Ubiquitous Transcription Factor Sp1 Research Articles

Redirection of the Transcription Factor SP1 to AT Rich Binding Sites by a Synthetic Adaptor Molecule

AbstractThe ubiquitous transcription factor SP1 binds to a GC rich consensus sequence. Here we describe an adaptor molecule that mediates binding of SP1 to a non‐cognate DNA site rich in AT. The adaptor is comprised of a Dervan‐type hairpin polyamide with high affinity to an AT rich hexamer duplex. It also carries a 27mer DNA that contains the SP1 consensus sequence. The synthesis and purification of the polyamide‐DNA conjugate is reported. Pulldown experiments and western blot analysis demonstrate adaptor mediated binding of SP1 to the hexamer duplex TTGTTA.

Gata3 cooperates with Gcm2 and MafB to activate parathyroid hormone gene expression by interacting with SP1

Haploinsufficiency of the Gata3 gene, which encodes a zinc-finger transcription factor, is associated with the disorder hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome in humans. However, the roles of Gata3 in transcriptional regulation in the parathyroid glands are not well-understood. In this study, we show that Gata3 activates transcription of parathyroid hormone (PTH), which is secreted from parathyroid glands and is critical for regulating serum calcium and phosphate homeostasis. Gata3 interacted with Gcm2 and MafB, two known transcriptional regulators of parathyroid development, and synergistically stimulated the PTH promoter. An SP1-binding element (GC box) located within the PTH-promoter proximal region was critical for activating transcription by Gata3. In addition, the ubiquitous transcription factor SP1 also interacted with Gata3 as well as MafB and Gcm2, and HDR syndrome-associated Gata3 mutants were defective in activating the PTH promoter. These results suggest that Gata3 is a critical regulator of PTH gene expression.

Spatial organization of housekeeping genes in interphase nuclei

Spatial organization of the eukaryotic genome is tightly connected to its functioning. In particular, the interaction of gene promoters with remote enhancer elements in active chromatin hubs, as well as the recruitment of genes to the common transcription factories plays an important role in regulation of gene transcription. Most of works related to the analysis of spatial interaction of genome regulatory elements relies on models of tissue-specific genes. Meanwhile, it remains unclear to which extent the spatial organization of chromosomes is guided by house-keeping genes that are transcribed in most of cell types and outnumber the transcribed tissue-specific genes. To address this question, we used the 4C technique to characterize genome-wide the spatial contacts of the chicken house-keeping genes CARHSP1 and TRAP1 situated on chromosome 14. The promoters of these genes had an increased frequency of interaction with chromosome regions enriched in CpG islands and binding motifs for the ubiquitous transcription factor Sp1, both of which mark promoters of house-keeping genes, and overall with transcriptionally active regions. By contrast, the analysis of interaction of a gene poor region of chromosome 14 revealed no such preferences. The evidence for the interaction of house-keeping gene promoters were also obtained in independent cytological experiments aimed at visualization of non-methylated CpG islands in individual nuclei of human cells, which showed clustering of CpG islands in the nuclear space. Altogether, the results of our work suggest that the interaction of house-keeping genes constitutes an important factor that determines the spatial organization of interphase chromosomes.

Sp1 and KLF15 regulate basal transcription of the human LRP5 gene

BackgroundLRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear.ResultsIn order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs.ConclusionsBoth KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.

Variable structure motifs for transcription factor binding sites

BackgroundClassically, models of DNA-transcription factor binding sites (TFBSs) have been based on relatively few known instances and have treated them as sites of fixed length using position weight matrices (PWMs). Various extensions to this model have been proposed, most of which take account of dependencies between the bases in the binding sites. However, some transcription factors are known to exhibit some flexibility and bind to DNA in more than one possible physical configuration. In some cases this variation is known to affect the function of binding sites. With the increasing volume of ChIP-seq data available it is now possible to investigate models that incorporate this flexibility. Previous work on variable length models has been constrained by: a focus on specific zinc finger proteins in yeast using restrictive models; a reliance on hand-crafted models for just one transcription factor at a time; and a lack of evaluation on realistically sized data sets.ResultsWe re-analysed binding sites from the TRANSFAC database and found motivating examples where our new variable length model provides a better fit. We analysed several ChIP-seq data sets with a novel motif search algorithm and compared the results to one of the best standard PWM finders and a recently developed alternative method for finding motifs of variable structure. All the methods performed comparably in held-out cross validation tests. Known motifs of variable structure were recovered for p53, Stat5a and Stat5b. In addition our method recovered a novel generalised version of an existing PWM for Sp1 that allows for variable length binding. This motif improved classification performance.ConclusionsWe have presented a new gapped PWM model for variable length DNA binding sites that is not too restrictive nor over-parameterised. Our comparison with existing tools shows that on average it does not have better predictive accuracy than existing methods. However, it does provide more interpretable models of motifs of variable structure that are suitable for follow-up structural studies. To our knowledge, we are the first to apply variable length motif models to eukaryotic ChIP-seq data sets and consequently the first to show their value in this domain. The results include a novel motif for the ubiquitous transcription factor Sp1.

Selective Activation of Trophoblast-specific PLAC1 in Breast Cancer by CCAAT/Enhancer-binding Protein β (C/EBPβ) Isoform 2

The trophoblast-specific gene PLAC1 (placenta-specific 1) is ectopically expressed in a wide range of human malignancies, most frequently in breast cancer, and is essentially involved in cancer cell proliferation, migration, and invasion. Here we show that basal activity of the PLAC1 promoter is selectively controlled by ubiquitous transcription factor SP1 and isoform 2 of CCAAT/enhancer-binding protein beta that we found to be selectively expressed in placental tissue and cancer cells. Binding of both factors to their respective elements within the PLAC1 promoter was essential to attain full promoter activity. Estrogen receptor alpha (ERalpha) signaling further augmented transcription and translation of PLAC1 and most likely accounts for the positive correlation between PLAC1 expression levels and the ERalpha status we observed in primary breast cancer specimens. DNA affinity precipitation and chromatin immunoprecipitation assays revealed that transactivation of the PLAC1 promoter by ligand-activated ERalpha is based on a nonclassical pathway independent of estrogen-response elements, by tethering of ERalpha to DNA-bound CCAAT/enhancer-binding protein beta-2, and SP1. Our findings provide first insight into a novel and hitherto unknown regulatory mechanism governing selective activation of trophoblast-specific gene expression in breast cancer.

Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

BackgroundThe ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression.Methodology and Principal FindingsWe made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis.ConclusionThis study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

Physical and Functional Interactions between Liver X Receptor/Retinoid X Receptor and Sp1 Modulate the Transcriptional Induction of the Human ATP Binding Cassette Transporter A1 Gene by Oxysterols and Retinoids

The lipid transporter ATP binding cassette transporter A1 (ABCA1) promotes the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I and thus initiates the biogenesis of high-density lipoprotein (HDL). The expression of the ABCA1 gene is controlled, coordinately with other genes of HDL metabolism, by liver X receptor/retinoid X receptor (LXR/RXR) heterodimers and their ligands oxysterols and retinoids. In the present study, we show that the oxysterol/retinoid-induced transcription of the ABCA1 gene is modulated by the ubiquitous transcription factor Sp1 that binds to the proximal ABCA1 promoter, adjacently to the LXR/RXR responsive element. The response of the ABCA1 gene to oxysterols/retinoids as well as the ligand-inducible recruitment of Sp1 and RXRalpha/LXRalpha heterodimers to the ABCA1 promoter was blocked by mithramycin A, a well-known Sp1 inhibitor. Using SL2 cells which lack endogenous Sp1, we showed that activation of the ABCA1 promoter by LXRalpha/RXRalpha heterodimers and their ligands requires Sp1. Functional interactions between these factors were demonstrated using the GAL4 transactivation system. Using both in vitro and in vivo assays, we show that physical interactions between Sp1 and LXRalpha require the N-terminal region of LXRalpha, which includes the AF1 and DNA binding domains and two different domains of Sp1: the transactivation domain B and the DNA binding domain. Overall, the present study revealed a novel mechanism of regulation of the human ABCA1 transporter which involves synergistic interactions between oxysterol/retinoid-inducible hormone nuclear receptors and the transcription factor Sp1.

Sp1 expression is disrupted in schizophrenia; a possible mechanism for the abnormal expression of mitochondrial complex I genes, NDUFV1 and NDUFV2.

BackgroundThe prevailing hypothesis regards schizophrenia as a polygenic disease, in which multiple genes combine with each other and with environmental stimuli to produce the variance of its clinical symptoms. We investigated whether the ubiquitous transcription factor Sp1 is abnormally expressed in schizophrenia, and consequently can affect the expression of genes implicated in this disorder.Methodology/Principal FindingsmRNA of Sp1 and of mitochondrial complex I subunits (NDUFV1, NDUFV2) was analyzed in three postmortem brain regions obtained from the Stanley Foundation Brain Collection, and in lymphocytes of schizophrenic patients and controls. Sp1 role in the transcription of these genes was studied as well. Sp1 was abnormally expressed in schizophrenia in both brain and periphery. Its mRNA alteration pattern paralleled that of NDUFV1 and NDUFV2, decreasing in the prefrontal cortex and the striatum, while increasing in the parieto-occipital cortex and in lymphocytes of schizophrenic patients as compared with controls. Moreover, a high and significant correlation between these genes existed in normal subjects, but was distorted in patients. Sp1 role in the regulation of complex I subunits, was demonstrated by the ability of the Sp1/DNA binding inhibitor, mithramycin, to inhibit the transcription of NDUFV1 and NDUFV2, in neuroblastoma cells. In addition, Sp1 activated NDUFV2 promoter by binding to its three GC-boxes. Both activation and binding were inhibited by mithramycin.Conclusions/SignificanceThese findings suggest that abnormality in Sp1, which can be the main activator/repressor or act in combination with additional transcription factors and is subjected to environmental stimuli, can contribute to the polygenic and clinically heterogeneous nature of schizophrenia.

Targeted Therapy of DNA Tumor Virus-Associated Cancers Using Virus-Activated Transcription Factors

DNA tumor virus-mediated tumorigenic processes typically involve functional inactivation of cellular tumor suppressors pRB and p53 by viral oncoproteins, with concomitant activation of oncogenic transcription factors such as E2Fs. This feature could be exploited to design a treatment for corresponding malignancies. Here, we report a gene therapy strategy for DNA tumor virus-associated cancers using a synthetic, E2F-regulated gene expression system named pESM6. This system contains multimerized E2F-responsive elements in combination with the binding sites for ubiquitous transcription factors Sp1 and CTF/NF1. pESM6 could drive a high-level transgene expression comparable to that of the CMV IE promoter and exert constitutive activity in cells expressing DNA tumor viral oncogenes. In contrast, it was effectively repressed by pRB and thus only minimally active in nontransformed cells. Expression of cytosine deaminase from pESM6 resulted in a highly efficient and specific killing of HPV-transformed fibroblasts (C3) after treatment with the prodrug 5-fluorocytosine. Also, an effective tumor mass reduction was observed when the vector was injected directly into C3 tumors implanted in C57BL/6 mice. pESM6 showed a superior performance throughout these experiments compared to the previously known E2F-regulated gene vector. These results clearly demonstrate the potential usability of pESM6 for the gene therapy of DNA tumor virus-associated cancers.

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells

The transactivation function of the human androgen receptor (AR) can be regulated by several coregulators that may be either positive or negative. Ubiquitous transcription factor Sp1 not only regulates the basal expression of the AR but also acts as its coregulator. Our previous study has shown that quercetin, one of the main polyphenols, can effectively inhibit the expression and function of the AR. The present study is to address if quercetin may affect Sp1's action on AR transactivation activity in human prostate adenocarcinoma cell lines, LNCaP and PC-3. First, we showed that indeed in transient transfections Sp1 could enhance transcriptional activity of the AR promoter and of androgen upregulated gene promoters, i.e. the prostate-specific antigen and the hK2 genes. Interestingly, the enhancing activity of Sp1 could be repressed by quercetin. The gel shift and western blot analyses indicated that the specific DNA motif binding activity of Sp1 and its protein levels were not altered by quercetin. However, the state of interaction of Sp1 with the AR treated by quercetin plus androgen was different from that by androgen treatment or none as demonstrated by coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down assays. Moreover, we showed that quercetin caused changes in post-translational modification of AR protein. The above findings strongly suggest that changes induced by quercetin in post-translational modification of the AR and in states of physical interaction of Sp1 with the AR may be critical for the attenuation of AR's function.

Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation

The silencer factor NRSF/REST has been reported to restrict expression to neurons of a variety of genes, including that encoding NMDA receptor subunit type 1 (NR1), by suppressing transcription in nonneuronal cells. However, we recently reported that in addition to the absence of NRSF/REST-binding activity, another neuron-specific mechanism is necessary for high level expression of the NR1 gene in neurons. In this study, we explored the mechanism of induction of NR1 promoter activity during neuronal differentiation of the P19 cell line. We identified a 27 base pair GC-rich region in the promoter as an important element responsible for induction of the NR1 gene after neuronal differentiation. We found that the ubiquitous transcription factors SP1 and MAZ bind to this GC-rich region. Surprisingly, the binding activities of SP1 and MAZ are not remarkably changed after neuronal differentiation. Mutations in the SP1 and MAZ sites impair binding of SP1 and MAZ proteins and also decrease NR1 promoter activity. These findings suggest that SP1 and MAZ mediate enhancement of NR1 promoter activity during neuronal differentiation despite the fact that their binding activity does not change.

Characterization of the human ABO gene promoter in erythroid cell lineage.

The human ABO blood group system is important in transfusion and organ transplantation. Although the molecular basis of the ABO gene has been established, recent studies have begun to characterize the mechanism of the ABO gene expression. Transient transfection assays were carried out in human erythroleukaemia HEL cells and human gastric cancer KATOIII cells. Electrophoretic mobility shift assays were performed using nuclear extracts derived from both cells. Our characterization of the 5'-upstream sequence of the ABO genes indicated that the region between -117 and +31 is essential to direct expression of a reporter gene in erythroid cells. We show that a sequence located between positions -22 and -14 of the ABO promoter binds a ubiquitous transcription factor Sp1 or Sp1-like protein(s). Mutation of this site abrogates binding of those factors and reduces the ability of the ABO promoter to function in erythroleukaemia cells and gastric cancer cells. The expression of the ABO promoter appears to be influenced by the binding of Sp1 or Sp1-like protein(s) in both erythroid and epithelial cell lineages.

Sp1 is Proapoptotic

Apoptosis of vascular smooth muscle cells (SMC) at the sites of atherosclerotic plaques may result in plaque rupture and thrombosis. Kavurma et al. used two immortalized, but phenotypically different, SMC cell lines (WKY12-22 and WKY3M-22) to study apoptosis. WKY12-22 cells are derived from rat pup arteries and will grow in the absence of exogenous growth factors; WKY3M-22 cells are derived from adult rat arteries and are dependent on plasma-derived serum for growth. WKY12-22 cells express higher levels of the ubiquitous transcription factor Sp1 and undergo spontaneous apoptosis at a greater frequency than seen in the WKY3M-22 cells. FasL is also expressed at higher levels in the WKY12-22 cells, suggesting a possible mechanism for the increased apoptosis. Overexpression of Sp1 in either cell type increases the expression of FasL. On the basis of reporter genes assays and electrophoretic mobility shift assays, Sp1 is a transcriptional activator of FasL and binds to the Sp1 consensus site in the FasL promoter. It is noteworthy that the inhibitor of DNA topisomerase I camptothecin (CAM), a known proapoptotic chemical, enhances the phosphorylation of Sp1 and increases the production of FasL and apoptosis in these cells. Thus, Sp1, a ubiquitous transcription factor, may represent a point of control for the apoptotic pathway. M. M. Kavurma, F. S. Santiago, E. Bonfoco, L. M. Khachigian, Sp1 phosphorylation regulates apoptosis via extracellular FasL-Fas engagement. J. Biol. Chem. 276 , 4964-4971 (2001). [Abstract] [Full Text]

SF-1 (Steroidogenic Factor-1), C/EBP (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells

SF-1 (Steroidogenic Factor-1), C/EBP (CCAAT/Enhancer Binding Protein), and Ubiquitous Transcription Factors NF1 (Nuclear Factor 1) and Sp1 (Selective Promoter Factor 1) Are Required for Regulation of the Mouse Aldose Reductase-Like Gene (AKR1B7) Expression in Adrenocortical Cells

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Role of Smad Proteins and Transcription Factor Sp1 in p21Waf1/Cip1 Regulation by Transforming Growth Factor-β

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21(WAF1/Cip1) (p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-beta, Smad3 and Smad4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that Smad3 and Smad4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaT p21 gene by TGF-beta1 is further enhanced after overexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad3 and Smad4 and the inhibitory Smad7 all inhibit p21 induction by TGF-beta1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas Smad proteins do not. In support of this finding, we show that DNA-binding domain mutants of Smad3 and Smad4 are capable of transactivating the p21 promoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4 and Sp1 in SL-2 cells or co-incubation of phosphorylated Smad3, Smad4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with Smad2, Smad3, and weakly with Smad4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with Smad proteins. In conclusion, Smad proteins play important roles in regulation of the p21 gene by TGF-beta, and the functional cooperation of Smad proteins with Sp1 involves the physical interaction of these two types of transcription factors.

Induction of c-Src in Human Blood Monocytes by Anti-CD98/FRP-1 mAb in an Sp1-Dependent Fashion

Freshly isolated human blood monocytes expressed neither c-src mRNA nor c-Src. However, when monocytes were incubated with anti-CD98 heavy chain (HC) mAb, expression of c-src mRNA, c-Src, and activated c-Src was induced. Many binding sites for the ubiquitous transcription factor Sp1 were identified in the promoter region of the c-src gene. Surprisingly, Sp1 and Sp1 mRNA were not found in monocytes that were freshly isolated or incubated with control antibody. Stimulation with anti-CD98HC mAb also resulted in the expression of Sp1 and its translocation to the nucleus. Herbimycin A, genistein, manumycin A, PD-98059, SB203580, and HBJ127 suppressed CD98HC-mediated c-src and Sp1 mRNA induction. On the contrary, H-7, Wortmannin, HA1077, and Y-27632 showed no effect on c-Src and Sp1 induction. Furthermore, anti-CD98HC mAb induced activation of tyrosine kinases and ERK kinases. These findings suggest that the tyrosine kinase(s)-Ras-MAPK-Sp1 pathway(s) is involved in CD98HC-mediated induction of c-Src in human blood monocytes.

Cloning and characterization of the promoter of murine cytohesin-1 gene

Mouse cytohesin-1 (CTH-1) is a guanine nucleotide exchange factor, specific for ADP ribosylation factors. The CTH-1 gene promoter was characterized by deletion mapping and mutational analysis. The region between −101 and −38 relative to the transcription start site showed the essential promoter activity when transfected into both NIH3T3 and COS7 cells. The nucleotides of the core promoter region contain three tandem GC boxes, which can offer potential binding sites for the ubiquitous transcription factor Sp-1 family. Mutational analysis revealed that these tandem GC boxes are indispensable for activation of the gene transcription.