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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., (© 2018 Völkel et al.)

Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.

For long it has been recognized that CCAAT boxes and GC-rich elements co-occur in many human and murine promoters within 100bp upstream of the transcription start site. The trimeric transcription factor NF-Y is the major CCAAT box-binding factor, and members of the SP family of transcription factors are the major GC box-binding proteins. Recent chromatin immunoprecipitations coupled with high throughput sequencing (ChIP-seq) have examined binding of NF-Y and the ubiquitous SP factors SP1, SP2 and SP3 genome-wide, allowing for comprehensive comparison of NF-Y and SP factor actions in the context of chromatin. Here, I attempt a synthesis of the earlier single-promoter type of analysis with the more recent genome-wide studies. In particular, I also discuss different modes of genomic interactions between SP factors and NF-Y that have emerged recently, and identify a key technical issue, which needs to be taken into account in a critical evaluation of genome-wide studies. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© 2016 International Society for Neurochemistry.)

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.

Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation., (© 2015 by The American Society of Hematology.)

Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it's binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR-DUB complex.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1) domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD) of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

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.

Objective: The transcription factor PAX5 is essential for the activation of B-cell-specific genes and for the silencing of myeloid-specific genes. We previously determined the molecular mechanism by which PAX5 silences the myeloid-specific colony-stimulating-factor-receptor (Csf1R) gene and showed that PAX5 directly binds to the Csf1r promoter as well as to an intronic enhancer that generates an antisense transcript in B cells. Here we examine the role of PAX5 in the regulation of sense and antisense transcription in B cells., Materials and Methods: We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5(-/-) pro-B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice., Results: PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain., Conclusions: We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

SUMO modification of transcription factors is linked to repression of transcription. The physiological significance of SUMO attachment to a particular transcriptional regulator, however, is largely unknown. We have employed the ubiquitously expressed murine transcription factor Sp3 to analyze the role of SUMOylation in vivo. We generated mice and mouse embryonic fibroblasts (MEFs) carrying a subtle point mutation in the SUMO attachment sequence of Sp3 (IKEE(553)D mutation). The E(553)D mutation impedes SUMOylation of Sp3 at K(551)in vivo, without affecting Sp3 protein levels. Expression profiling revealed that spermatocyte-specific genes, such as Dmc1 and Dnahc8, and neuronal genes, including Paqr6, Rims3, and Robo3, are de-repressed in non-testicular and extra-neuronal mouse tissues and in mouse embryonic fibroblasts expressing the SUMOylation-deficient Sp3E(553)D mutant protein. Chromatin immunoprecipitation experiments show that transcriptional de-repression of these genes is accompanied by the loss of repressive heterochromatic marks such as H3K9 and H4K20 tri-methylation and impaired recruitment of repressive chromatin-modifying enzymes. Finally, analysis of the DNA methylation state of the Dmc1, Paqr6, and Rims3 promoters by bisulfite sequencing revealed that these genes are highly methylated in Sp3wt MEFs but are unmethylated in Sp3E(553)D MEFs linking SUMOylation of Sp3 to tissue-specific CpG methylation. Our results establish SUMO conjugation to Sp3 as a molecular beacon for the assembly of repression machineries to maintain tissue-specific transcriptional gene silencing., Competing Interests: The authors have declared that no competing interests exist.

Background: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown., Methodology/principal Findings: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation., Conclusions/significance: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.

To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNgamma-inducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response. Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with the decline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNgamma. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1 activation that controls PIAS1-mediated repression by arginine methylation.

Modification of many transcription factors including Sp3 and steroidogenic factor 1 with the small ubiquitin-like modifier (SUMO) is associated with transcriptional repression. Here, we show that SUMOylation of transcription factors bound to DNA provokes the establishment of compacted repressive chromatin with characteristics of heterochromatin. Chromatin immunoprecipitation experiments revealed SUMO-dependent recruitment of the chromatin remodeller Mi-2, MBT-domain proteins, heterochromatic protein 1, and the histone methyltransferases SETDB1 and SUV4-20H, concomitant with the establishment of histone modifications associated with repressed genes, including H3K9 and H4K20 trimethylation. These results indicate that SUMOylation has a crucial role in regulating gene expression by initiating chromatin structure changes that render DNA inaccessible to the transcription machinery.

SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.

Mice lacking the zinc finger transcription factor specificity protein 3 (Sp3) die prenatally in the C57BL/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at embryonic day 10.5 (E10.5), and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analyzed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. Chromatin immunoprecipitation analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small-molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development and suggest that it has a crucial role in myocardial differentiation.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

The ubiquitously expressed zinc finger transcription factors Sp1 and Sp3 play critical roles in embryonic development. Sp1 knockout mice die around embryonic day 10.5. Mice lacking Sp3 are postnatal lethal. Mice heterozygous for either Sp1 or Sp3 are apparently normal, although slightly smaller. Here, we show that compound heterozygosity of Sp1 and Sp3 results in embryonic lethality accompanied by a spectrum of developmental abnormalities, including growth retardation, morphological alterations of the lung, impaired ossification, anemia, and placental defects. Anemia in Sp1/Sp3 compound heterozygous mutant embryos is associated with impaired maturation of erythrocytes. Analyses of the placenta revealed a markedly reduced spongiotrophoblast layer and a severe disorganization of the labyrinth layer in Sp1/Sp3 compound heterozygous as well as in Sp3-deficient mutant embryos. Our findings demonstrate that a threshold of Sp1 and Sp3 activity is required for normal embryonic development, suggesting that Sp1 and Sp3 act cooperatively to regulate downstream targets., ((c) 2007 Wiley-Liss, Inc.)

The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1-Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-zeta expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to approximately 50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1- and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.

The advent of the genome projects has provided new avenues to explore the question of how DNA sequence information is used appropriately by mammalian cells. Regulation of transcription is not the only, but is certainly a very important, mechanism involved in this process. We can now identify all the genes encoding transcription factors belonging to a certain class and study their biological functions in unprecedented detail through the use of an array of biomolecular tools. It is important to use rigorous and uniform definitions for the classification of transcription factors, because this helps us to comprehend the functions of transcription factor families in biological networks. Here, we propose an unambiguous nomenclature for the members of the Specificity Protein/Krüppel-like Factor (SP/KLF) transcription factor family.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Both proteins contain a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich domains in the N-terminal moiety. Immunoblot analyses of Sp3 reveal a striking complex protein pattern of up to eight distinct species. This pattern is not observed in Sp3-deficient cell lines showing that all signals reflect Sp3 antigen. In this study, we have unraveled the complexity of Sp3 expression. We show that four isoforms of Sp3 that retain different parts of the N terminus are expressed in vivo. The four isoforms derive from alternative translational start sites at positions 1, 37, 856, and 907. An upstream open reading frame located at position -47 to -18 regulates expression of the two long isoforms. Unlike Sp1, none of the Sp3 isoforms is glycosylated. However, all four isoforms become SUMO-modified in vivo and in vitro specifically and exclusively at lysine residue 551. The transcriptional activity of the two long isoforms strongly depends on the promoter settings, whereas the small isoforms appear to be inactive. The transcriptional activity of all the Sp3 isoforms is regulated by SUMO modification. Our results demonstrate that Sp3 has many unique features and is not simply a functional equivalent of Sp1.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known antiinflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. In this study, PPARalpha agonists are found to inhibit endothelial VEGFR2 expression, whereas predominant PPARgamma ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. Inhibitory effects of PPARalpha agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between base pairs -60 and -37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1-containing complex formation to this sequence is decreased by PPARalpha treatment, indicating that VEGFR2 gene expression is inhibited by repressing Sp1 site-dependent DNA binding and transactivation. Our coimmunoprecipitation experiments revealed enhanced protein interactions between PPARalpha and Sp1 on PPARalpha activation, thus constituting a probable mechanism by which PPARalpha activators decrease Sp-dependent binding activity to the VEGFR2 promoter. Hence, molecular mechanisms by which PPARs modulate the rate of gene transcription may include direct interactions between specific transcription factors and PPARs that ultimately result in reduced DNA binding to their respective response elements.

Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. We recently identified MYCN as a target gene of the E2F transcription factors. Here we show that Sp1 and Sp3 cooperate with E2F-1 to activate the MYCN promoter. However, in a neuroblastoma cell line that does not express MYCN, overexpression of E2F-1 was not sufficient to activate the MYCN promoter even in the presence of trichostatin A and 5-aza-cytidine. This was because of a failure of E2F-1 to bind to the MYCN promoter in these cells, although access of E2F-1 to the inactive MYCN promoter was not blocked by a nucleosome. Differences in nucleosomal organization of the MYCN promoter in different cell lines did not correlate with gene activation per se but with the switch from basal to activated transcription. Binding of E2F and Sp1/Sp3 to the MYCN promoter in vivo correlated with acetylation of histones H3 and H4 and recruitment of RNA polymerase II and the protein acetyltransferase Tip60 but not with nucleosome remodeling. Our results define distinct chromatin states of the MYCN promoter, indicate that factors in addition to E2F and Sp1/Sp3 are required to activate MYCN in neuroblastomas, and provide evidence for a novel mechanism of controlling access of E2F to selected target genes.

Interleukin-1 beta (IL-1 beta) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1 beta inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1 beta down-regulates COL2A1 gene transcription through a -41/-33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1 beta effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type -50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1 beta inhibition of a -63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the -41/-33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the -63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1 beta down-regulation of that gene, as we found previously for transforming growth factor-beta 1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1 beta increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1 beta decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.

As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre-B-cell proliferation and differentiation into immunoglobulin M-positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.

The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.

Enhanced VEGF-A (vascular endothelial growth factor A) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies including gastric cancer. Oxidative stress has been linked to tumor-associated neoangiogenesis; underlying mechanisms, however, remained poorly understood. Therefore, we studied the effect of oxidative stress on VEGF-A gene expression in gastric cancer cells. Oxidative stress generated by H(2)O(2) application potently stimulated VEGF-A protein and mRNA levels as determined by enzyme-linked immunosorbent assay and real-time PCR techniques, respectively, and elevated the activity of a transfected (-2018) VEGF-A promoter reporter gene construct in a time- and dose-dependent manner (4-8-fold). These effects were abolished by the antioxidant N-acetylcysteine, demonstrating specificity of oxidative stress responses. Functional 5' deletion analysis mapped the oxidative stress response element of the human VEGF-A promoter to the sequence -88/-50, and a single copy of this element was sufficient to confer basal promoter activity as well as oxidative stress responsiveness to a heterologous promoter system. Combination of EMSA studies, Sp1/Sp3 overexpression experiments in Drosophila SL-2 cells, and systematic promoter mutagenesis identified enhanced Sp1 and Sp3 binding to two GC-boxes at -73/-66 and -58/-52 as the core mechanism of oxidative stress-triggered VEGF-A transactivation. Additionally, in Gal4-Sp1/-Sp3-Gal4-luciferase assays, oxidative stress increased Sp1 but not Sp3 transactivating capacity, indicating additional mechanism(s) of VEGF-A gene regulation. Signaling studies identified a cascade comprising Ras --> Raf --> MEK1 --> ERK1/2 as the main pathway mediating oxidative stress-stimulated VEGF-A transcription. This study for the first time delineates the mechanisms underlying regulation of VEGF-A gene transcription by oxidative stress and thereby further elucidates potential pathways underlying redox control of neoangiogenesis.

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.

Although transforming growth factor beta1 (TGF-beta1) is generally considered as a stimulator of type I collagen production in smooth organs, we found that it can inhibit type II collagen biosynthesis in primary rabbit articular chondrocytes (RAC) at transcriptional levels. Constructs of promoter and first intron sequences associated with the luciferase reporter gene were used to delineate the gene sequences involved in TGF-beta1 control of human COL2A1 gene transcription. Cotransfection of these DNA fragments with a TbetaRII/I cDNA hybrid receptor, capable of inducing a TGF-beta1 dominant negative effect, showed that TGF-beta1 inhibits specifically COL2A1 gene transcription in RAC by a 63-bp proximal promoter. Footprint and gel retardation analyses revealed that the TGF-beta1-induced inhibition effect exerted through the 63-bp promoter sequence implies a multimeric complex that binds to the -41/-33 sequence and involves Sp1 and Sp3 transcription factors. Transfection of decoy Sp-binding oligonucleotides corroborated the implication of the proximal promoter in the TGF-beta1-induced inhibition of COL2A1 gene transcription. In addition, TGF-beta1 was found to increase the expression of Sp3 without significant changes to its binding level, but repressed both the biosynthesis and binding activity of Sp1. In functional assays, Sp3 inhibited the 63-bp promoter activity and prevented Sp1 induction of transcription. These findings suggest that TGF-beta1 inhibition of COL2A1 gene transcription in RAC is mediated by an increase of the Sp3/Sp1 ratio and by the repression of Sp1 transactivating effects on that gene.

The basic region-leucine zipper transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) and the homeodomain transcription factor Nkx2.1/thyroid transcription factor-1 are essential for normal lung morphogenesis. Nkx2.1 is expressed from the onset of lung development, whereas C/EBPalpha expression is turned on at later stages. The expression of C/EBPalpha correlates to the appearance of lung-specific proteins with differentiation-dependent expression patterns, such as the Clara cell secretory protein (secretoglobin 1a1 (Scgb1a1), CCSP). In this study, we demonstrate synergistic transactivation by C/EBPalpha and Nkx2.1 in the regulation of the CCSP gene. We show that the synergistic activity of C/EBPalpha and Nkx2.1 originates from cis-acting elements in the proximal promoter of CCSP and that the synergism is dependent on NH(2)-terminal transactivation domains of C/EBPalpha and Nkx2.1. Our results suggest that the cooperation of C/EBPalpha and Nkx2.1 is a major determinant for the high level, lung epithelial-specific expression of CCSP during the later stages of lung development and in the adult lung.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Here we show that Sp3 is a target for SUMO modification in vivo and in vitro. SUMO modification of Sp3 occurs at a single lysine located between the second glutamine-rich activation domain and the DNA-binding domain. Mutational analyses identified the sequence IKXE as essential for SUMO conjugation to Sp3. We identified the protein inhibitor of activated STAT1 (PIAS1) as an interaction partner of Sp3 and Ubc9. Moreover, PIAS1 strongly stimulated SUMO conjugation to Sp3, thus acting as an E3 ligase for SUMO conjugation to Sp3. All mutations that prevented SUMO modification in vitro strongly enhanced the transcriptional activity of Sp3, showing that SUMO modification silences Sp3 activity. SUMO-modified Sp3 bound to DNA with similar specificity and affinity as unmodified Sp3. However, DNA-bound Sp3 did not act as a substrate for SUMO modification.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Previous analyses showed that, unlike Sp1, Sp3 fails to activate transcription in certain promoter settings. This is due to the presence of an inhibitory domain located between the second glutamine-rich activation domain and the DNA-binding domain. To further analyze the transcriptional properties of Sp3, we have expressed and purified recombinant Sp3 and Sp1 as epitope-tagged proteins from stable transfected insect cells. We found that Sp3 does act as a strong activator similar to Sp1 in an in vitro transcription assay using Sp1/Sp3-depleted HeLa nuclear extract. However, on the same promoter Sp3 is almost inactive when transfected into cells. Mutational studies demonstrate that a single lysine residue is responsible for the low transcriptional activity of Sp3 in vivo. We show that Sp3, but not a mutant of Sp3 that lacks this lysine residue, is highly acetylated in vivo. Our results strongly suggest that the transcriptional activity of Sp3 is regulated by acetylation. The consequences of acetylation for the activity of Sp3 are discussed.

Sp1 and Sp3 effects on the transcription of the human alpha1(II) procollagen gene (COL2A1) were investigated in both differentiated and de-differentiated rabbit articular chondrocytes. Transient transfection with constructs of deleted COL2A1 promoter sequences driving the luciferase reporter gene revealed that the region spanning -266 to +121 base pairs showed Sp1-enhancing effects, whatever the differentiation state. In contrast, Sp3 did not influence COL2A1 gene transcription. Concomitant overexpression of the two Sp proteins demonstrated that Sp3 blocked the Sp1 induction of COL2A1 promoter activity. Moreover, inhibition of Sp1/Sp3 binding to their target DNA sequence decreased both COL2A1 gene transcription and Sp1-enhancing effects. DNase I footprinting and gel retardation assays revealed that Sp1 and Sp3 bind specifically to cis-sequences of the COL2A1 gene promoter whereby they exert their transcriptional effects. Sp1 and Sp3 levels were found to be reduced in de-differentiated chondrocytes, as revealed by DNA-binding and immunochemical study. Sp1 specifically activated collagen neosynthesis whatever the differentiation state of chondrocytes, suggesting that this factor exerts a major role in the expression of collagen type II. However, our data indicate that type II collagen-specific expression in chondrocytes depend on both the Sp1/Sp3 ratio and cooperation of Sp1 with other transcription factors, the amounts of which are also modulated by phenotype alteration.

Background: Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutamine-rich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues., Results: We have disrupted the mouse Sp4 gene by a targeted deletion of the exons encoding the N-terminal activation domains. Sp4 knockout mice show a complete absence of Sp4 expression. They develop until birth without obvious abnormalities. After birth, two-thirds die within 4 weeks. Surviving mice are growth retarded. Male Sp4null mice do not breed. The cause for the breeding defect remains obscure since they show complete spermatogenesis. In addition, pheromone receptor genes in the vomeronasal organ appear unaffected. Female Sp4null mice have a smaller thymus, spleen and uterus. In addition, they exhibit a pronounced delay in sexual maturation., Conclusions: The phenotype of the Sp4null mice differs significantly from those described for Sp1-/- and Sp3-/- mice. Thus, the structural similarities, the common recognition motif and the overlapping expression pattern of these three transcription factors do not reflect similar physiological functions.

Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. Recently, the mouse Sp3 gene has been disrupted by homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced defect in late tooth formation. Here we show that Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. The transcription factor - core binding factor 1 (Cbfa1)--that is essential for bone formation, however, is expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is significantly diminished. Our studies establish Sp3 as an essential transcription factor for late bone development.

Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner.

Sp3 is a ubiquitously expressed transcription factor closely related to Sp1 (specificity protein 1). We have disrupted the mouse Sp3 gene by homologous recombination. Sp3-deficient embryos are growth retarded and invariably die at birth of respiratory failure. The cause for the observed breathing defect remains obscure since only minor morphological alterations were observed in the lung, and surfactant protein expression is indistinguishable from that in wild-type mice. Histological examinations of individual organs in Sp3(-/-) mice show a pronounced defect in late tooth formation. In Sp3 null mice, the dentin/enamel layer of the developing teeth is impaired due to the lack of ameloblast-specific gene products. Comparison of the Sp1 and Sp3 knockout phenotype shows that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy.

Clara cell secretory protein/uteroglobin (CCSP/UG) is specifically expressed in the conducting airway epithelium of the lung in a differentiation-dependent manner. The proximal promoter region of the rodent CCSP/UG gene directs Clara cell specificity. Previously, it was shown that the forkhead transcription factors HNF-3 alpha and beta and the homeodomain factor TTF-1 are important transcription factors acting through this region, suggesting that they contribute to cell specificity of the CCSP/UG gene. Members of the C/EBP family of transcription factors can also interact with elements of the proximal rat and mouse CCSP/UG promoters. The onset of C/EBP alpha expression in Clara cells correlates with the strong increase of CCSP/UG expression. Thus, C/EBP alpha may play a crucial role for differentiation-dependent CCSP/UG expression. Transfection studies demonstrate that C/EBP alpha and TTF-1 can synergistically activate the murine CCSP/UG promoter. Altogether, these results suggest that C/EBP alpha, TTF-1, and HNF-3 determine the Clara cell-specific, differentiation-dependent expression of the CCSP/UG gene in murine lung. The relative importance of these three transcription factors, however, differs in rabbits and humans.

GC-boxes and related motifs are frequently occurring DNA-elements present in many promoters and enhancers. In contrast to other elements it was generally thought that the transcription factor Sp1 is the only factor acting through these motifs. The cloning of paralogous genes of the Sp1 factor uncovered the existence of a small protein family consisting of Sp1, Sp2, Sp3 and Sp4. All four proteins exhibit very similar structural features. They contain a highly conserved DNA-binding domain composed of three zinc fingers close the C-terminus and serine/threonine- and glutamine-rich domains in their N-terminal regions. The high degree of structural conservation between these four proteins suggested that they do exert similar functions. Molecular, genetic and biochemical analyses, however, demonstrated that Sp2, Sp3 and Sp4 are not simply functional equivalents of Sp1. Here, I will summarize and discuss recent advances which have been made towards understanding the mode of action and biological function of individual family members.

One of the most common regulatory elements is the GC box and the related GT/CACC box, which are widely distributed in promoters, enhancers and locus control regions of housekeeping as well as tissue-specific genes. For long it was generally thought that Sp1 is the major factor acting through these motifs. Recent discoveries have shown that Sp1 is only one of many transcription factors binding and acting through these elements. Sp1 simply represents the first identified and cloned protein of a family of transcription factors characterised by a highly conserved DNA-binding domain consisting of three zinc fingers. Currently this new family of transcription factors has at least 16 different mammalian members. Here, we will summarise and discuss recent advances that have been directed towards understanding the biological role of these proteins.

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.

The pituitary peptide hormone ACTH regulates transcription of the cholesterol side chain cleavage cytochrome P450 (CYP11A) gene via cAMP and activation of cAMP-dependent protein kinase. A G-rich sequence element conferring cAMP-dependent regulation has been found to reside within region -118 to -100 of the bovine CYP11A promoter. Previous studies have suggested that it binds a protein antigenically related to the transcription factor Sp1. We now report that the -118/-100 element binds both Sp1 and Sp3, members of the Sp family of transcription factors. We have made use of Drosophila SL2 cells, which lack endogenous Sp factors, to dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors stimulated the activity of cotransfected reporter constructs in which the promoter of the bovine CYP11A gene regulates luciferase expression. Sp3 did not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of the -118/-100 element of CYP11A abolished Sp1-mediated activation of a CYP11A reporter gene in SL2 cells as well as cAMP responsiveness in human H295R cells. Furthermore, cotransfection of SL2 cells with the catalytic subunit of cAMP-dependent protein kinase together with Sp1 and a CYP11A reporter construct enhanced Sp1-dependent activation of the reporter 4.2-fold, demonstrating that Sp1 confers cAMP responsiveness in these cells. Thus, we show that introduction of Sp1 alone in an Sp-negative cell such as SL2 is sufficient to achieve the cAMP-dependent regulation observed using the -118/-100 element of CYP11A in adrenocortical cells.

Analysis of the human Bruton's agammaglobulinemia tyrosine kinase (Btk) gene promoter revealed that 280 bp upstream of the transcriptional start site is sufficient for a cell restricted expression pattern. Here, the interplay of the transcription factors Sp1, Sp3, and PU.1 binding to this promoter area was analysed. All three proteins are able to independently activate the promoter in Drosophila Schneider (SL2) cells lacking endogenous Sp- or PU.1-like activities. Furthermore, PU.1 is able to act synergistically with Sp1 as well as Sp3 to transactivate the promoter. This transactivation is mediated through adjacent binding sites rather than through the more distant Sp binding site, suggesting a possible direct interaction between PU.1 and Sp1/3. Expression of Btk was found in ES cells and levels of expression were the same as in ES cells with a targeted deletion of the Sp1 gene, suggesting that Sp3 acts as a positive regulator of Btk in vivo, in the absence of Sp1., (Copyright 1999 Academic Press.)