buttonhead
Id family helix-loop-helix (HLH) proteins (Drosophila homolog: Extramacrochaetae) are involved in the regulation of proliferation and differentiation for several cell
types. To identify cis- and trans-acting factors that regulate Id4 gene expression, the promoter regulatory sequences of the human Id4 gene have been analyzed in transient transfections and gel mobility shift assays. Two functional elements, both located downstream from the TATA motif, have been identified that control Id4 promoter activity. One element contains a consensus E-box: the protein complex binding to the E-box contains the bHLH-zip upstream stimulatory factor (USF) transcription factor. Enforced expression of USF1 leads to E-box-mediated stimulation of promoter activity. The E-box also mediates stimulatory effects of several bHLH transcription factors, and co-expression of
Id4 blocks the stimulatory effect mediated by the bHLH factors. A second element is a GA motif, located downstream from the transcriptional start sites. A 20-fold increase in transcriptional activity is seen as a result of the mutation of this GA motif. Gel-shift analysis and transfections into Drosophila Schneider SL2 cells show that the repressor element is recognized by both Sp1 and Sp3 factors. These data suggest that Id4 transcription control is highly complex, involving both negative and positive regulatory elements, including a novel inhibitory function exerted by Sp1 and Sp3 transcription factors (Pagliuca, 1998).
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays important roles in neuronal development, plasticity, and cell death. NMDA receptor subunit 1 (NR1) is an essential subunit of the NMDA receptor and is developmentally expressed in postnatal neurons of the central nervous system. A binding site has been identified on the NR1 promoter for myocyte enhancer factor 2C (MEF2C), a developmentally expressed neuron/muscle transcription factor found in cerebrocortical neurons. Co-expression of MEF2C and Sp1 cDNAs in primary neurons or cell lines synergistically activates the NR1 promoter. Disruption of the MEF2 site or the MEF2C DNA binding domain moderately reduces this synergism. Mutation of the Sp1 sites or the activation domains of Sp1 protein strongly reduces the synergism. Results of yeast two-hybrid and co-immunoprecipitation experiments reveal a physical interaction between MEF2C and Sp1 proteins. The MEF2C DNA binding domain is sufficient for this interaction. Dominant-negative MEF2C interferes with expression of NR1 mRNA in neuronally differentiated P19 cells. Growth factors, including epidermal growth factor and basic fibroblast growth factor, can up-regulate NR1 promoter activity in stably transfected PC12 cells, even in the absence of the MEF2 site, but the Sp1 sites are necessary for this growth factor regulation, suggesting that Sp1 sites may mediate these effects (Krainc, 1998).
The 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene has been cloned and the cis- and trans-activating regulatory elements responsible for its tissue specific activity have been characterized. By using a native epsilon3-promoter/lacZ-construct and various 5'-deletion constructs, beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells were compared. Large parts of the epsilon3 promoter are shown to be responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences leads to an enhancement of epsilon3 transcription, suggesting a role for the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region reveals potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologs, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirm specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site (Pieri, 1999).
Cellular cholesterol and fatty acid levels are coordinately regulated by a family of transcriptional regulatory proteins designated sterol regulatory element binding proteins (SREBPs). SREBP-dependent transcriptional activation from all promoters examined thus far is dependent on the presence of an additional binding site for a ubiquitous coactivator. In the low-density lipoprotein (LDL) receptor, acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) promoters, which are all regulated by SREBP, the coactivator is the transcription factor Sp1. In this report, Sp3, another member of the Sp1 family, is shown to be capable of substituting for Sp1 in coactivating transcription from all three of these promoters. Efficient activation of transcription from the LDL receptor promoter requires domain C of Sp1; however, this domain is not crucial for activation of the simian virus 40 promoter, where synergistic activation occurs through multiple Sp1 binding sites and does not require SREBP. The critical determinant of the C domain required for activation of the LDL receptor is localized to a small region that is highly conserved between Sp1 and Sp3. This crucial domain encompasses the buttonhead box, which is a 10-amino-acid stretch that is present in several Sp1 family members, including the Drosophila buttonhead gene product. Interestingly, neither the buttonhead box nor the entire C domain is required for the activation of the FAS and ACC promoters even though both SREBP and Sp1 are critical players. ACC and FAS each contain two critical SREBP sites, whereas there is only one in the LDL receptor promoter. This finding suggested that buttonhead-dependent activation by SREBP and Sp1 may be limited to promoters that naturally contain a single SREBP recognition site. Consistent with this model, a synthetic construct containing three tandem copies of the native LDL receptor SREBP site linked to a single Sp1 site is also significantly activated in a buttonhead-independent fashion. Taken together, these studies indicate that transcriptional activation through the concerted action of SREBP and Sp1 can occur by at least two different mechanisms, and that promoters activated by either one can potentially be identified by the number of critical SREBP binding sites they contain (Athanikar, 1997).
Gene activation by NF-kappaB/Rel transcription factors is modulated by synergistic or antagonistic interactions with other promoter-bound transcription factors. For example, Sp1 sites are often found in NF-kappaB-regulated genes, and Sp1 can activate certain promoters in synergism with NF-kappaB through nonoverlapping binding sites. Sp1 acts directly through a subset of NF-kappaB binding sites. The DNA binding affinity of Sp1 to these NF-kappaB sites, as determined by their relative dissociation constants and their relative efficiencies as competitor DNAs or as binding site probes, is on the order of that for a consensus GC box Sp1 site. In contrast, NF-kappaB does not bind to a GC box Sp1 site. Sp1 can activate transcription through an immunoglobulin kappa-chain enhancer or P-selectin promoter NF-kappaB sites. p50 homodimers replace Sp1 from the P-selectin promoter by binding site competition and thereby either inhibit basal Sp1-driven expression or, in concert with Bcl-3, stimulate expression. The interaction of Sp1 with NF-kappaB sites thus provides a means to keep an elevated basal expression of NF-kappaB-dependent genes in the absence of activated nuclear NF-kappaB/Rel (Hirano, 1998).
It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and that AP1 is not. The Sp1 inhibitor mithramycin blocks stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin has no effect. Furthermore, antibodies against Jun-B and c-Jun fail to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they are purified from untreated or TGF-beta-treated cells. AP1 does bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, it is concluded that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays exclude that COL1A2 transactivation involves the retinoblastoma gene product (pRb) ( an activator of Sp1), the pRb-related protein p107 (an inhibitor of Sp1), or the Sp1-related repressor, Sp3 (Greenwel, 1997).
Electrical stimulation of contractions (pacing) of primary neonatal rat ventricular myocytes increases intracellular calcium and activates a hypertrophic growth program that includes expression of the cardiac-specific gene, atrial natriuretic factor (ANF). To investigate the mechanism whereby pacing increases ANF, pacing was tested for its ability to regulate mitogen-activated protein kinase family members, ANF promoter activity, and the trans-activation domain of the transcription factor, Sp1. Pacing and the calcium channel agonist BAYK 8644 activate c-Jun N-terminal kinase (See Drosophila JNK or Basket) but not extracellular signal-regulated kinase. Pacing stimulates ANF-promoter activity approximately 10-fold. Transfection with an expression vector for c-Jun, a substrate for JNK, also activates the ANF promoter; the combination of pacing and c-Jun is synergistic, consistent with roles for JNK and c-Jun in calcium-activated ANF expression. Proximal serum response factor and Sp1 binding sites are required for the effects of pacing or c-Jun on the ANF promoter. Pacing and c-Jun activate a GAL4-Sp1 fusion protein by 3- and 12-fold, respectively, whereas the two stimuli together activate GAL4-Sp1 synergistically, similar to their effect on the ANF promoter. Transfection with an expression vector for c-Fos inhibits the effects of c-Jun, suggesting that c-Jun acts independently of AP-1. These results demonstrate an interaction between c-Jun and Sp1 and are consistent with a novel mechanism of calcium-mediated transcriptional activation involving the collaborative actions of JNK, c-Jun, serum response factor, and Sp1 (McDonough, 1997).
The ability of C/EBPbeta but not C/EBPalpha (Homologs of Drosophila Slbo) to synergize with an SP1 protein is specified by the leucine zipper and activation domain. The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. Liver-specific transcription fo the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5 that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBPalpha and C/EBPbeta are expressed abundantly in liver, only C/EBPbeta is capable of stimulating the 2D5 promoter in HepG2 cells. In addition, activation of the 2D5 promoter by C/EBPbeta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBPbeta proteins containing either the leucine zipper or the activation domain of C/EBPalpha are unable to stimulate the 2D5 promoter. The serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperativity with C/EBPbeta. These findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein (Lee, 1997).
The human alcohol dehydrogenase 5 gene (also known as the formaldehyde dehydrogenase gene, ADH5/FDH) has a GC-rich promoter with many sites at which transcription factors bind. A minimal promoter extending from -34 base pairs (bp) to +61 bp directs high levels of transcription in several different cells, consistent with the ubiquitous expression of the gene. Nearly the entire minimal promoter can be bound by Sp1. The transcriptional regulation of ADH5/FDH by members of the Sp1 multigene family was analyzed. Two core cis-elements (-22 bp to +22 bp) have the highest affinity for Sp1. Mutagenesis reveals that these cis-elements are critical for transcriptional activation. The zinc-finger domains of Sp3 and Sp4 also bind selectively to the core cis-elements. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activates transcription over 50-fold, suggesting that Sp1 is critical in the initiation of transcription. Neither Sp3 nor Sp4 is able to activate transcription in those cells, and transcriptional activation by Sp1 is repressed by either Sp3 or Sp4. These data suggest that Sp3 and Sp4 can repress transcription by competing with Sp1 for binding to the core cis-elements. The content of Sp1, Sp3, and Sp4 in different cells may be critical factors regulating transcription of the ADH5/FDH gene (Kwon, 1999).
The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains a consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter is strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogates Sp1-dependent transactivation. In gel shift assays, the same mutations abolish Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter is dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells reveals that cell lines overexpressing Sp1 have increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bounds Egr-1 in gel shift assays and Egr-1 competitively displaces bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppresses pgp2/mdr1b promoter activity; mutations in the Egr-1 site decrease Egr-1 binding and correlate with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreases Pgp expression and selectively increases vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells (Thottassery, 1999).
CD18, the beta chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. The ets factors, PU.1 and GABP, bind to three ets sites in the CD18 promoter, which are essential for high level myeloid expression of CD18. Two binding sites have been identified for the transcription factor, Sp1; these binding sites flank the ets sites. Sp1 is the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 into Drosophila Schneider cells, which otherwise lack Sp1, dramatically activates the CD18 promoter. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors (Rosmarin, 1998).
Maximal gene expression driven by the promoter for the transforming growth factor beta type I receptor (TGF-betaRI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBFalpha binding sites among other possible cis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Nuclear transcription factors that bind to PEBP2/CBFalpha-related cis-acting elements in the basal promoter sequence bind in osteoblasts. These studies reveal that constitutive expression of TGF-betaRI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-betaRI levels that parallel changes in bone cell differentiation or activity (Ji, 1997).
Neuronal nicotinic acetylcholine receptors play important roles in signal transduction within the nervous system. The receptors exist in a variety of functionally distinct subtypes, determined by their subunit structures. The subunits are encoded by 11 genes, alpha2-alpha9 and beta2-beta4. Three of the genes, alpha3, alpha5, and beta4, are tightly clustered, and their encoded proteins make up the predominant receptor subtype in the peripheral nervous system. The tight linkage of the genes suggests there may be a common regulatory mechanism underlying their expression. However, although their expression patterns significantly overlap, they are not identical, indicating that independent regulatory mechanisms must also exist. There are several transcriptional regulatory elements encoding the beta4 subunit. One of these elements, E2, specifically interacts with the general transcription factor Sp1. Another member of the Sp family of factors, Sp3, can specifically interact with E2; however, two other SP family members, Sp2 and Sp4, cannot. Co-transfection experiments indicate that Sp3 can transactivate a beta4 promoter/reporter gene construct and, furthermore, that Sp1 and Sp3 can transactivate the beta4 reporter construct synergistically. The transactivation is dependent upon an intact E2 and may involve direct interactions between Sp1 and Sp3 (Bigger, 1997).
The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). Factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter have been characterized. Both Sp1 and Sp3 bind to the 5' portion of this region, whereas a PMA-inducible factor (PIF) binds mainly to its 3' portion and to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity is rapidly induced, required de novo protein synthesis, and is sustained at a high level for at least 23 h. Interestingly, PIF is constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. PIF is Egr-1 (Drosophila homolog: Stripe) based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differs substantially from the canonical Egr-1 binding site. In addition, Egr-1 binds to the Sp binding site. In Jurkat cells, both sites are required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 can drive activity of a reporter construct containing both sites. Sp1 and Egr-1 can form a complex with kinetics that correlate with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity (Lin, 1997).
The growth hormone (GH) receptor is essential for the actions of growth hormone on postnatal growth and metabolism. GH receptor transcripts are characterized by the presence of disparate 5'-untranslated exons. Factors regulating the expression of the GC rich L2 transcript of the murine GH receptor gene have hitherto remained unidentified. To characterize the mechanisms regulating expression of the L2 transcript, primer extension and ribonuclease protection assays were used to identify transcription start sites in RNA from adult mouse liver. Transient transfection experiments revealed that 2.0 kilobase pairs of the L2 5'-flanking sequence exhibit promoter activity in BNL CL.2 (mouse liver) cells, CV-1 (monkey kidney) cells, and HRP.1 trophoblasts. Deletional analysis localizes a major regulatory region to within 75 base pairs of the 5' transcription start site. Sequence analysis reveals that the region contains consensus binding sites for the Sp family of transcription factors. Standard gel shift and supershift analysis using liver nuclear extracts has established that Sp1 and Sp3 bind this regulatory element. Transfection of wild type but not mutant decoy oligonucleotides into BNL CL.2 cells decreases the activity of the L2 promoter. Overexpression of Sp1 and Sp3 protein in Drosophila Schneider cells establishes that Sp3 is more potent than Sp1 in transactivating the L2 promoter. Co-transfection experiments further established that Sp1 antagonizes the activity of Sp3 to transactivate the L2 promoter. Western blot analysis of liver nuclear extracts reveals that the levels of Sp3 increase significantly after birth, suggesting a role for the Sp family of transcription factors in controlling the fetal to postnatal increase in GH receptor gene expression (Yu, 1999).
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that contribute to pathological conditions associated with angiogenesis and tumor invasion. MMP-2 is highly expressed in human astroglioma cells, and contributes to the invasiveness of these cells. The human MMP-2 promoter contains potential cis-acting regulatory elements including cAMP response element-binding protein, AP-1, AP-2, PEA3, C/EBP, and Sp1. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrates that the Sp1 site at -91 to -84 base pairs and the AP-2 site at -61 to -53 base pairs are critical for constitutive activity of this gene in invasive astroglioma cell lines. Electrophoretic gel shift analysis demonstrates binding of specific DNA-protein complexes to the Sp1 and AP-2 sites: Sp1 and Sp3 bind to the Sp1 site, while the AP-2 transcription factor binds the AP-2 element. Co-transfection expression experiments in Drosophila SL2 cells lacking endogenous Sp factors demonstrate that Sp1 and Sp3 function as activators of the MMP-2 promoter and synergize for enhanced MMP-2 activation. Overexpression of AP-2 in AP-2-deficient HepG2 cells enhances MMP-2 promoter activation. These findings document the functional importance of Sp1, Sp3, and AP-2 in regulating constitutive expression of MMP-2. Delineation of MMP-2 regulation may have implications for the development of new therapeutic strategies to arrest glioma invasion (Qin, 1999).
Transforming growth factor-beta arrests growth of epithelial cells by inducing the transcription of p15Ink4B, a cyclin-dependent kinase inhibitor. p15Ink4B induction is mediated by a TGF-beta-induced complex of Smad2, Smad3, Smad4 and Sp1. Mutations in the Sp1- or Smad-binding sequences decrease or abolish the TGF-beta responsiveness of the p15Ink4B promoter. Interference with, or deficiency in, Smad2, Smad3 or Smad4 functions also reduces or abolishes the TGF-beta-dependent p15Ink4B induction, whereas the absence of Sp1 reduces the basal and TGF-beta-induced p15Ink4B transcription. In the nucleoprotein complex, Smad2 interacts through its C-domain with Sp1 and enhances the DNA binding and transcriptional activity of Sp1. Smad3 interacts indirectly with Sp1 through its association with Smad2 and/or Smad4, and binds directly to the p15Ink4B promoter. Finally, Smad4 interacts through its N-domain with Sp1. These data demonstrate the physical interactions and functional cooperativity of Sp1 with a complex of Smad2, Smad3 and Smad4 in the induction of the p15Ink4B gene. These findings explain the tumor suppressor roles of Smad2 and Smad4 in growth arrest signaling by TGF-beta (Feng, 2000).
Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. The 5' regulatory region of the human Phox2a gene has been isolated and the transcriptional mechanisms underlying its expression have been studied. The minimal gene promoter was identified by means of molecular and functional criteria: its activity relies on a degenerate TATA box and a canonical Sp1 site. The region immediately upstream of the promoter stimulates transcription in a neurospecific manner because its deletion causes a substantial decline in reporter gene expression only in neuronal cells. This DNA region contains a putative binding site for homeodomain transcription factors, and its mutation severely affects the transcriptional activity of the entire 5' regulatory region, thus indicating that this site is necessary for the expression of Phox2a in this cellular context. The use of the electrophoretic mobility shift assay has shown that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrate that it is capable of transactivating the human Phox2a promoter. Many data obtained from knock-out mice support the hypothesis that Phox2a acts downstream of Phox2b during the development of most of the autonomic nervous system. The first molecular evidence has been provided that Phox2b can regulate the expression of Phox2a by directly binding to its 5' regulatory region (Flora, 2001).
The regulatory mechanisms mediating basal and inducible platelet-derived growth factor (PDGF)-A expression have been the focus of intense recent investigation, but repression of PDGF-A expression is largely unexplored. A nuclear factor that interacts with the proximal region of the PDGF-A
promoter has been isolated using bulk binding assays and chromatography techniques. Peptide mass fingerprint and supershift
analysis has revealed this DNA-binding protein to be NF1/X. NF1/X represses PDGF-A promoter-dependent transcription and endogenous mRNA expression, which is reversible by oligonucleotide decoys bearing an
NF1/X-binding site. Mutation in the DNA-binding domain of NF1/X abolishes its repression of PDGF-A promoter. NF1/X antagonizes
the activity of a known activator of the PDGF-A chain, Sp1, by inhibiting its occupancy of the proximal PDGF-A promoter. NF1/X
physically and specifically interacts with Sp1 via its subtype-specific domain and blocks Sp1 induction of the promoter. NF1/X residues
311-416 mediate NF1/X suppression of basal PDGF-A transcription, whereas residues 243-416 are required for NF1/X repression of Sp1-inducible promoter activity. These findings demonstrate that repression of PDGF-A gene transcription is governed by interplay between NF1/X and Sp1 (Rafty, 2002).
The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. This study shows that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, the role of SCL is defined as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, it was demonstrated by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. It is therefore concluded that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, these observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells (Lécuyer, 2002).
Tail formation in vertebrates involves the specification of a population of
multipotent precursors, the tailbud, which will give rise to all of the
posterior structures of the embryo. Wnts are signaling proteins that are
candidates for promoting tail outgrowth in zebrafish, although which Wnts are
involved, what genes they regulate, and whether Wnts are required for
initiation or maintenance steps in tail formation has not been resolved.
Both wnt3a and wnt8 are shown to be expressed in the
zebrafish tailbud. Simultaneous inhibition of both wnt3a and
wnt8 using morpholino oligonucleotides can completely block tail
formation. In embryos injected with wnt3a and wnt8
morpholinos, expression of genes in undifferentiated presomitic mesoderm is
initiated, but not maintained. To identify genes that might function
downstream of Wnts in tail formation, a DNA microarray screen was conducted,
revealing that sp5l, a member of the Sp1 family of zinc-finger
transcription factors, is activated by Wnt signaling. Moreover,
sp5l expression in the developing tail is dependent on both
wnt3a and wnt8 function. Supporting a role for sp5l
in tail formation, it was found that inhibition of sp5l strongly
enhances the effects of wnt3a inhibition, and overexpression of
sp5l RNA is able to completely restore normal tail development in
wnt3a morphants. These data place sp5l downstream of
wnt3a and wnt8 in a Wnt/ß-catenin signaling pathway
that controls tail development in zebrafish (Thorpe, 2005).
The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE (Late cornified envelope) multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). A complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. It is concluded that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers (Jackson, 2011).
Orderly cell cycle progression is regulated by coordinated interactions among cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylates the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending on the cell type. In these studies, overexpression of E2F-1 inhibits cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 are required for full repression of cyclin D1. Overexpression of pRB activates the cyclin D1 promoter, and a dominant interfering pRB mutant is defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter are required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at -127 binds Sp1, Sp3, and Sp4, and the distal region at -143 binds E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induces cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell (Watanabe,1998).
Cyclin A1 is a recently cloned cyclin with high level expression in meiotic cells in the testis. However, it is also frequently expressed at high levels in acute myeloid leukemia. To elucidate the regulation of cyclin A1 gene expression, the genomic structure of cyclin A1 was cloned and analyzed. It consists of 9 exons within 13 kilobase pairs. The TATA-less promoter initiates transcription from several start sites, with the majority of transcripts beginning within a 4-base pair stretch. A construct containing a fragment from -190 to +145 shows the highest transcriptional activity. Transfection of cyclin A1 promoter constructs into S2 Drosophila cells demonstrates that Sp1 is essential for the activity of the promoter. Sp1, as well as Sp3, binds to four GC boxes between nucleotides -130 and -80 as observed by gel shift analysis. Mutations in two or more of the four GC boxes decreases promoter activity by >80%. The promoter is cell cycle-regulated with highest activities found in late S and G2/M phase. Further analyses suggests that cell cycle regulation is accomplished by periodic repression of the GC boxes in G1 phase. Taken together, these data show that cyclin A1 promoter activity critically depends on four GC boxes, and members of the Sp1 family appear to be involved in directing expression of cyclin A1 in both a tissue- and cell cycle-specific manner (Muller, 1999).
The cell cycle inhibitor p21/WAF1/Cip1 (Drosophila homolog: Dacapo) is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. The p21 gene is shown to be constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter shows that it contains a distal (positions -2,300/-210) and a proximal (positions -124 to -61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important roles in the transduction of extracellular signals, such as transforming growth factor beta, activin, etc. Constitutive expression of p21 is severely reduced by a C-terminally truncated form of Smad4 that has been shown previously to block signaling through Smads. Smad3/4, and to a much lesser extent Smad2/4, causes high levels of transcriptional activation of the p21 promoter. Transactivation is compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, it has been shown that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells (Moustakas, 1998).
Calcium functions as a trigger for the switch between epithelial cell growth and differentiation. The calcium/calmodulin-dependent phosphatase calcineurin is involved in this process. Treatment of primary mouse keratinocytes with cyclosporin A, an inhibitor of calcineurin activity, suppresses the expression of terminal differentiation markers and of p21WAF1/Cip1 and p27KIP1, two cyclin-dependent kinase inhibitors that are usually induced with differentiation. In parallel with down-modulation of the endogenous genes, suppression of calcineurin function blocks induction of the promoters for the p21WAF1/Cip1 and loricrin differentiation marker genes, whereas activity of these promoters is enhanced by calcineurin overexpression. The calcineurin-responsive region of the p21 promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA box, and calcineurin induces activity of the p21 promoter through Sp1/Sp3-dependent transcription. The endogenous NFAT-1 and -2 transcription factors, major downstream targets of calcineurin, associate with Sp1 in keratinocytes in a calcineurin-dependent manner, and calcineurin up-regulates Sp1/Sp3-dependent transcription and p21 promoter activity in synergism with NFAT1/2. Thus, this study reveals an important role for calcineurin in control of keratinocyte differentiation and p21 expression, and points to a so-far-unsuspected interconnection among this phosphatase, NFATs, and Sp1/Sp3-dependent transcription (Santini, 2001).
An unresolved question relating to the current understanding of erythroid cell-specific gene expression asks how a limited number of transcriptional factors cooperate to direct high-level expression, mediated by cis-regulatory elements, yet separated over large distances within globin loci. GATA-1 (see Drosophila Serpent), the major erythroid transcription factor, activates transcription in a synergistic fashion with two Kruppel family factors: the ubiquitous protein Sp1 and the erythroid-restricted factor EKLF
(erythroid Kruppel-like factor), both of which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Kruppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. GATA-1 interacts physically with Sp1 and EKLF; interactions are mediated through their respective DNA-binding domains. GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. The formation of GATA-1-SP1 complexes has been demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops might be formed or stabilized between locus control regions and globin promoters and/or enhancers (Merika, 1995).
The cell cycle inhibitor protein p21WAF1/Cip1 (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. The transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. The Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. Gel electrophoretic mobility shift assays show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun is unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells results in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene is transactivated by overexpressed c-Jun in HepG2 cells. The above data suggest functional cooperation between c-Jun and Sp1. Physical interactions between the two factors have been demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 maps within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 have been demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivates only a chimeric promoter consisting of five tandem GAL4-binding sites when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, it was found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) is sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, these data support a mechanism for superactivation of Sp1 by c-Jun that is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters (Kardassis, 1999).
Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. This study reports that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes (Li, 2002).
Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. This study reports that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms (Dunah, 2002).
Huntington's disease (HD) is a neurodegenerative disease caused by expansion of a polyglutamine tract within the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis; recent evidence suggests a defect in Sp1-mediated transcription. Chromatin immunoprecipitation (ChIP) assays followed by real-time PCR were used to quantify the association of Sp1 with individual genes. Despite normal protein levels and normal to increased overall nuclear binding activity, Sp1 has decreased binding to specific promoters of susceptible genes in transgenic HD mouse brain, in striatal HD cells, and in human HD brain. Genes whose mRNA levels are decreased in HD have abnormal Sp1-DNA binding, whereas genes with unchanged mRNA levels have normal levels of Sp1 association. Moreover, the altered binding seen with Sp1 is not found with another transcription factor, NF-Y. These findings suggest that mutant huntingtin dissociates Sp1 from target promoters, inhibiting transcription of specific genes (Chen-Plotkin, 2006).
The forebrain consists of multiple structures necessary to achieve elaborate functions. Proper patterning is, therefore, a prerequisite for the generation of optimal functional areas. Only a few factors have been shown to control the genetic networks that establish early forebrain patterning. Using conditional inactivation, this study shows that the transcription factor Sp8 has an essential role in the molecular and functional patterning of the developing telencephalon along the anteroposterior axis by modulating the expression gradients of Emx2 and Pax6. Moreover, Sp8 is essential for the maintenance of ventral cell identity in the septum and medial ganglionic eminence (MGE). This is probably mediated through a positive regulatory interaction with Fgf8 in the medial wall, and Nkx2.1 in the rostral MGE anlage, and independent of SHH and WNT signaling. Furthermore, Sp8 is required during corticogenesis to sustain a normal progenitor pool, and to control preplate splitting, as well as the specification of cellular diversity within distinct cortical layers (Zembrzycki, 2007).
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