Sp1 Expression Plasmid Research Articles

Sp1 and Sp3 transactivate the human lipoprotein lipase gene promoter through binding to a CT element: synergy with the sterol regulatory element binding protein and reduced transactivation of a naturally occurring promoter variant

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and energy metabolism and, therefore, regulation of its expression could have an important bearing on these processes. We have identified an evolutionarily conserved 5′-CCTCCCCCC-3′ motif (from −91 to −83, CT element) in the human LPL gene promoter, deletion or mutation of which caused approximately 70–80% decrease in promoter activity. We found that Sp1 and Sp3 in THP-1 nuclear protein extracts bind specifically to this element. Co-transfection with Sp1 and Sp3 expression plasmids transactivated the LPL promoter via the CT element in Drosophila SL2 cells devoid of Sp proteins. Sp3 moderately repressed Sp1-mediated LPL promoter activation when both were co-expressed in SL2 cells. Furthermore, co-expression of an active sterol regulatory element binding protein (SREBP-1), with Sp1, but not with Sp3, synergistically activated the LPL promoter in SL2 cells. We previously reported a naturally occurring T→G substitution at position −93 of the human LPL promoter which reduces promoter activity by 40–50% in transient transfection assays. In this study, we showed that this substitution results in reduced binding affinity to Sp1/Sp3 and in diminished transactivation by Sp1/Sp3 alone and by the synergistic action of Sp1 and SREBP-1. In conclusion, recruitment of Sp1/Sp3 by the CT element may play an important role in expression of the human lipoprotein lipase gene. Synergistic transcriptional activation by Sp1 and SREBP-1 may provide a mechanism for cross-talk between cholesterol and triglyceride metabolic pathways.—Yang, W-S., and S. S. Deeb. Sp1 and Sp3 transactivate the human lipoprotein lipase gene promoter through binding to a CT element: synergy with the sterol regulatory element binding protein and reduced transactivation of a naturally occurring promoter variant. J. Lipid Res. 1998. 39: 2054–2064.

Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase kinase [corrected

The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5' region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603-609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA-protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.

Characterization of the Human Transcobalamin II Promoter: A PROXIMAL GC/GT BOX IS A DOMINANT NEGATIVE ELEMENT

Deletion and mutagenesis of the 5'-flanking region of the human transcobalamin II (TC II) transfected in human intestinal epithelial Caco-2 cells have revealed that TC II promoter activity is: (a) very weak; (b) restricted to a core region (-29 to -163) that contained multiple transcription initiation sites; (c) not dependent on other potential elements, such as a distally localized CCAAT box, a CF1, a HIP1 binding motif and a MED-1 element; (d) modulated weakly by a positive-acting GC box (-568-GAGGCGGTGC) and strongly by a proximal GC/GT overlapping box (-179 CCCCCGCCCCACCCC). Gel shift and immunosupershift analyses demonstrated that both the positive-acting GC box and the negative-acting GC/GT box were recognized by Sp1 and Sp3. Co-transfection studies using Sp1 and/or Sp3 expression plasmids revealed that while Sp1 stimulated, Sp3 repressed Sp1-mediated transactivation of TC II transcription. The proximal GC/GT box also acted as a negative element in human chronic myelogenous leukemia K-562 and HeLa cells. These results suggest that tissue/cell specific expression of the TC II gene may be controlled by the relative ratios of Sp1 and Sp3 that bind to the GC/GT box and the weak promoter activity of TC II is due to the transcriptional repression caused by the binding of Sp3 to the proximal GC/GT box.

Sp family transcription factors regulate expression of rat D2 dopamine receptor gene.

The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. We previously reported that a negative modulator is located between nucleotides -116 and -76 (D2Neg-B) in this gene and that Sp1 as well as another unknown factor bind to this region (Minowa et al., J. Biol. Chem. 269, 11656, 1994). In the present investigation employing the in situ filter detection method, we identified this factor as Sp3. Anti-Sp3 antiserum used in gel-shift assays also revealed that Sp3 binds to the D2Neg-B sequence. Cotransfection of Drosophila Schneider's SL2 cells with Sp3 or Sp1 expression plasmids in the presence of CAT reporter plasmids containing D2 promoter regions demonstrated that Sp1 increased CAT activity in a dose-dependent manner, whereas Sp3, either alone or in the presence of Sp1, failed to activate or repress transcription. On the other hand, using the TATA-containing reporter plasmid BCAT-2, Sp3 coexpression significantly repressed Sp1-induced trans-activation, although Sp3 alone was ineffective. Thus, the transcriptional activity of Sp3 is dependent on the promoter context, and the negative regulation of D2 gene expression appears quite complex and may not depend simply on known DNA-protein interactions involving only Sp1 and Sp3.

A CACCC box in the proximal exon 2 promoter of the rat insulin-like growth factor I gene is required for basal promoter activity.

The insulin-like growth factor I gene is transcribed from two promoter regions, resulting in alternative first exons in insulin-like growth factor I messenger RNAs. A previous study showed that the sequence from -73 to +44 (where +1 is the first nucleotide in the exon 2 transcription initiation cluster) contained an active exon 2 promoter, and that sequences between -73 and -36 were required for promoter activity. In the current study, the roles of two putative cis-acting elements within the -73 to +44 region in basal exon 2 promoter activity were evaluated using mutagenesis and nuclear protein-DNA binding assays. Mutation of the CCCCACCC sequence at position -53 to GAAATCCC resulted in a complete loss of promoter activity in transient transfection assays in GH3, OVCAR-3, C6, and Chinese hamster ovary (CHO) cells. A -73/+24 exon 2 promoter-luciferase construct had partial promoter activity. Mutation of a putative initiator motif surrounding the major exon 2 start site did not alter the activity of this construct. In electrophoretic mobility shift assays, a 32P-labeled oligomer extending from -73 to +44 in the exon 2 promoter was specifically bound by GH3 cell nuclear extracts. A 32P-labeled oligomer which extended from -63 to -37 in the exon 2 promoter was specifically bound by GH3 and OVCAR-3 cell nuclear extracts. These unlabeled oligomers inhibited the binding of a labeled -236/+44 exon 2 promoter fragment to OVCAR-3 nuclear extracts. Mutation of the CCCCACCC sequence prevented the unlabeled -73/+44 oligomer from inhibiting the binding of the -236/+44 fragment. An unlabeled oligomer containing a consensus activating protein-2 (AP-2)-binding site inhibited labeled -236/+44, -73/+44, and -63/-37 exon 2 promoter binding with a much lower affinity than did the respective unlabeled oligomers. Purified AP-2 protein did not bind to the exon 2 promoter fragment, nor did anti-AP-2 antibody alter the binding. Cotransfection of AP-2 expression vector did not significantly increase exon 2 promoter activity. On the other hand, an oligomer containing a consensus Sp1-binding site inhibited labeled -63/-37 exon 2 promoter binding by GH3 cell nuclear extracts with an affinity similar to that of the unlabeled -63/-37 oligomer. A mutation in the Sp1-binding site in this same oligomer resulted in a complete loss of binding affinity. Purified Sp1 bound to the -63/-37 exon 2 promoter oligomer. Addition of polyclonal antibody to Sp1 resulted in a partial supershift of the complex formed between GH3 cell and OVCAR-3 cell nuclear extracts and the labeled -63/-37 oligomer. However, in Drosophila Schneider cells, which are an experimental model for studying the ability of Sp1 to activate transcription, the -73/+44 exon 2 promoter construct was not stimulated by cotransfection with an Sp1 expression plasmid. UV cross-linking studies indicated that proteins of approximate molecular mass 125, 76, 47, and 38 kDa are bound to the proximal (-236/+44) exon 2 promoter region. It is concluded that the CCCCACCC sequence at -53 is required for exon 2 promoter activity. Moreover, specific binding of nuclear proteins to the proximal exon 2 promoter region requires the CCCCACCC sequence. Sequences downstream of the exon 2 initiation site from +24 to +44 are required for full promoter activity. However, the putative initiator surrounding the major transcription start site at +1 does not appear to be important for the strength of the proximal promoter. The CCCCACCC sequence at -53 appears to be a CACCC box, which binds zinc finger transcription factors of the Kruppel family such as Sp1, although protein factors in addition to Sp1 are required to activate exon 2 transcription through the -73/+44 proximal promoter region.

Activation of the adenovirus major late promoter by transcription factors MAZ and Sp1.

Multiple binding sites for the transcription factors MAZ and Sp1 within the adenovirus type 5 major late promoter have been identified by DNase I protection studies. In the proximal region of the promoter, both MAZ and Sp1 interact with GC-rich sequences flanking the TATA box. Two MAZ binding sites are centered at -18 and -36 relative to the transcriptional initiation site. Sp1 bound only to the -18 GC-rich sequence. Several sites of interaction were also evident in the distal region of the promoter. Both MAZ and Sp1 interacted with a sequence centered at -166, and MAZ bound weakly to an additional site centered at -130. Overexpression of MAZ or Sp1 activated expression from the major late promoter in transient expression assays. Mutational analysis of the GC-rich sequences in the major late promoter suggested that a primary target of MAZ activation is the GC-rich sequences flanking the TATA sequence, whereas Sp1 requires the distal GC-rich sequence elements to stimulate gene expression. This activation is enhanced by the adenovirus E1A protein, and evidence for interaction between E1A and both transcription factors was obtained by using an immunoprecipitation assay. Activation by MAZ and Sp1 also was observed in transfection studies using the complete adenovirus type 5 genome as the target. Increased levels of late mRNA from both the L1 and L5 regions were observed when MAZ or Sp1 expression plasmids were transfected with viral DNA. Unexpectedly, activation of the major late promoter by MAZ and Sp1 was detected irrespective of whether the viral DNA could replicate.

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.

Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.

Transcriptional Regulation of the Gene Coding for Human Protein C

The promoter for the gene coding for human protein C has been characterized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream from the first intron/exon boundary along with two minor sites. Functional characterization of 1528 base pairs at the 5'-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (nucleotides -25 to +9) contained binding sites for at least two different transcription factors, including a hepatic nuclear factor 1-binding site (-10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (-25 to -11). A second major region (PCE1) (+12 to +30) appeared to be a unique, liver-specific regulatory sequence. An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Sp1 expression plasmid. Specific mutations in these promoter elements reduced transcriptional activity and abolished the binding of hepatic nuclear proteins. Finally, a strong silencer element (PCS1) (between -162 and -82) and two possible liver-specific enhancer regions (PCE2 and PCE3), which interact coordinately with the promoter elements, were also found (between -1462 and -162).

Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif.

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from -53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483-8489]. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5' flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA-protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1-binding consensus sequence. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.

Interaction of Sp1 with the human gamma globin promoter: binding and transactivation of normal and mutant promoters

We have analyzed the binding of Sp1, a ubiquitously expressed transactivator, to the promoter region of the gamma genes. Low-affinity Sp1 sites were found at -50 and -200. A high-affinity site was detected at -140, over the CACCC sequence. To analyze the function of these sites, Drosophila SL-2 cells, which lack Sp1, were cotransfected with an Sp1 expression plasmid and gamma globin promoter-CAT constructs. In these assays, the gamma promoter was significantly stronger in the presence than in the absence of Sp1. Thus, the three Sp1 sites in the gamma promoter allow binding as well as transactivation of the promoter. The majority of this transactivation was due to the strong binding site at -140 because introduction of a point mutation at -144 (CACCC----AACCC) reduced Sp1-dependent promoter strength by 57%. Analysis of the -200 region suggested that in the wild-type promoter, Sp1 binding at this site contributes little to promoter strength. However, a point mutation (-198 T----C) associated with hereditary persistence of fetal hemoglobin (HPFH) dramatically increased the affinity of this site for Sp1 and significantly increased Sp1 dependent promoter strength in SL-2 cells. Three other point mutations associated with HPFH did not significantly affect the interaction of Sp1 with the - 200 region.

Interaction of Sp1 With the Human γ Globin Promoter: Binding and Transactivation of Normal and Mutant Promoters

AbstractWe have analyzed the binding of Sp1, a ubiquitously expressed transactivator, to the promoter region of the γ genes. Low-affinity Sp1 sites were found at -50 and -200. A high-affinity site was detected at -140, over the CACCC sequence. To analyze the function of these sites, Drosophila SL-2 cells, which lack Sp1, were cotransfected with an Sp1 expression plasmid and γ globin promoter-CAT constructs. In these assays, the γ promoter was significantly stronger in the presence than in the absence of Sp1. Thus, the three Sp1 sites in the γ promoter allow binding as well as transactivation of the promoter. The majority of this transactivation was due to the strong binding site at -140 because introduction of a point mutation at -144 (CACCC → AACCC) reduced Sp1-dependent promoter strength by 57%. Analysis of the -200 region suggested that in the wild-type promoter, Sp1 binding at this site contributes little to promoter strength. However, a point mutation (-198 T→C) associated with hereditary persistence of fetal hemoglobin (HPFH) dramatically increased the affinity of this site for Sp1 and significantly increased Sp1 dependent promoter strength in SL-2 cells. Three other point mutations associated with HPFH did not significantly affect the interaction of Sp1 with the -200 region.