brain weights in our sample. We found significant associations between four SNP-phenotype pairs, after controlling for multiple testing. Specificity protein 2 (Sp2, rs3708840), tryptophan hydroxylase 1 (Tph1, rs262731280) and serotonin receptor 3A (Htr3a, rs50670893) were associated with activity/anxiety behaviours, and microtubule-associated protein 2 (Map2, rs13475902) was associated with cognitive performance. All these genes except for Tph1 were expressed in the brain above the array median, and remained significantly associated with relevant behaviours after controlling for the family structure. Additionally, we found evidence for a correlation between Htr3a expression and activity. We discuss our findings in the light of the advantages and limitations of currently available mouse genetic tools, suggesting further directions for association studies in rodents.

Faithful progression through the cell cycle is crucial to the maintenance and developmental potential of stem cells. Here, we demonstrate that neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs) employ a zinc-finger transcription factor specificity protein 2 (Sp2) as a cell cycle regulator in two temporally and spatially distinct progenitor domains. Differential conditional deletion of Sp2 in early embryonic cerebral cortical progenitors, and perinatal olfactory bulb progenitors disrupted transitions through G1, G2 and M phases, whereas DNA synthesis appeared intact. Cell-autonomous function of Sp2 was identified by deletion of Sp2 using mosaic analysis with double markers, which clearly established that conditional Sp2-null NSCs and NPCs are M phase arrested in vivo. Importantly, conditional deletion of Sp2 led to a decline in the generation of NPCs and neurons in the developing and postnatal brains. Our findings implicate Sp2-dependent mechanisms as novel regulators of cell cycle progression, the absence of which disrupts neurogenesis in the embryonic and postnatal brain.

Although the search for quantitative trait loci for behaviour remains a considerable challenge, the complicated genetic architecture of quantitative traits is beginning to be understood. The current project utilised heterogeneous stock (HS) male mice (n = 580) to investigate the genetic basis for brain weights, activity, anxiety and cognitive phenotypes. We identified 126 single nucleotide polymorphisms (SNPs) in genes involved in regulation of neurotransmitter systems, nerve growth/death and gene expression, and subsequently investigated their associations with changes in behaviour and/or brain weights in our sample. We found significant associations between four SNP-phenotype pairs, after controlling for multiple testing. Specificity protein 2 (Sp2, rs3708840), tryptophan hydroxylase 1 (Tph1, rs262731280) and serotonin receptor 3A (Htr3a, rs50670893) were associated with activity/anxiety behaviours, and microtubule-associated protein 2 (Map2, rs13475902) was associated with cognitive performance. All these genes except for Tph1 were expressed in the brain above the array median, and remained significantly associated with relevant behaviours after controlling for the family structure. Additionally, we found evidence for a correlation between Htr3a expression and activity. We discuss our findings in the light of the advantages and limitations of currently available mouse genetic tools, suggesting further directions for association studies in rodents.

Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of "omics" and "big data." Here, we provide an integrated approach for systematic analysis of the "lipidome" in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos.

The Sp family of transcription factors is required for the expression of cell cycle- and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.

Mixed lineage leukemias (MLLs) are histone-methylating enzymes with critical roles in gene expression, epigenetics and cancer. Although MLLs are important gene regulators little is known about their own regulation. Herein, to understand the effects of toxic stress on MLL gene regulation, we treated human cells with a common food contaminant mycotoxin, deoxynivalenol (DON). Our results demonstrate that MLLs and Hox genes are overexpressed upon exposure to DON. Studies using specific inhibitors demonstrated that Src kinase families are involved in upstream events in DON-mediated upregulation of MLL1. Sequence analysis demonstrated that the MLL1 promoter contains multiple Sp1-binding sites and importantly, the binding of Sp1 is enriched in the MLL1 promoter upon exposure to DON. Moreover, antisense-mediated knockdown of Sp1 diminished DON-induced MLL1 upregulation. These results demonstrated that MLL1 gene expression is sensitive to toxic stress and Sp1 plays crucial roles in the stress-induced upregulation of MLL1.

Genetic analysis in the IL10-deficient mouse model revealed a modifier locus of experimental inflammatory bowel disease (IBD) on chromosome 18, with the allele of the strain C3H/HeJBir (C3Bir) conferring resistance and the allele of C57BL/6J (B6) conferring susceptibility. Differential Cd14 expression was associated with this background specific susceptibility to intestinal inflammation. Polymorphisms of the Cd14 promoter were found to be likely causative for strain specific expression, and Cd14-knockout mice revealed a protective role of this gene-product in experimental IBD. In this study, luciferase reporter assays confirmed an increased activity of the C3Bir derived Cd14 promoter compared to the one of B6. Promoter truncation experiments and site-directed mutagenesis in both strains resulted in reduced Cd14 promoter activity and confirmed that a central AP1 and the proximal SP1 transcription factor binding sites mediated the basal activity of the Cd14 promoter in the mouse. Moreover, a T to C exchange at position -259 replaced putative STAT1 and CDX1 sites in the Cd14 promoter from B6 by a SP2 site in C3Bir. Ablation of the Sp2 site through truncation was associated with a decreased promoter activity. Site-directed mutagenesis also demonstrated that the inactivation of SP2 led to a substantial loss of promoter activity in C3Bir. Performing electrophoretic mobility shift and supershift assays demonstrated interaction of SP2 with its potential binding site. In addition, retroviral—mediated overexpression of the SP2 transcription factor in primary bone marrow macrophages derived from C3Bir mice caused a significant increase in Cd14 transcription. These data characterized SP2 as important factor responsible for higher Cd14 expression and reduced IBD susceptibility mediated by the C3Bir allele.

The complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 has been determined. This information when taken together with N-terminal sequencing and mass spectroscopy data indicates that these two proteins share a product precursor relationship in which SP2 results from cleavage of a short N-terminal peptide of SP1. The secondary and tertiary structures of SP2 have been characterized using circular dichroism and three dimension structure prediction. These structural analyses have conclusively shown that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x. Hence, they can be considered bona fide members of the protamine-like- I (PL-I) group of sperm nuclear basic proteins (SNBPs) that have been described in other vertebrate and invertebrate groups. SP2 binds to nucleosomal DNA in a way that is very similar to that of histone H1. However, its interaction with circular DNA does not exhibit an enhanced preference for the supercoiled conformation, and it appears to be mainly driven by ionic interactions.

The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome.

Th17 cells are involved in the immune response against pathogens, autoimmunity, and tumor progression. The differentiation of human Th17 cells requires the upregulation of RORγT, which in human cells is still not well understood. We identified 2 putative binding motifs for specificity protein transcription factors from the specificity protein/Kruppel-like factor family in the promoter of human RORγT and investigated the involvement of specificity proteins in the transcriptional regulation of this gene. To this end, a human lymphocytic cell line and in vitro-differentiated Th17 cells were used in promoter activity assays, in situ mutagenesis, chromatin immunoprecipitation, and real-time RT-PCR assays. In some experiments, specificity protein expression and activity was inhibited by siRNA and mithramycin A. The results showed that the transcription factor specificity protein 2 recognized binding motifs in the human RORγT promoter, which was critical for maintaining expression. Furthermore, specificity protein 2 was necessary for maximum IL-17 expression in in vitro-differentiated Th17 cells. These observations demonstrate the significant role of specificity protein 2 in the regulation of the Th17 signature transcription factor RORγT and the maintenance of the Th17 phenotype. The findings also suggest that specificity protein 2 plays a role in Th17-dependent physiologic and pathologic immune responses and might serve as a potential novel target for their modulation.

Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo., Author Summary A major question in eukaryotic gene regulation is how transcription factors with similar structural features elicit specific biological responses. We used the three transcription factors Sp1, Sp2 and Sp3 as a paradigm for investigating this question. All three proteins are ubiquitously expressed, and they share glutamine-rich domains as well as a conserved bona fide zinc finger DNA binding domain. Yet, each of the three proteins carries out unique functions in vivo, and each is absolutely essential for mouse development. By genome-wide binding analysis, we found that Sp1 and Sp3 on the one hand, and Sp2 on the other hand engage completely different protein domains for their genomic binding site selection. Most strikingly, the zinc finger domain of Sp2 is dispensable for recruitment to its target sites in vivo. Moreover, we provide strong evidence that the histone-fold protein Nf-y is necessary for recruitment of Sp2. Conversely, Sp2 potentiates Nf-y binding showing that binding of Sp2 and Nf-y to shared sites is mutually dependent. Our findings uncover an unexpected mechanistic diversity in promoter recognition by seemingly similar transcription factors. This work has broader implications for our understanding of how members of other multi-protein transcription factor families could achieve specificity.

Combinatorial interactions between cis elements and trans-acting factors are required for regulation of cardiac gene expression during normal cardiac development and pathological cardiac hypertrophy. Sp factors bind GC boxes and are implicated in recruitment and assembly of the basal transcriptional complex. In this study, we show that the cardiac troponin T (cTnT) promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC box in a sequence-specific fashion and consist of Sp1, Sp2, and Sp3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter “in vivo.” Cotransfected Sp1 trans-activates the cTnT promoter in cardiomyocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of the cTnT gene in embryonic cardiomyocytes. Sp3 repression of Sp1-mediated cTnT promoter activation is dose dependent, inferring a mechanism of competitive binding/inhibition. To evaluate the role of Sp factors in cardiac gene expression in vivo, we have established a clinically relevant animal model of pathological cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle, and pressure loading of the right ventricle. Sp1 expression is increased in all four hypertrophied cardiac chambers, whereas Sp3 expression is diminished. This observation is consistent with the in vitro activating function of Sp1 and inhibitory effects of Sp3 on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program in vivo.

We have reported that extracts prepared from many human and mouse cell lines show little or no Sp2 DNA-binding activity and that Sp2 has little or no capacity to stimulate transcription of promoters that are activated by Sp1, Sp3, and Sp4. Using an array of chimeric Sp1/Sp2 proteins we showed further that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. As part of an ongoing effort to study Sp2 function and regulation we characterized its subcellular localization in comparison with other Sp-family members in fixed and live cells. We report that 1) Sp2 localizes largely within subnuclear foci associated with the nuclear matrix, and 2) these foci are distinct from promyelocytic oncogenic domains and appear to be stable during an 18-h time course of observation. Deletion analyses identified a 37 amino acid sequence spanning the first zinc-“finger” that is sufficient to direct nuclear matrix association, and this region also encodes a bipartite nuclear localization sequence. A second nuclear matrix targeting sequence is encoded within the Sp2 trans-activation domain. We conclude that Sp2 preferentially associates with the nuclear matrix and speculate that this subcellular localization plays an important role in the regulation of Sp2 function.

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at -266/-216 bp 5' to the ATG codon. The minimal promoter domain resides within -254/-217 bp 5' to ATG codon, and upstream sequences to -404 bp (-1035/-405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of -217/-1 bp, containing a 23 nt GC rich sequence (-112/-90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.

The steroid/sterol sulfotransferase gene (SULT2B1) encodes for two isozymes of which one (SULT2B1b) sulfonates cholesterol and is selectively expressed in skin. The human SULT2B1 gene contains neither a TATAAA nor a CCAAT motif upstream of the coding region for SULT2B1b; however, this area is GC-rich. Of five Sp1 elements identified two had regulatory activity utilizing immortalized human keratinocytes: one element is located above the ostensible transcription initiation site, whereas the other is located within the 5′-untranslated region of the SULT2B1b mRNA. Sp1 and Sp2 transcription factors identified by supershift analyses induced reporter gene activity, an effect markedly augmented by histone deacetylase inhibition.

Down-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) tumor suppressor gene expression is common in several malignancies including prostate, colon, and breast cancer. The mechanism that mediates this down-regulation is not known. Here, we report that down-regulation of CEACAM1 expression in prostate cancer cells occurs primarily at the transcriptional level and is mediated by Sp2, a member of the Sp family of transcription factors. Sp2 binds to the CEACAM1 promoter in vitro and in vivo, and transient overexpression of Sp2 down-regulates endogenous CEACAM1 expression in normal prostate epithelial cells. Sp2 appears to repress CEACAM1 gene expression by recruiting histone deacetylase activity to the CEACAM1 promoter. In human prostate cancer specimens, Sp2 expression is high in prostate cancer cells but low in normal prostate epithelial cells and is inversely correlated with CEACAM1 expression. Our studies show that transcriptional repression by Sp2 represents one mechanism by which CEACAM1 tumor suppressor gene is down-regulated in prostate cancer.

The transcription factor Ets2 has a role in cancer development and represents an attractive therapeutic target. In this study, we designed a triplex-forming oligonucleotide (TFO) directed to a homopurine:homopyrimidine sequence in the Ets2 promoter. Transcription factors of the Sp family bound to this sequence and mutation of the Sp1 site reduced Ets2 promoter activity. The Ets2-TFO had high binding affinity for the target sequence and inhibited binding of Sp1/Sp3 to the overlapping site. This effect occurred with a high degree of sequence specificity. Mismatched oligonucleotides did not inhibit Sp1/Sp3 binding and mutations in the target sequence that abolished triplex formation prevented inhibition of Sp1/Sp3 binding by the TFO. The Ets2-TFO inhibited Ets2 promoter activity and expression of the endogenous gene in prostate cancer cells at nanomolar concentrations. The TFO did not affect reporter constructs with mutations in the TFO binding site and promoters of non-targeted genes. Expression of non-targeted genes was also not affected in TFO-treated cells. Collectively, these data demonstrated that the anti-transcriptional activity of the Ets2-TFO was sequence- and target-specific, and ruled out alternative, non-triplex mediated mechanisms of action. This anti-transcriptional approach may be useful to examine the effects of selective downregulation of Ets2 expression and may have therapeutic applications.

In this report, we explored the mechanisms underlying keratinocyte-specific and differentiation-specific gene expression in the skin. We have identified five keratinocyte-specific, open chromatin regions that exist within the 6 kb of 5' upstream regulatory sequence known to faithfully recapitulate the strong endogenous keratin 5 (K5) promoter and/or enhancer activity. One of these, DNase I-hypersensitive site (HSs) 4, was unique in that it acted independently to drive abundant and keratinocyte-specific reporter gene activity in culture and in transgenic mice, despite the fact that it was not essential for K5 enhancer activity. We have identified evolutionarily conserved regulatory elements and a number of their associated proteins that bind to this compact and complex enhancer element. The 125-bp 3' half of this element (referred to as 4.2) is by far the smallest known strong enhancer element possessing keratinocyte-specific activity in vivo. Interestingly, its activity is restricted to a subset of progeny of K5-expressing cells located within the sebaceous gland. The other half of HSs 4 (termed 4.1) possesses activity to suppress sebocyte-specific expression and induce expression in the channel (inner root sheath) cells surrounding the hair shaft. Our findings lead us to a view of keratinocyte gene expression which is determined by multiple regulatory modules, many of which contain AP-2 and/or Sp1/Sp3 binding sites for enhancing expression in skin epithelium, but which also harbor one or more unique sites for the binding of factors which determine specificity. Through mixing and matching of these modules, additional levels of specificity are obtained, indicating that both transcriptional repressors and activators govern the specificity.

CD14 is a pattern recognition receptor that plays a central role in innate immunity through recognition of bacterial lipoglycans, primarily LPS. Recently, our group has identified a common single nucleotide polymorphism, −159C→T, in the CD14 proximal promoter. Homozygous carriers of the T allele have a significant increase in soluble CD14, but a decreased total serum IgE. This epidemiologic evidence led us to investigate the molecular basis for the effects of CD14/−159C→T on CD14 regulation in monocytes and hepatocytes, the two major cell types known to express this gene in vivo. EMSA analysis showed that the T allele results in decreased affinity of DNA/protein interactions at a GC box that contains a binding site for Sp1, Sp2, and Sp3 transcription factors. In reporter assays, the transcriptional activity of the T allele was increased in monocytic Mono Mac 6 cells, which express low levels of Sp3, a member of the Sp family with inhibitory potential relative to activating Sp1 and Sp2. By contrast, both alleles were transcribed equivalently in Sp3-rich hepatocytic HepG2 cells. Our data indicate that the interplay between CD14 promoter affinity and the [Sp3]:[Sp1 + Sp2] ratio plays a critical mechanistic role in regulating transcription of the two CD14 alleles. Variation in a key gene of innate immunity may be important for the pathogenesis of allergy and inflammatory disease through gene-by-gene and/or gene-by-environment interactions.

Abstract —A new polymorphism located at position −629 (CETP/−629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The −629A allele was associated with lower CETP mass ( P P P P

Cyclooxygenase-2 (COX-2) has been implicated in neuronal survival and death. However, the precise regulatory mechanisms involved in COX-2 function are unclear. In the present study we found that COX-2 is induced in response to glutathione depletion-induced oxidative stress in primary cortical neurons. Two proximal specific Sp1 and Sp3 binding sites are responsible for the COX-2 promoter activity under normal as well as oxidative stress conditions through enhanced Sp1 and Sp3 DNA binding activity. Site-directed mutagenesis confirmed that -268/-267 positions serve as specific Sp1 and Sp3 recognition sites under oxidative stress. Enforced expression of Sp1 and Sp3 using HSV vectors increased the promoter activity, transcription, and protein level of COX-2 in cortical neurons. The dominant negative form of Sp1 abrogated the oxidative stress-induced promoter activity and expression of COX-2. We also demonstrated that adenovirus-mediated COX-2 gene delivery protected neurons from DNA damage induced by oxidative, genotoxic, and excitotoxic stresses and by ischemic injury. Moreover, COX-2(-/-) cortical neurons were more susceptible to DNA damage-induced cell death. These results indicate that in primary neurons Sp1 and Sp3 play an essential role in the modulation of COX-2 transcription, which mediates neuronal homeostasis and survival by preventing DNA damage in response to neuronal stress.

Na,K-ATPase α subunit has three isoforms whose expression is regulated developmentally and hormonally. Na,K-ATPase α 3 subunit gene ( Atp1a3 ) is expressed only in brain and neonatal heart in a rat. The purpose of this study is to analyze cis -acting elements and trans -acting factors regulating the transcription of Atp1a3 in cultured neonatal rat cardiocytes. Transient transfection assays with Atp1a3 -luciferase chimeric construct and a series of 5′ sequential deletion mutations revealed the existence of positive regulatory elements from −74 to −59 and from −59 to −39. A factor was identified to bind across −59 by gel retardation assay. Methylation interference and DNase I footprinting analyses revealed the binding region from −74 to −53 (positive regulatory element (PRE) 1). The binding factor was identified to be NF-Y by gel retardation assay using specific antibody. Gel retardation and methylation interference analyses revealed that factors bind to two other elements from −54 to −43 (PRE2) and from −25 to −13 (PRE3). The binding factors were identified to be Sp1/Sp3 using specific antibodies. The functions of above-mentioned three elements were examined by transient transfection assay with various combinations of mutations. They all regulated the transcription positively and a synergistic enhancement of it was observed. Roles of NF-Y in the transcriptional activation and synergy are discussed.

MicroRNAs play important roles in the development and progression of various cancers. Recent studies have shown that miR-638 was downregulated in several tumors; however, its role in gastric cancer (GC) has not been investigated in detail.The purpose of this study was to determine the role of miR-638 and to elucidate its regulatory mechanism in GC.The expression levels of miR-638 and specificity protein 2 (Sp2) were detected by real-time PCR and Western blotting in GC. After pcDNA6.2-GW/EmGFP-miR-638 vector, miR-638 inhibitor and Sp2-siRNA transfection, the AGS cell proliferation was investigated by MTT assay and cell cycle, and apoptosis was detected using the Annexin V/PI. In addition, the regulation of Sp2 by miR-638 was evaluated by real-time RT-PCR, Western blot and luciferase reporter assays; cyclin D1 expression was measured by Western blotting.The expression of miR-638 is dramatically down-regulated and Sp2 expression is remarkably up-regulated in GC tissues. Luciferase assays revealed that miR-638 inhibited Sp2 expression by targeting the 3'-UTR of Sp2 mRNA. Overexpression of miR-638 and Sp2-siRNA reduced Sp2 expression at both the mRNA and protein levels in vitro, and inhibition of miR-638 increased Sp2 expression. Moreover, we found that miR-638 overexpression and Sp2-siRNA markedly suppressed cell proliferation with decreasing expression of cyclin D1 and inducing G1-phase cell-cycle arrest in vitro; inhibition of miR-638 significantly promoted cell proliferation by increasing expression of cyclin D1 and leading more cells into the S and G2/M phase.Our results demonstrated that miR-638 suppressed GC cell proliferation by targeting Sp2 with influence on the expression of cyclin D1. We suggest that miR-638 might be a candidate predictor or an anticancer therapeutic target for GC patients.

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.

MicroRNA-27a (miR-27a) is expressed in MCF-7 breast cancer cells, and antisense miR-27a (as-miR-27a) induces ZBTB10, a specificity protein (Sp) repressor. Both as-miR-27a and overexpression of ZBTB10 decreased Sp1, Sp3, and Sp4 mRNA and protein expression in MCF-7 cells, and this was also accompanied by decreased levels of estrogen receptor alpha (ERalpha) mRNA and protein. RNA interference studies confirmed that basal expression of ERalpha was dependent on Sp1 but not Sp3 or Sp4 in MCF-7 cells. as-miR-27a and overexpression of ZBTB10 inhibited 17beta-estradiol (E2)-induced transactivation in MCF-7 cells, and this was accompanied by decreased binding of Sp and ER proteins in cell lysates to oligonucleotides containing GC-rich motifs or estrogen-responsive elements, respectively. as-miR-27a and overexpression of ZBTB10 arrested MCF-7 cells in G(0)/G(1) and inhibited E2-induced G(0)/G(1) to S phase progression. as-miR-27a induced only a minimal increase in Myt-1, another miR-27a regulated gene, and this was not accompanied by Myt-1-dependent G(2)/M arrest as observed previously in ER-negative MDA-MB-231 breast cancer cells. Thus, miR-27a indirectly regulates E2-responsiveness in MCF-7 cells through suppression of ZBTB10, thereby enhancing expression of ERalpha.

The Sp-family of transcription factors is comprised by nine members, Sp1-9, that share a highly conserved DNA-binding domain. Sp2 is a poorly characterized member of this transcription factor family that is widely expressed in murine and human cell lines yet exhibits little DNA-binding or trans-activation activity in these settings. As a prelude to the generation of a "knock-out" mouse strain, we isolated a mouse Sp2 cDNA and performed a detailed analysis of Sp2 transcription in embryonic and adult mouse tissues. We report that (1) the 5' untranslated region of Sp2 is subject to alternative splicing, (2) Sp2 transcription is regulated by at least two promoters that differ in their cell-type specificity, (3) one Sp2 promoter is highly active in nine mammalian cell lines and strains and is regulated by at least five discrete stimulatory and inhibitory elements, (4) a variety of sub-genomic messages are synthesized from the Sp2 locus in a tissue- and cell-type-specific fashion and these transcripts have the capacity to encode a novel partial-Sp2 protein, and (5) RNA in situ hybridization assays indicate that Sp2 is widely expressed during mouse embryogenesis, particularly in the embryonic brain, and robust Sp2 expression occurs in neurogenic regions of the post-natal and adult brain.

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.

Suppressor of cytokine signalling (SOCS) proteins are inducible feedback inhibitors of Janus kinase (JAK) and signal transducers and activators of transcription signalling (STAT) pathways. Interferon (IFN)-gamma induces the expression of the socs1 gene in several cell types through several cis elements present in its promoter and their binding proteins. Socs1 expression is induced in the human keratinocytes HaCaT cell line through sequential activation of STAT1 and IRF-1. Comparison of the 5'-upstream sequences of the mouse and human socs1 genes identified conserved binding sites for IRF-1 regulatory elements. Although this response element is able to bind IRF-1 in human cells, no IFN-gamma responsiveness was observed with human socs1 promoter reporter constructs containing this element. In contrast the mouse socs1 promoter was fully responsive. The mouse promoter contains two cis-acting elements which modulate its expression and are recognized by IRF-1 and Sp2. Despite the absence of Sp2 in the 5'-upstream sequence of the human promoter, silencing of Sp2 by RNA interference clearly demonstrated that Sp2 is required for IFN-gamma-induced regulation of socs1 mRNA both in human and mouse.

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency., Author Summary After HIV genome integration into the host chromosome, the viral promoter coordinates a complex set of inputs to control the establishment of viral latency, the onset of viral gene expression, and the ensuing gene expression levels. Among these inputs are chromatin structure at the site of integration, host transcription factors, and the virally encoded transcriptional regulator Tat. Importantly, transcriptional noise from host and viral transcriptional regulators may play a critical role in the decision between replication versus latency, because stochastic fluctuations in gene expression are amplified by a Tat-mediated positive transcriptional feedback loop. To evaluate the individual contributions of key transcription factor binding elements in gene expression dynamics, we employ model HIV viruses with mutations introduced into numerous promoter elements. Extensive analysis of gene expression dynamics and transcription factor recruitment to the viral promoter reveals that each site differentially contributes to viral gene expression and to the establishment of a low expression state that may contribute to viral latency. This systems-level approach elucidates the synergistic contributions of host and viral factors to the dynamics, magnitudes, and stochastic effects in viral gene expression, as well as provides insights into mechanisms that contribute to proviral latency.

We have developed a novel method to detect PCR products from pathogen genome DNA by Zinc finger protein that can bind to double strand DNA (dsDNA) in sequence specific manner. In this study, we tried to detect Legionella pneumophila strain Philadelphia 1 using Zinc finger protein. We found the specific target DNA sequence for zinc finger protein Sp2 in L. pneumophila strain Philadelphia 1 genome DNA. Specific PCR product was successfully amplified from L. pneumophila strain Philadelphia 1 genome DNA and we used Zinc finger protein Sp2 to detect it. We succeeded in detecting the PCR products from L. pneumophila strain Philadelphia 1 genome DNA with Sp2.

Id3 is a member of the Id family of transcriptional regulators that have been implicated in the development of multiple tissues. Altered expression of the Id genes and proteins contribute to carcinogenesis and atherosclerosis. Id3 is highly expressed in proliferating skeletal muscle cells but becomes downregulated upon terminal differentiation. We have identified several DNase I protected footprints within a proximal region of the mouse Id3 promoter that has been shown previously to support high levels of transcriptional activity in proliferating skeletal muscle cells. Two of these sites interacted, respectively, in vitro with Sp2 and Egr-1 proteins present in muscle cell nuclear extracts. Mutation analysis revealed that the Sp2 site accounted for a major part of the Id3 promoter activity in proliferating muscle cells whereas the Egr-1 site was dispensable. Consistent with the previously observed downregulation of the endogenous Id3 gene, protein binding to the Sp2 site was substantially reduced with extracts from differentiated muscle cells. Our results reveal Id3 as a potential target for Sp2 and raise the possibility that acute activation and the chronic and maintained expression of Id3 gene might be regulated by different mechanisms.

Previous studies have indicated that Sp2 binds poorly to GC-rich sequences bound by Sp1 and Sp3, and further functional analyses of Sp2 have been limited. To study Sp2-mediated transcription, we employed a PCR-based protocol to determine the Sp2 consensus DNA-binding sequence (5′-GGGCGGGAC-3′) and performed kinetic experiments to show that Sp2 binds this consensus sequence with high affinity (225 pm) in vitro. To determine the functional consequence of Sp2 interaction with this sequence in vivo, we transformed well characterized Sp-binding sites within the dihydrofolate reductase (DHFR) promoter to consensus Sp2-binding sites. Incorporation of Sp2-binding sites within the DHFR promoter increased Sp2-mediated trans-activation in transient co-transfection experiments but also revealed Sp2 to be a relatively weak trans-activator with little or no capacity for additive or synergistic trans-activation. Using chimeric molecules prepared with portions of Sp1 and Sp2 and the human prostate-specific antigen promoter, we show that Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells. Taken together, our data indicate that Sp2 is functionally distinct relative to other Sp family members and suggest that Sp2 may play a unique role in cell physiology.

Steroid hormones are synthesized in adrenals, gonads, placenta, and the central and peripheral nervous systems (neurosteroids). Neurosteroidogenesis, like conventional steroidogenesis, begins with the conversion of cholesterol to pregnenolone, catalyzed by mitochondrial P450 side-chain cleavage enzyme (P450scc). Transcription of the P450scc gene in the adrenals and gonads requires steroidogenic factor-1, which is not expressed in the nervous system cells that express P450scc. A crucial transcriptional regulatory region of the rat P450scc gene is at -130/-94. We have purified two nuclear proteins (70 and 86 kDa) from rat glial C6 cells that specifically bind to the -130/-94 region of the rat P450scc promoter and identified them as the DNA-binding subunits of autoimmune antigen Ku. Ku colocalized with P450scc in several regions of the nervous system, but its overexpression in C6 cells did not augment transcription from a -130/-94 Luciferase construct. Members of the Sp family of transcription factors also bind to the same DNA sequence as Ku. Sp4 and Sp2 colocalize with P450scc in the nervous system early in development, whereas Sp1 and Sp4 colocalize later in development. Sp1 robustly increased transcription from this element in Sp-deficient Drosophila SL2 cells, and Ku synergistically enhanced this Sp1-stimulated transcription. Thus, members of the Sp transcription family play a role in activating P450scc gene transcription in the nervous system, and Ku may further augment this activation.

Topo IIalpha (topoisomerase IIalpha) is a major target of several commonly used anticancer drugs and is subject to down-regulation at the transcriptional level in some drug-resistant cell lines and tumours in response to chemotherapy. Clinical resistance to such drugs has been correlated with down-regulation of topo IIalpha at transcription in some drug-resistant cell lines and tumours. Putative binding sites for a variety of transcription factors, including Sp1 (specificity protein 1) and NF-Y (nuclear factor Y) have previously been identified in the topo IIalpha promoter, but their functional significance and interactions have not been described following exposure to anti-cancer drugs. The binding of these factors to specific putative regulatory elements in the topo IIalpha promoter was studied using electrophoretic-mobility-shift assays. Sp1 was found to bind strongly to both distal and proximal GC-rich elements and NF-Y to ICB1 (the first inverted CCAAT box). The functional significance of transcription-factor binding was studied using transient transfection of HeLa cells using a luciferase reporter driven by a 617-bp minimal promoter containing point mutations in putative regulatory elements. Sp1 and NF-Y were both found to be transcriptional modulators with activator or repressor functions depending on protein/DNA context. Moreover, a functional interaction between Sp1 and NF-Y bound at proximal elements was observed.