Sp Family Members Research Articles

Transcriptional regulation of the human toll-like receptor 2 gene in monocytes and macrophages.

This report investigates the molecular basis for tissue-restricted and regulated expression of the pattern recognition receptor Toll-like receptor (TLR)2 in human monocytes and macrophages. To define the proximal promoter, the full 5'-sequence and transcriptional start sites of TLR2 mRNA were determined. The human TLR2 gene was found to consist of two 5' noncoding exons followed by a third coding exon. Alternative splicing of exon II was detected primarily in human blood monocytes. The proximal promoter, exon I, and part of intron I were found to be located in a CpG island. Although CpG methylation of the proximal human TLR2 promoter in cell lines correlated with TLR2 repression, the promoter was unmethylated in primary cells, indicating that CpG methylation does not contribute to the cell-type specific expression of human TLR2 in normal tissues. The promoter sequence contains putative binding sites for several transcription factors, including Sp1 and Ets family members. Reporter gene analysis revealed a minimal promoter of 220 bp that was found to be regulated by Sp1, Sp3, and possibly PU.1. Interestingly, no sequence homology was detected between human and murine TLR2 promoter regions. In contrast to murine TLR2, expression of human TLR2 in monocytes/macrophages is not induced by the proinflammatory stimuli LPS or macrophage-activating lipopeptide-2, and reporter activity of the promoter was not enhanced by stimuli-induced NF-kappaB activation in THP-1 or MonoMac-6 cells. Our findings provide an initial definition of the human TLR2 promoter and reveal profound differences in the regulation of an important pattern recognition molecule in humans and mice.

Molecular cloning and functional analysis of the human Na(+)/H(+) exchanger NHE3 promoter.

Na(+)/H(+) exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5'-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5'-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in -95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from -78 to -56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2alpha antibody. Our studies suggest that the NHE3 expression is regulated by a combination of cis elements and their cognate transcription factors that include the AP-2 and Sp1 family members.

Reciprocal regulation of beta(1)-adrenergic receptor gene transcription by Sp1 and early growth response gene 1: induction of EGR-1 inhibits the expression of the beta(1)-adrenergic receptor gene.

The beta(1)-adrenergic receptor (beta(1)-AR) plays a key role in regulating heart rate and contractility in response to catecholamines. Our studies have focused on defining the factors that regulate the expression of the beta(1)-AR gene. We determined that a 65-base-pair (bp) region in the beta(1)-AR promoter between bp -394 and bp -330 directs basal transcription. An element located between -377 and -365 can bind Sp1 and Sp3. In Drosophila melanogaster SL2 cells, Sp1 stimulated the expression of the beta(1)-AR promoter, whereas Sp3 was unable to activate transcription. Site-directed mutagenesis indicated that an intact Sp1-binding site is essential for maintaining the activity of the basal promoter. In addition to binding Sp family members, the nucleotides between -381 and -367 can bind the zinc-finger transcription factor Egr-1. The Egr-1 and Sp1 binding sites are partially overlapping and their binding sequence is conserved among mammalian beta(1)-AR genes. The induction of Egr-1 in rat neonatal ventricular myocytes with phorbol-12-myristate-13-acetate or in HeLa S3 cells by regulated expression of Egr-1 in a tetracycline-responsive promoter, suppressed expression from the beta(1)-AR promoter. Overexpression of Sp1 in SK-N-MC cells increased beta(1)-AR mRNA by 2.4-fold, whereas overexpression of Egr-1 reduced beta(1)-AR mRNA by 40%. Coexpression of Egr-1 with Sp1 reduced Sp1-mediated up-regulation of beta(1)-AR mRNA by 60%. Mutagenesis revealed that an intact Sp1-binding site is essential for observing transcriptional repression by Egr-1 and that Egr-1 suppressed the transcription of the beta(1)-AR gene by competing with Sp1 for binding to their overlapping sites. These results reveal a novel physiologically relevant transcriptional mechanism for reciprocal regulation of beta(1)-AR gene expression.

Regulation of Human Osteocalcin Promoter in Hormone-independent Human Prostate Cancer Cells

Osteocalcin (OC) is a small (6 kDa) polypeptide whose expression was thought to be limited to mature osteoblasts. The discovery of OC expression in prostate cancer specimens led us to study the regulation of OC gene in androgen-independent metastatic human prostate PC3 cells. An 800-bp human OC (hOC) promoter-luciferase construct exhibited strong basal and vitamin D-induced activity in OC-positive human prostate and osteosarcoma cell lines. Through deletion analysis of the hOC promoter, the functional hierarchy of the cis-acting elements, OSE1, OSE2, and AP-1/VDRE, was established in PC3 cells (OSE1 > AP-1/VDRE > OSE2). By juxtaposing dimers of these 3 cis-elements, we produced a minimal hOC promoter capable of displaying high tissue specific activity in prostate cancer cells. Our study demonstrated three groups of transcription factors, Runx2, JunD/Fra-2, and Sp1, responsible for the high hOC promoter activity in PC3 cells by binding to the OSE2, AP-1/VDRE, and OSE1 elements, respectively. Among the three groups of transcription factors, the expression levels of Runx2 and Fra-2 are higher in the OC-positive PC3 cells and osteoblasts, compared with the OC-negative LNCaP cells. Interestingly, unlike the mouse OC promoter, the OSE1 site in hOC promoter is regulated by members of Sp1 family instead of the osteoblast-specific factor Osf1. The molecular basis for androgen-independent prostate cancer cells behaving like mature osteoblasts may be explained by the interplay and coordination of these transcription factors under the tight regulation of autocrine and paracrine mediators.

Progesterone receptors and Sp/Krüppel-like family members in the uterine endometrium .

Sp/KLF family members, of which at least 20 distinct nuclear proteins are known to date, serve as transcription factors by binding to GC-enriched sites within target gene promoters. These molecules regulate diverse cellular processes such as proliferation, differentiation, and apoptosis, via their interactions with various other nuclear proteins in promoter- and cell-context-dependent pathways to effect transactivation or transrepression. Recent studies indicate that Sp-family members may functionally interact with ligand-activated nuclear receptors, especially those for steroid hormones. This review provides a discussion of the putative mechanisms and important participants involved in the crosstalk between selected members of this family and the receptor for the pregnancy hormone progesterone, and the relevant outcomes to the control of the transcriptional network in the uterine endometrium.

Sp family members stimulate transcription of the hex gene via interactions with GC boxes.

The 5'-flanking region of the mouse Hex gene was examined in order to identify transcription factors regulating its expression in hepatocytes and haematopoietic cells. We have identified two further GC boxes (GC boxes 3 and 4 at nucleotide positions -149 to -140 and -79 to -70, respectively), i.e. in addition to the two previously determined ones (GC boxes 1 and 2 at nucleotide positions -197 to -188 and -176 to -167, respectively). Luciferase reporter assays revealed that all four GC boxes are transcriptionally active in both MH(1)C(1) rat hepatoma and K562 human chronic myelogenous leukemia cells. Electrophoretic mobility shift assays with specific competitors and antibodies showed that members of the Sp family, namely Sp1 and Sp3, bind to these GC boxes. Overexpression of Sp1 and Sp3 in Drosophila SL2 cells stimulated transcription of the Hex gene through interactions with GC boxes 1 to 4, Sp1 being a more potent activator than Sp3. Thus, we conclude that Sp1 and Sp3 stimulate transcription of the Hex gene in both MH(1)C(1) and K562 cells.

Characterization of Sp1, AP-1, CBF and KRC binding sites and minisatellite DNA as functional elements of the metastasis-associated mts1/S100A4 gene intronic enhancer.

The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.

Cooperative Regulation of CYP3A5 Gene Transcription by NF-Y and Sp Family Members

The purpose of this study was to clarify the mechanism(s) responsible for the transcriptional regulation of the human CYP3A5 gene. It was found that a region from nucleotides −90 to −40 was involved in the transcriptional activity of the CYP3A5 gene by transfection of a series of 5′-truncated promoter–luciferase chimeric genes into human hepatoma HepG2 cells. A gel shift assay using nuclear extracts prepared from HepG2 cells showed that nuclear factor-Y (NF-Y) and specificity protein (Sp) 1 and Sp3 bound to CCAAT box (−78/−68) and a basic transcription element (BTE) (−67/−46) in the CYP3A5 gene. Furthermore, introduction of mutations in the CCAAT box, the BTE, or both elements decreased the transcriptional activity to 10, 21, or 4% of that seen with the intact gene. Thus, we conclude that the transcription of the CYP3A5 gene is cooperatively regulated by NF-Y, Sp1, and Sp3 in HepG2 cells.

Necdin acts as a transcriptional repressor that interacts with multiple guanosine clusters

Necdin is a growth suppressor expressed predominantly in postmitotic neurons, and ectopic expression of this protein suppresses cell growth. Here we report that Necdin directly binds to specific DNA sequences and serves as a transcriptional repressor. Polyhistidine-tagged Necdin was used for selection of random-sequence oligonucleotides by polymerase chain reaction-based amplification. Necdin recognized guanosine (G)-rich sequences that encompass multiple G clusters and intervening mono- or di-nucleotides of A, T and C. These sequences, termed GN boxes, resemble multiply aligned forms of the canonical GC box which is recognized by Sp family members. Necdin directly bound to a GN box consisting of contiguous two GC boxes with four G clusters, but not to a single GC box with two G clusters, whereas Sp1 bound to both. In a reporter system using Drosophila Schneider Line 2 cells, Necdin repressed Sp1-dependent activity of mouse c-myc P1 promoter that contains a typical GN box. Deletion of the GN box from the c-myc P1 promoter or its conversion to the single GC box abolished the Necdin-dependent repression. These results suggest that Necdin modulates gene transcription via the GN box that is potentially recognized by GC box-targeting Sp family members.

Characterization of the Promoter Region of the Human Peroxisomal Multifunctional Enzyme Type 2 Gene

Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3α,7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24-keto)-3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (−151/−142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2α and AP2γ were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.

Regulation of human monoamine oxidase B gene by Sp1 and Sp3.

The human monoamine oxidase (MAO) B plays a major role in the degradation of biogenic and dietary amines such as phenylethylamine, benzylamine, dopamine, and tyramine. We previously showed that the -246/-99 MAO B promoter region exhibited the highest activity and contained two clusters of overlapping Sp1 sites, a CACCC element and a TATA box. Here, using a series of 10 deletion constructs of the 2-kilobase pair 5'-flanking sequence, we identified additional potential regulatory elements, including activator proteins 1 and 4, CAAT, GATA, upstream stimulatory factor (USF), estrogen receptor (ER), and sex-determining region Y-box 5 (SOX5). Analysis of nine site-directed mutations of -246/-99 region reveals that both clusters of Sp1 sites contribute positively whereas the CACCC element contributes negatively to the transcriptional activity. Gel shift analysis demonstrates that in addition to Sp1, Sp3 can interact with both clusters of Sp1 sites. Cotransfection experiments show that Sp1 and its closely related family member Sp4 can trans-activate MAO B promoter activity through the proximal cluster of Sp1 sites and its activation can be repressed by the over-expression of Sp3 and a related family member BTEB2. These results suggest that the binding to the overlapping Sp1 sites by various members of Sp family is important for the regulation of the MAO B gene expression.

Interaction of EGR-1 and Sp family members is involved in the transcriptional regulation of the human NHE-2 gene promoter

Interaction of EGR-1 and Sp family members is involved in the transcriptional regulation of the human NHE-2 gene promoter

Interaction of EGR-1 and Sp family members is involved in the transcriptional regulation of the human NHE-2 gene promoter

Interaction of EGR-1 and Sp family members is involved in the transcriptional regulation of the human NHE-2 gene promoter

Identification and dissection of an enhancer controlling epithelial gene expression in skin.

Keratins 14 and 5 are the structural hallmarks of the basal keratinocytes of the epidermis and outer root sheath (ORS) of the hair follicle. Their genes are controlled in a tissue-specific manner and thus serve as useful tools to elucidate the regulatory mechanisms involved in keratinocyte-specific transcription. Previously we identified several keratinocyte-specific DNase I hypersensitive sites (HSs) in the 5' regulatory sequences of the K14 gene and showed that a 700-bp regulatory domain encompassing HSs II and III can confer epidermal and ORS-specific gene expression in transgenic mice in vivo. Although HS II harbored much of the transactivation activity in vitro, it was not sufficient to restrict expression to keratinocytes in vivo. We now explore the HS III regulatory element. Surprisingly, this element on its own confers gene expression to the keratinocytes of the inner root sheath (IRS) of the hair follicle, whereas a 275-bp DNA fragment containing both HSs II and III shifts the expression from the IRS to the basal keratinocytes and ORS in vivo. Electrophoretic mobility-shift assays and mutational studies of HSs III reveal a role for CACCC-box binding proteins, Sp1 family members, and other factors adding to the list of previously described factors that are involved in keratinocyte-specific gene expression. These studies highlight a cooperative interaction of the two HSs domains and strengthen the importance of combinatorial play of transcription factors that govern keratinocyte-specific gene regulation.

Modulation of Sp1 activity by a cyclin A/CDK complex

Transcription factors of the Sp1 family are targets of several regulatory pathways and can induce or inhibit gene expression. Here we show that Sp1 is associated with a histone 1 kinase activity. This activity is growth regulated and correlates with the expression of cyclin A. Co-immunoprecipitation experiments demonstrate, that Sp1 interacts with cyclin A and can be phosphorylated by a cyclin A associated kinase. The interaction is direct and requires the zinc-finger region of Sp1 and the amino-terminal domain of cyclin A. Over-expression of cyclin A enhances the expression of a reporter gene controlled by an Sp1 responsive promoter. Addition of olomoucine, a specific inhibitor of CDK2 and CDC2 activity on the other hand reduces the expression of the reporter. Electrophoretic mobility shift assays suggest that this is due to a reduction of the DNA-binding ability of Sp1 family members. Our results indicate that phosphorylation of Sp1 and other members of the family by a cyclin A/CDK complex may play a role in the growth and cell cycle regulation of its transcriptional activity.

Synergistic transcriptional activation by Sox10 and Sp1 family members

Neuronal nicotinic acetylcholine receptors (nAChR) are expressed at specific times during development and in discrete neuronal populations. Transcriptional regulation of the receptor genes clearly plays a key role in the molecular pathway underlying the expression of these critical synaptic components. In an effort to understand this regulation, we focus upon the genes encoding three receptor subunits: α3, α5 and β4. These subunits are genomically clustered and constitute the predominant nAChR subtype expressed in the peripheral nervous system. We and others demonstrated that the general transcription factors, Sp1 and Sp3, can transactivate the promoter of each subunit gene. Further, we showed that the regulatory factor Sox10 transactivates the α3 and β4 promoters and does so in a cell-type-specific manner. Interestingly, the Sp- and Sox10-binding sites on the β4 promoter are located immediately adjacent to each other, raising the possibility that the two sets of factors functionally interact to regulate receptor gene expression. Consistent with this hypothesis, we demonstrated that the proteins can directly interact. Here, we extend these observations and show that Sox10 and the Sp factors functionally interact, leading to synergistic transcriptional activation in a cholinergic cell line. Finally, evidence for the existence of cell-type-specific co-regulators for Sp1 and Sox10 is presented.

Analysis of the HIV-1 LTR NF-κB-Proximal Sp Site III: Evidence for Cell Type-Specific Gene Regulation and Viral Replication

It has been widely demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope, specifically the V3 loop of the gp120 spike, evolves to facilitate adaptation to different cellular populations within an infected host. Less energy has been directed at determining whether the viral promoter, designated the long terminal repeat (LTR), also exhibits this adaptive quality. Because of the unique nature of the cell populations infected during the course of HIV-1 infection, one might expect the opportunity for such adaptation to exist. This would permit select viral species to take advantage of the different array of conditions and factors influencing transcription within a given cell type. To investigate this hypothesis, the function of natural variants of the NF-κB-proximal Sp element (Sp site III) was examined in human cell line models of the two major cell types infected during the natural course of HIV-1 infection, T cells and monocytes. Utilizing the HIV-1 LAI molecular clone, which naturally contains a high-affinity Sp site III, substitution of low-affinity Sp sites in place of the natural site III element markedly decreased viral replication in Jurkat T cells. However, these substitutions had relatively small effects on viral replication in U-937 monocytic cells. Transient transfections of HIV-1 LAI-based LTR-luciferase constructs into these cell lines suggest that the large reduction in viral replication in Jurkat T cells, caused by low-affinity Sp site III variants, may result from reduced basal as well as Vpr- and Tat-activated LTR activities in Jurkat T cells compared to those in U-937 monocytic cells. When the function of Sp site III was examined in the context of HIV-1 YU-2-based LTR-luciferase constructs, substitution of a high-affinity element in place of the natural low-affinity element resulted in increased basal YU-2 LTR activity in Jurkat T cells and reduced activity in U-937 monocytic cells. These observations suggest that recruitment of Sp family members to Sp site III is of greater importance to the function of the viral promoter in the Jurkat T cell line as compared to the U-937 monocytic cell line. These observations also suggest that other regions of the LTR may compensate for Sp recruitment defects in specific cell populations.

Sp1 and Sp3 function as key regulators of leukotriene C(4) synthase gene expression in the monocyte-like cell line, THP-1.

The goal of this study was to examine the mechanisms of leukotriene C(4) (LTC(4)) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC(4) synthase promoter-reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between -92 and -23 bp, which contains a signal protein (Sp)1 consensus site at -42 to -37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from -56 to -17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 cell line with a construct containing the -92 to -23 bp promoter region and Sp expression vectors revealed that Sp1 and Sp3 transactivate gene transcription. We conclude that the Sp1 site is a necessary element for LTC(4) synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC(4) synthase gene is transcriptionally regulated in mononuclear phagocytes.

Transcriptional Regulation of Rat Scavenger Receptor Class B Type I Gene

The scavenger receptor class B type I (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells and plays an important role in the reverse uptake of cholesterol to the liver and in the delivery of substrates for steroidogenesis in steroidogenic organs. We report here on the isolation and characterization of the upstream promoter region of the rat SR-BI gene. The transcription start site for rat SR-BI was mapped, and DNA sequence analysis revealed the presence of binding sites for the Sp1 family in the proximal 5'-flanking region. Analysis of deletion mutants with different 5' lengths revealed that the region between -121 and -90 base pairs from the transcription start site is essential for the efficient transcription of SR-BI. Both Sp1 and Sp3 bind to three GC boxes in the region (-141 to -1 base pairs) in a sequence-specific manner. Mutations in any of the GC boxes decreased efficient transcription from this promoter in MA-10 mouse Leydig tumor cells. The overexpression of Sp1 or Sp3 protein enhanced the rat SR-BI promoter activity. These results indicate that Sp1 family members of transcription factors are essential for transcription of the rat SR-BI gene.

Sp Family Members and Nuclear Factor-Y Cooperatively Stimulate Transcription from the Rat Pyruvate Kinase M Gene Distal Promoter Region via Their Direct Interactions

The three distal transcriptional regulatory elements of the rat pyruvate kinase M gene, referred to as boxes A, B, and C, are located around -270 base pairs upstream from the transcriptional initiation site. Electrophoretic mobility shift assays with specific competitors and antibodies show that both box A and box B bind to Sp1 and Sp3 and that box C binds nuclear factor-Y (NF-Y). Luciferase reporter assays revealed that although box A and box B alone have no independent effect on luciferase activities, box C alone stimulates transcription. However, the inclusion of all three elements lead to maximal activity because of a synergistic effect, mainly between box B and box C, suggesting that functional synergism between Sp1/Sp3 and NF-Y is critical for the pyruvate kinase M (PKM) gene distal promoter activity. In fact, co-transfection of a dominant negative mutant of NF-YA (NF-YA29) resulted in a decrease in reporter activity in a box C-dependent manner. In addition, the overexpression of Sp1 or Sp3 and NF-Y in Drosophila SL2 cells synergistically stimulated PKM gene distal promoter activity. Using a mammalian two-hybrid system in HeLa cells, it was shown that both Sp1 and Sp3 interacted with NF-YA but not NF-YB and NF-YC. Moreover, glutathione S-transferase pull-down assays revealed that only in vitro translated (35)S-labeled NF-YA interacted with both Sp1 and Sp3 in vitro. A subunit interaction domain of NF-YA, which forms a heterotrimer with NF-YB and NF-YC, is not required for these interactions with Sp1 or Sp3. Thus, we conclude that Sp1, Sp3, and NF-Y stimulate the transcription of the PKM gene via their interactions.