Circ_0047835 Combines with miR-144-3p to Promote the Proliferation, Invasion, Migration, and Fibrosis of TGF-ß1-Treated Human Tenon's Capsule Fibroblasts by Upregulating SP1.

PURPOSE: Glaucoma is the leading cause of blindness worldwide with complex pathogenesis. Circular RNAs (circRNAs) play critical roles in various diseases, including glaucoma. The purpose of this study was to investigate the role of circ_0047835 and underlying mechanisms in the development of fibrosis after glaucoma filtration surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were stimulated using transforming growth factor-ß1 (TGF-ß1) to mimic a cellular model of glaucoma in vitro. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell invasion and migration were detected by transwell assay and wound healing assay, respectively. Western blot assay was used to measure protein levels. The expression levels of circ_0047835, microRNA-144-3p (miR-144-3p) and specific protein 1 (SP1) mRNA were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-144-3p and circ_0047835 or SP1 was confirmed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. RESULTS: Circ_0047835 expression was elevated in glaucoma tissues and TGF-ß1-treated HTFs. Circ_0047835 or SP1 knockdown suppressed the proliferation, migration, invasion, and fibrosis of TGF-ß1-treated HTFs. MiR-144-3p was a target of circ_0047835, and miR-144-3p inhibition reversed the effects of circ_0047835 knockdown in TGF-ß1-treated HTFs. Moreover, SP1 was identified as a target of miR-144-3p, and miR-144-3p overexpression weakened TGF-ß1-induced proliferation, migration, invasion, and fibrosis by targeting SP1 in HTFs. Furthermore, circ_0047835 combined with miR-144-3p to regulate SP1 expression. CONCLUSION: Circ_0047835 might contribute to fibrosis progression after glaucoma surgery by regulating the miR-144-3p/SP1 axis.

Bone Marrow-Derived Mesenchymal Stem Cell-Derived Exosomes Containing Gli1 Alleviate Microglial Activation and Neuronal Apoptosis In Vitro and in a Mouse Parkinson Disease Model by Direct Inhibition of Sp1 Signaling.

This study investigated possible therapeutic effect mechanisms of exosomes from bone marrow-derived mesenchymal stem cells (BMSC) in neuronal and microglial cells and in a Parkinson disease (PD) model. Neuronal SH-SY5Y cells and microglial HMC3 cells were subjected to 1-methyl-4-phenylpyridinium (MPP+) or LPS, respectively. The mRNA and protein expression was assessed using qRT-PCR, Western blotting, and enzyme-linked immunosorbent assay. Cell viability and apoptosis of SH-SY5Y cells were examined using the MTT assay and flow cytometry. Chromatin immunoprecipitation assays were performed to assess the binding relationship between glioma-associated oncogene homolog 1 (Gli1) and the Sp1 transcription factor promoter. BMSC-derived exosomes promoted cell proliferation and inhibited apoptosis in MPP+-treated SH-SY5Y cells and suppressed inflammatory markers in LPS-treated HMC3 cells. Sp1 knockdown decreased SH-SY5Y cell damage and HMC3 immune activation. Gli1 carried by BMSC exosomes directly bound with Sp1 to inhibit Sp1-mediated LRRK2 activation whereas exosomes secreted by Gli1-knockdown in BMSC did not. In a PD mouse model induced with MPTP, BMSC exosomes decreased neuron loss injury and the inflammatory response by inhibiting Sp1 signaling. Thus, BMSC-derived exosomal Gli1 alleviates inflammatory damage and neuronal apoptosis by inhibiting Sp1 in vitro and in vivo. These findings provide the basis for the potential clinical use of BMSC-derived exosomes in PD.

Inhibition of myeloid cell leukemia-1 by tolfenamic acid induces apoptosis in mucoepidermoid carcinoma.

OBJECTIVES: The aim of this study was to evaluate the role of tolfenamic acid (Tol) and ampiroxicam (Amp) in the apoptotic regulation of YD-15 salivary mucoepidermoid carcinoma (MEC). MATERIALS AND METHODS: The effect of Tol on apoptosis and its mechanism were examined using a 3-(4,5-dimethylthiazol-2-yl)-5-(2,4-disulfophenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, Sub-G(1) population, Western blot analysis, 4'-6-Diamidino-2-phenylindole staining, reverse transcriptase polymerase chain reaction, immunostaining and small interfering RNA transfection. RESULTS: Tol inhibited cell growth of YD-15 cells but Amp did not. Tol induces apoptosis in YD-15 cells as evidenced by nuclear fragmentation, accumulation of the sub-G1 phase and the activation of caspase 3. Tol inhibited myeloid cell leukemia-1 (MCL-1) at the protein and mRNA levels. The treatment of MCL-1 siRNA to YD-15 cells resulted in the activation of caspase 3 and the inhibition of cell growth. Moreover, MCL-1 was regulated by specificity protein 1, but not by mitogen-activated protein kinases. CONCLUSION: These results suggest that Tol could be a potent anti-cancer drug for YD-15 MEC cells that acts by regulating the MCL-1 protein.

Indole-3-carbinol inhibits Sp1-induced matrix metalloproteinase-2 expression to attenuate migration and invasion of breast cancer cells.

Indole-3-carbinol (I3C), a major indole metabolite in cruciferous vegetables, has been shown to inhibit invasion of breast cancer cells. This study addressed the effect of I3C on the expression of matrix metalloproteinases (MMPs) and clarified the underlying mechanism. Migration, invasion, and MMP-2 activity of MCF-7 breast cancer cells were dose-dependently inhibited by I3C. In addition, the MMP-2 mRNA level was also reduced by I3C. Promoter deletion and mutation analysis suggested that I3C inhibited MMP-2 gene transcription via the -85/-7 bp promoter region and the Sp1 transcription factor binding site located within the -72/-64 bp promoter region was important for the inhibition. Chromatin immunoprecipitation assay confirmed that Sp1 proteins constitutively bound to this consensus sequence in vivo and that the binding was attenuated by I3C. In addition, I3C inhibited the extracellular signal-regulated kinase (ERK) signaling pathway in MCF-7 cells. The results suggest that I3C inhibits MMP-2 expression by blocking the ERK/Sp1-mediated gene transcription to attenuate migration and invasion of breast cancer cells.

Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors.

Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (-402/-350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region -152/-135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.

Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes.

Amyloid beta protein (Abeta) is the principal component of neuritic plaques in Alzheimer's disease (AD). Abeta is derived from beta amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) has been identified as the major beta-secretase. BACE2 is the homolog of BACE1. The BACE2 gene is on chromosome 21 and has been implicated in the pathogenesis of AD. However, the function of BACE2 in Abeta generation is controversial. Some studies have shown that BACE2 cleaved APP at the beta-site whereas other studies showed it cleaved around the alpha-secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Abeta generation. We cloned and functionally characterized the human BACE2 promoter. The BACE2 gene is controlled by a TATA-less promoter. Though Sp1 can regulate both BACE1 and BACE2 genes, comparative sequence analysis and transcription factor prediction showed little similarity between the two promoters. BACE1 increased APP cleavage at the beta-site and Abeta production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Abeta production. Knockdown of BACE2 resulted in increased APP C83. Our data indicate that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a beta-secretase, but processes APP within the Abeta domain at a site downstream of the alpha-secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.

v-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3.

Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

Regulation of human glucocorticoid receptor gene transcription by Sp1 and p53.

The human glucocorticoid receptor (hGR) gene has several GC boxes in the promoter 1C region. We studied the effects of Sp1 and p53 on promoter 1C in HepG2 and HEK293 cells using luciferase (Lu) reporter assay. The results showed that the first GC box upstream of the transcription site activated the hGR promoter and over-expression of Sp1 obviously enhanced the activity. A mutant Lu-hGR vector, whose first GC box was defective, lost promoter activity nearly completely. Further, over-expression of p53 strongly suppressed the stimulating effect of Sp1 on hGR promoter activity. We concluded that Sp1 activates the hGR gene promoter, at least in part, by acting on the first GC box in promoter 1C, while p53 suppresses the transactivation by Sp1. These phenomena, demonstrated in cultured cells, may be important for the expression of hGR in vivo.

DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter.

Changes in DNA methylation patterns are frequently observed in human cancers and are associated with a decrease in tumor suppressor gene expression. Hypermethylation of the BRCA1 promoter has been reported in a portion of sporadic breast tumours that correspond to a reduction in BRCA1 transcription and expression. Questions remain concerning the maintenance of methylation free zones in promoter regions of tumor suppressor genes in normal tissues. Sodium bisulfite based analysis of the BRCA1 promoter defines a methylation free zone in normal breast tissue that starts 650 bp upstream of the transcription start site and extends for 1.4 kb through most of the BRCA1 CpG island. We provide data implicating 2 proteins, Sp1 and CTCF, in the maintenance of this methylation-free zone. Both of these proteins have been shown to function as methylation boundaries in other genes. Four Sp1 sites have been identified in the BRCA1 promoter by gel shift assays. In vivo binding of Sp1 has been confirmed at 2 of these sites in the BRCA1 promoter and at 2 CTCF sites that flank the unmethylated region. Our data suggest that these DNA binding sites for Sp1 and CTCF may act as boundary elements that are important in maintaining genomic integrity by delineating the normal methylation free BRCA1 promoter. Inactivation or disruption of these boundaries may facilitate an epigenetic "hit", in this case DNA methylation, leading to BRCA1 downregulation and contributing to tumorigenesis, particularly the genesis of sporadic breast tumours.

Okadaic acid augments utrophin in myogenic cells.

Duchenne muscular dystrophy is a fatal childhood disease caused by mutations that abolish the expression of dystrophin in muscle. Utrophin is a paralogue of dystrophin and can functionally replace it in skeletal muscle. A potential therapeutic approach is to increase utrophin levels in muscle. One way to achieve this aim is to increase the expression of the utrophin gene at a transcriptional level via promoter activation. In this study, we have shown that utrophin A mRNA levels can be induced by okadaic acid in murine myogenic C2C12 cells. We have found that a utrophin A promoter reporter can be induced by Sp1 in C2C12 myoblasts, but not in myotubes. This activation can be enhanced by okadaic acid treatment. Our data suggest that this induction is due to Sp1 phosphorylation during myogenesis and thus, utrophin expression in muscle could be regulated by treatment with phosphatase inhibitors. Control of utrophin promoter activation could then be used to increase the expression of utrophin, and thus ameliorate the symptoms of Duchenne muscular dystrophy.

Regulation of the estrogen receptor alpha minimal promoter by Sp1, USF-1 and ERalpha.

The exact molecular mechanisms regulating estrogen receptor alpha (ERalpha) expression in breast tumors are unclear, but studies suggest that they are partly at the level of transcription. We have focused on the transcription factors that regulate the ERalpha minimal promoter, which we have previously shown to reside within the first 245 bp of the 5'-flanking region of the gene. Within this region are several elements essential for full ERalpha promoter transcriptional activity, including a GC box and an imperfect E box. In earlier studies we demonstrated an essential function for the Sp1 family of transcription factors in the regulation of ERalpha expression. We have now identified both USF-1 and ERalpha itself as components of a multi-protein complex of transcription factors that interacts at the ERalpha minimal promoter and is essential for its full transcriptional activity. Electrophoretic mobility shift assays demonstrated that Sp1 and USF-1, but not ERalpha, bind directly to the ERalpha minimal promoter. We showed by GST pull-down assays that ERalpha is able to interact in vitro with USF-1, suggesting, in addition to a possible interaction between ERalpha and Sp1, a mechanism whereby ERalpha is able to interact with the protein complex. Combined exogenous expression of the components of the complex in MCF-7 breast cancer cells resulted in a synergistic effect on transactivation of the ERalpha minimal promoter, suggesting that the importance of the protein complex is in the interactions among the components. Based upon these findings, we propose a possible model for transcription from the ERalpha minimal promoter.

Transcriptional regulation of P450scc gene expression in the embryonic rodent nervous system.

Steroid hormones are synthesized in adrenals, gonads, placenta, and the central and peripheral nervous systems (neurosteroids). Neurosteroidogenesis, like conventional steroidogenesis, begins with the conversion of cholesterol to pregnenolone, catalyzed by mitochondrial P450 side-chain cleavage enzyme (P450scc). Transcription of the P450scc gene in the adrenals and gonads requires steroidogenic factor-1, which is not expressed in the nervous system cells that express P450scc. A crucial transcriptional regulatory region of the rat P450scc gene is at -130/-94. We have purified two nuclear proteins (70 and 86 kDa) from rat glial C6 cells that specifically bind to the -130/-94 region of the rat P450scc promoter and identified them as the DNA-binding subunits of autoimmune antigen Ku. Ku colocalized with P450scc in several regions of the nervous system, but its overexpression in C6 cells did not augment transcription from a -130/-94 Luciferase construct. Members of the Sp family of transcription factors also bind to the same DNA sequence as Ku. Sp4 and Sp2 colocalize with P450scc in the nervous system early in development, whereas Sp1 and Sp4 colocalize later in development. Sp1 robustly increased transcription from this element in Sp-deficient Drosophila SL2 cells, and Ku synergistically enhanced this Sp1-stimulated transcription. Thus, members of the Sp transcription family play a role in activating P450scc gene transcription in the nervous system, and Ku may further augment this activation.

Sp1 and Sp3 are oxidative stress-inducible, antideath transcription factors in cortical neurons.

Neuronal cell death in response to oxidative stress may reflect the failure of endogenous adaptive mechanisms. However, the transcriptional activators induced by oxidative stress in neurons that trigger adaptive genetic responses have yet to be fully elucidated. We report that basal DNA binding of the zinc finger transcription factors Sp1 and Sp3 is unexpectedly low in cortical neurons in vitro and is significantly induced by glutathione depletion-induced or hydrogen peroxide-induced oxidative stress in these cells. The increases in Sp1/Sp3 DNA binding reflect, in part, increased levels of Sp1 and Sp3 protein in the nuclei of cortical neurons. Similar induction of Sp1 and Sp3 protein is also observed in neurons in vivo in a chemical or a genetic model of Huntington's disease, two rodent models in which neuronal loss has been attributed to oxidative stress. Sustained high-level expression of full-length Sp1 or full-length Sp3, but not the Sp1 zinc finger DNA-binding domain alone, prevents death in response to oxidative stress, DNA damage, or both. Taken together, these results establish Sp1 and Sp3 as oxidative stress-induced transcription factors in cortical neurons that positively regulate neuronal survival.

Synergistic regulation of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1.

Cystathionine-beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously described essential transactivating roles for specificity protein 1 (Sp1), Sp3, nuclear factor Y (NF-Y), and USF-1 in the regulation of the CBS-1b promoter. Differential binding of Sp1/Sp3 to the CBS-1b promoter due to differences in Sp1/Sp3 phosphorylation, and Sp1/Sp3 synergism with NF-Y might, in part, explain cell-specific patterns of CBS expression. In this report, the roles of various NF-YA isoforms in influencing cell-specific differences in CBS gene expression were determined in HT1080 and HepG2 cells. Seven unique NF-YA isoforms were detected in HT1080 by reverse transcriptase-PCR (RT-PCR) and DNA sequencing, characterized by deletions in the glutamine-rich and/or serine/threonine-rich domains. Only four of the seven NF-YA isoforms were found in HepG2 cells. The six alternatively spliced NF-YA isoforms all showed significantly less synergistic transactivation of the CBS-1b promoter with Sp1 than wild-type NF-YA, as determined by cotransfections in Drosophila SL2 cells with NF-YB and NF-YC. Further, all six alternatively spliced NF-YA isoforms inhibited the synergistic transactivation of the CBS-1b promoter by wild-type NF-Y and Sp1. Thus, the cellular distributions of these alternatively spliced NF-YA isoforms could impart an important cell-specific component to CBS transcriptional regulation, by virtue of their abilities to directly synergize with Sp1/Sp3 and interfere with transactivation of the CBS-1b promoter by wild-type NF-Y. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down's syndrome (DS).

Regulation of cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) expression by Sp1 in the mammalian testis.

In the mammalian testis, the binding of FSH to Sertoli cells activates the cAMP-dependent protein kinase A signaling pathway, resulting in the phosphorylation of the cAMP response element binding protein (CREB). Previous studies have also shown that CREB gene expression is activated by cAMP in Sertoli cells and that 2 cAMP response elements (CREs) that bind CREB and a neighboring Sp1 binding site are required for basal and cAMP-inducible CREB promoter activity. In contrast, CREB expression has been less well characterized in testis germ cells. We demonstrated that CREB and Sp1 are expressed in early germ cells only through the midpachytene stage of spermatogenesis. Furthermore, CREB promoter activity was induced over 70-fold by transient overexpression of Sp1 in SL2 cells, suggesting that Sp1 is an important regulator of CREB expression. Further studies of the CREB promoter revealed an additional regulatory element in the -130 region between the Sp1 and CREB transcription factor binding sites that is necessary for full promoter activity. Proteins expressed in Sertoli cells and germ cells bind specifically to the newly identified regulatory region. These studies suggest that proteins binding to Sp1 motifs and the -130 region are required to activate the CREB promoter.

Sp1 and Egr1 regulate transcription of the Dmrt1 gene in Sertoli cells.

Dmrt1 is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating the Dmrt1 proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the rat Dmrt1 gene and more than 9 kilobases of 5' flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. The Dmrt1 5' flanking region from -5000 to +74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for full Dmrt1 promoter function. These regions were located between -3200 and -2000 base pairs (bp) and downstream of -150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of -150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate the Dmrt1 promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role in Dmrt1 regulation in the testis.

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.

Sp proteins and Phox2b regulate the expression of the human Phox2a gene.

Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. We have isolated the 5' regulatory region of the human Phox2a gene and studied the transcriptional mechanisms underlying its expression. We first identified the minimal gene promoter by means of molecular and functional criteria and demonstrated that its activity relies on a degenerate TATA box and a canonical Sp1 site. We then concentrated on the region immediately upstream of the promoter and found that it stimulates transcription in a neurospecific manner because its deletion caused 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 showed that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrated 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. We have provided the first molecular evidence that Phox2b can regulate the expression of Phox2a by directly binding to its 5' regulatory region.

CD99 expression is positively regulated by Sp1 and is negatively regulated by Epstein-Barr virus latent membrane protein 1 through nuclear factor-kappaB.

Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is highly expressed in Hodgkin and Reed-Sternberg (H-RS) cells from patients with EBV-associated Hodgkin disease. It was previously demonstrated that CD99 can be negatively regulated by LMP1 at the transcriptional level, and the decreased expression of CD99 in a B lymphocyte cell line generates H-RS-like cells. In this study, detailed dissection of the CD99 promoter region was performed to search regulatory factor(s) involved in the expression of the gene. Using various mutant constructs containing deletions in the promoter region, it was revealed that the maximal promoter activity was retained on 5'-deletion to the position -137 from the transcriptional initiation site. Despite the presence of multiple putative Sp1-binding sites in the promoter region, the site located at -95 contributes heavily as a positive cis-acting element to its basal promoter activity. However, on examination of the involvement of the positive-acting Sp1-binding site of the promoter for the repressive activity of LMP1, it appeared to be dispensable. Instead, the repressive effect was mapped to the nuclear factor (NF)-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP1 despite the absence of the NF-kappaB consensus sequences in the CD99 promoter region. Furthermore, the decreased CD99 promoter activity by LMP1 was markedly restored when NF-kappaB activity was inhibited. Taken together, these data suggest that Sp1 activates, whereas LMP1 represses, transcription from the CD99 promoter through the NF-kappaB signaling pathway, and they might aid in the understanding of the molecular mechanisms of viral pathogenesis in EBV-positive Hodgkin disease. (Blood. 2001;97:3596-3604)

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.