Putative Sp1 Binding Sites Research Articles

생쥐 도파민 수용쳬 조절인자 (DRRF) 유전자의 전사조절

생쥐의 도파민 수용체 조절인자 (DRRF)유전자는 몇몇 Spl 결합부위를 가지고 TATA가 없는 프로모터로부터 전사된다. 본 연구에서는 이 유전자의 발현을 조절하는 기능성 조절인자들을 밝힌다. <TEX>$D_2$</TEX> 도파민 수용체를 발현하는 NB41A3 세포에서 Spl은 pCAT-DRRF-l153/+17에 포함된 DRRF 프로모터부터 의 전사를 촉진시키지만 DRRF는 전사를 억제시켰다. -1153과 -1122 사이의 31 bp 단편의 결손에 의해 전사활성은 약 <TEX>$60\%$</TEX> 정도 감소하였다. 이 단편은 기능성 AP1 결합부위를 포함하고 있다 게다가, -901과 -772사이의 129 bp영역의 결손에 의해 전사활성이 더욱 더 감소하였다. 이 영역은 기능성 AP2 결합부위를 가진다. DRRF_AP1 (bases -1153 to -1121) 탐침을 이용한 gel shift실험에서 특정 벤드가 관찰되었고, 이 벤드는 API 상보성 경쟁자에 의해 효과적으로 사라졌다. 더욱이, DRRF_AP2 (bases -873 to -846) 탐침을 이용한 gel shift실험에서도 특정 벤드가 관찰되었고, 이 벤드도 AP2상보성 경쟁자에 의해 효과적으로 사라졌다. 본 연구결과로, Spl과 DRRF가 DRRF 프로모터를 효과적으로 조절한다는 사실과, AP1과 AP2 역시 이 유전자를 조절한다는 사실을 알 수 있었다. The murine dopamine receptor regulating factor (DRRF) gene is transcribed from a TATA-less promoter that has several putative Sp1 binding sites. The present investigation identifies functional transcription factors that modulate the expression of this gene, In the <TEX>$D_2-expressing$</TEX> NB41A3 cells, Spl potently activates transcription from the DRRF promoter in pCAT-DRRF-1153/+17, but DRRF effectively inhibits it. Deletion of the 31 bp fragment between -1153 and -1122 decreased transcription down to about <TEX>$60\%$</TEX>. This fragment contains a functional API binding site. In addition, deletion of the 129 bp region between -901 and -772 further decreased transcription. The latter region has a functional AP2 binding site. Using a DRRF_AP1 (bases -1153 to -1121) probe, a specific retarded band was observed, and the unlabeled AP1 consensus competitor could effectively compete away this retarded band. In addition, using a DRRF_AP2 (bases -873 to -846), a specific retarded band was observed, and the unlabeled AP2 consensus competitor could effectively compete away this retarded band. The present observations suggest that Spl and DRRF regulate the DRRF promoter and that both API and AP2 also modulate this gene.

Activator Protein Accelerates Dihydropyrimidine Dehydrogenase Gene Transcription in Cancer Cells

Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for individual response to 5-fluorouracil. Clinical responses to the anticancer agent, along with various reports, have clearly shown that dihydropyrimidine dehydrogenase activity is closely correlated to its mRNA levels, but the regulatory mechanisms of its expression have remained unclear. We attempted to clarify the mechanisms and found that activator protein (AP-1) is probably one of the key factors in the transcriptional regulation of DPYD in cancer cells, and that phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment enhances transcription of DPYD via AP-1 activation. In this study, we characterized our previously subcloned 5' region of human DPYD, an approximately 3.0-kb fragment (accession no. AB162145). Luciferase reporter assay showed that the clone showed strong promoter activities in 293T and HSC42 cells, and comparative analysis using 5' deletion mutants suggested the existence of several positive and negative regulatory regions, including putative binding sites for AP-1, SP-1, and nuclear factor-kappaB. PMA/ionomycin treatment increased the mRNA level of DPYD in HSC42 cells, and electrophoretic gel mobility shift assay showed that the complex on the putative AP-1 binding site was drastically induced by PMA/ionomycin treatment. The complexes formed were competed out by preincubation with the cold-consensus AP-1 binding site, and the DNA binding complex formed on the site contained c-Jun and c-Fos, which are components of AP-1 transcription factor. We further identified the functional AP-1 binding site (nucleotide positions from -290 to -280), whose nucleotide mutations abolished PMA/ionomycin-induced DPYD promoter activation.

A Retroviral Promoter and a Cellular Enhancer Define a Bipartite Element Which Controls env ERVWE1 Placental Expression

The HERV-W family contains hundreds of loci diversely expressed in several physiological and pathological contexts. A unique locus termed ERVWE1 encodes an envelope glycoprotein (syncytin) involved in hominoid placental physiology. Here we show that syncytin expression is regulated by a bipartite element consisting of a cyclic AMP (cAMP)-inducible long terminal repeat (LTR) retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism. Deletion mutant analysis showed that the ERVWE1 5' LTR contains binding sites essential for basal placental activity in the region from positions +1 to +125. The region from positions +125 to +310 represents a cAMP-responsive core HERV-W promoter active in all cell types. Site-directed mutagenesis analysis highlighted the complexity of U3 regulation. ERVWE1 placenta-specific positive (e.g., T240) and negative (e.g., G71) regulatory sites were identified, as were essential sites required for basic activity (e.g., A247). The flanking sequences of the ERVWE1 provirus contain several putative regulatory elements. The upstream HERV-H and HERV-P LTRs were found to be inactive. Conversely, the 436-bp region located between the HERV-P LTR and ERVWE1 was shown to be an upstream regulatory element (URE) which is significantly active in placenta cells. This URE acts as a tissue-specific enhancer. Genetic and functional analyses of hominoid UREs revealed large differences between UREs of members of the Hominidae and the Hylobatidae. These data allowed the identification of a positive regulatory region from positions -436 to -128, a mammalian apparent LTR retrotransposon negative regulatory region from positions -128 to -67, and a trophoblast-specific enhancer (TSE) from positions -67 to -35. Putative AP-2, Sp-1, and GCMa binding sites are essential constituents of the 33-bp TSE.

Transcriptional Regulation of the Human β-1,4-Galactosyltransferase V Gene in Cancer Cells: ESSENTIAL ROLE OF TRANSCRIPTION FACTOR Sp1

Beta-1,4-galactosyltransferase (beta-1,4-GalT) V is a constitutively expressed enzyme that can effectively galactosylate the GlcNAcbeta1-->6Man group of the highly branched N-glycans that are characteristic of tumor cells. Upon malignant transformation of cells, the expression of the beta-1,4-GalT V gene increases in accordance with the increase in the amounts of highly branched N-glycans. Lectin blot analysis showed that the galactosylation of highly branched N-glycans is inhibited significantly in SH-SY5Y human neuroblastoma cells by the transfection of the antisense beta-1,4-GalT V cDNA, indicating the biological importance of the beta-1,4-GalT V for the functions of highly branched N-glycans. We cloned the 2.3-kb 5'-flanking region of the human beta-1,4-GalT V gene, and we identified the region -116/-18 relative to the transcription start site as that having promoter activity. The region was found to contain several putative binding sites for transcription factors, including AP2, AP4, N-Myc, Sp1, and upstream stimulatory factor. Electrophoretic mobility shift assay showed that Sp1 binds to nucleotide positions -81/-69 of the promoter region. Mutations induced in the Sp1-binding site showed that the promoter activity of the beta-1,4-GalT V gene is impaired completely in cancer cells. In contrast, the promoter activity increased significantly by the transfection of the Sp1 cDNA into A549 human lung carcinoma cells. Mithramycin A, which inhibits the binding of Sp1 to its binding site, reduced the promoter activation and expression of the beta-1,4-GalT V gene in A549 cells. These results indicate that Sp1 plays an essential role in the transcriptional activity of the beta-1,4-GalT V gene in cancer cells.

The SANT Domain of Human MI-ER1 Interacts with Sp1 to Interfere with GC Box Recognition and Repress Transcription from Its Own Promoter

To gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within-68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters.

Role of Sp1, C/EBP alpha, HNF3, and PXR in the basal- and xenobiotic-mediated regulation of the CYP3A4 gene.

Cytochrome P450 3A4 (CYP3A4) is the major cytochrome P450 present in adult human liver and is involved in the metabolism of over 50% of therapeutic compounds currently in use. Since expression levels of CYP3A4 are regulated by many of these compounds, this raises the potential for drug-drug interactions and subsequent altered efficacy or toxicity of the individual compounds at the dose prescribed. Hence, understanding the molecular mechanisms of CYP3A4 regulation is of key importance in predicting and understanding such interactions. To examine this we have used DNase I footprinting and bioinformatic analysis to identify putative transcription factor binding sites within the 250 base pairs of promoter proximal to the transcription start site. We identified several protected fragments within this region that corresponded to putative binding sites for Sp1, AP2, CCAAT/enhancer binding protein (C/EBPalpha), and hepatic nuclear factor-3 (HNF3), as well as confirming previously identified C/EBPalpha, pregnane X receptor (PXR), and HNF3 binding sites. Sequential site-directed mutagenesis of C/EBPalpha, Sp1, HNF3, and PXR binding sites was next used to examine the role of these sites in basal CYP3A4 expression. Disruption of the C/EBPalpha, HNF3, and PXR binding sites all affected basal expression. Finally, the role of these sites was examined in activation of CYP3A4 expression by rifampicin, metyrapone, clotrimazole, and phenobarbital. Disruption of any of these sites either led to an altered pattern of activation by the xenobiotic, as altered maximal activation, or altered the EC(50) value of activation. Such effects were xenobiotic-specific, with each disrupted site playing a role in the activation of some of the xenobiotics.

Cloning, characterisation and identification of several polymorphisms in the promoter region of the human α 2B-adrenergic receptor gene

Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human α 2B-adrenergic receptor, that contained 5.5 kb of the 5′-flanking region, the open reading frame and 2.9 kb of the 3′-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5′-flanking region included AP2, USF, Stat-6, NFκB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase contructs containing various truncated fragments of the 5′-region showed that the fragment −3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (−603/−411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5′-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position −98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect α 2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the −98 G/C single nucleotide polymorphism was located within a portion of the 5′-UTR (−127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.

Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region

An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region.

NFY interacts with the promoter region of two genes involved in the rat peroxisomal fatty acid β-oxidation: the multifunctional protein type 1 and the 3-ketoacyl-CoA B thiolase

Backgroundβ-oxidation of long and very long chain fatty acyl-CoA derivatives occurs in peroxisomes, which are ubiquitous subcellular organelles of eukaryotic cells. This pathway releases acetyl-CoA as precursor for several key molecules such as cholesterol. Numerous enzymes participating to cholesterol and fatty acids biosynthesis pathways are co-localized in peroxisomes and some of their encoding genes are known as targets of the NFY transcriptional regulator. However, until now no interaction between NFY transcription factor and genes encoding peroxisomal β-oxidation has been reported.ResultsThis work studied the interactions between NFY factor with the rat gene promoters of two enzymes of the fatty acid β-oxidation, MFP-1 (multifunctional protein type 1) and ThB (thiolase B) and their involvement in the cholesterol dependent-gene regulation. Binding of this nuclear factor to the ATTGG motif of the MFP-1 and of the ThB promoters was demonstrated by EMSA (Electrophoretic Mobility Shift Assay) and super shift assay. In contrast, in spite of the presence of putative Sp1 binding sites in these promoters, competitive EMSA did not reveal any binding. The promoter-dependent luciferase gene expression was downregulated by cholesterol in MFP-1 and ThB promoters harbouring constructs.ConclusionsThis work describes for the first time a NFY interaction with promoter sequences of the peroxisomal β-oxidation encoding genes. It suggests that cholesterol would negatively regulate the expression of genes involved in β-oxidation, which generates the initial precursor for its own biosynthesis, via at least the NFY transcription factor.

Insulin-like growth factor I increases rat peptide YY promoter activity through Sp1 binding sites.

Studies in rodents demonstrate that the mitogen, IGF-I, stimulates intestinal peptide YY (PYY) expression. To investigate whether the stimulatory influence of IGF-I is exerted at the level of gene transcription, rat PYY 5'-upstream sequences (-2800/+37 bp, -770/+37 bp, -127/+37 bp) fused to the firefly luciferase (luc) reporter gene were transfected into rat pheochromocytoma cells (PC12) and luc activity measured after IGF-I treatment. IGF-I increased transcriptional activity of all constructs similarly; the PYY (-127/+37 bp)-luc construct was used in subsequent experiments. IGF-I increased PYY (-127/+37 bp)-luc activity in a time- and dose-dependent fashion. Sequence analysis detected five putative Sp1 binding sites in the -127/+37-bp sequence. EMSA and supershift experiments using two oligonucleotide fragments of the -127/+37 region showed that Sp1 and Sp3 proteins bound to putative Sp1 sites. Overexpression of Sp1 greatly increased PYY (-127/+37 bp)-luc activity and site-directed mutagenesis of putative Sp1 binding sites decreased basal and IGF-I-induced elevations in PYY (-127/+37 bp)-luc activity. IGF-I treatment also increased Sp1 protein levels and binding activity. Blockade of the IGF-I receptor (IGF-IR) with an IGF-IR antibody decreased the stimulatory influence of IGF-I on Sp1 protein levels and PYY (-127/+37 bp)-luc activity. Together, these findings indicate that IGF-I functions as a positive regulator of PYY gene expression and that the stimulatory effect may be mediated by Sp1 proteins that bind to the proximal PYY promoter region.

Autoregulation of mouse histone deacetylase 1 expression.

Histone deacetylase 1 (HDAC1) is a major regulator of chromatin structure and gene expression. Tight control of HDAC1 expression is essential for development and normal cell cycle progression. In this report, we analyzed the regulation of the mouse HDAC1 gene by deacetylases and acetyltransferases. The murine HDAC1 promoter lacks a TATA box consensus sequence but contains several putative SP1 binding sites and a CCAAT box, which is recognized by the transcription factor NF-Y. HDAC1 promoter-reporter studies revealed that the distal SP1 site and the CCAAT box are crucial for HDAC1 promoter activity and act synergistically to constitute HDAC1 promoter activity. Furthermore, these sites are essential for activation of the HDAC1 promoter by the deacetylase inhibitor trichostatin A (TSA). Chromatin immunoprecipitation assays showed that HDAC1 is recruited to the promoter by SP1 and NF-Y, thereby regulating its own expression. Coexpression of acetyltransferases elevates HDAC1 promoter activity when the SP1 site and the CCAAT box are intact. Increased histone acetylation at the HDAC1 promoter region in response to TSA treatment is dependent on binding sites for SP1 and NF-Y. Taken together, our results demonstrate for the first time the autoregulation of a histone-modifying enzyme in mammalian cells.

Functional analysis of the endogenous retroviral promoter of the human endothelin B receptor gene.

We previously reported that the long terminal repeats (LTRs) of retroviral elements belonging to the HERV-E family contribute to the expression of the human apolipoprotein C1 (APOC1) and endothelin B receptor (EDNRB) genes by providing alternative promoters. While both LTRs were shown to promote transcription in vivo and in vitro, their respective activity and tissue specificity appeared to differ even though they shared a high degree of sequence identity. In the present study, we further characterized the promoter of the EDNRB LTR and delineated the regions and motifs required for strong activity. We confirmed the placenta-restricted expression of the LTR by transient transfections and quantitative real-time PCR and determined that the retroviral promoter contributes significantly to the level of EDNRB transcripts in placenta, where chimeric mRNAs were found to represent 15% of overall EDNRB mRNAs. Transient transfection of 5' deletion constructs in cells of placental origin identified a motif, named LPE1, between positions 111 and 122 of the EDNRB LTR necessary for transcriptional activity. Removal of this region, which contains a putative SP1 binding site, abolished promoter activity. A second enhancing region resides between positions 175 and 215 of the LTR and was termed LPE2. Interestingly, this section contained three binding sites that were not present in the APOC1 LTR due to minor nucleotide differences. The predicted motifs in the EDNRB LTR were found to likely act in symbiosis as modifications to any of the three sites reduced transcription by one-third while alterations to all three eliminated promoter activity. The results from this study illustrate how slight variations in transcriptional regulatory sequences can have a profound effect on promoter activity and demonstrate the complex regulatory effects of human endogenous retrovirus elements on human gene expression.

Characterization and analysis of the proximal Janus kinase 3 promoter.

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common gamma-chain (gammac), i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially expressed in hemopoietic cells and is up-regulated upon cell differentiation and activation. Despite the importance of Jak3 in lymphoid development and immune function, the mechanisms that govern its expression have not been defined. To gain insight into this issue, we set out to characterize the Jak3 promoter. The 5'-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron. Upstream of this intron and the transcription initiation site, we identified an approximately 1-kb segment that exhibited lymphoid-specific promoter activity and was responsive to TCR signals. Truncation of this fragment revealed that core promoter activity resided in a 267-bp fragment that contains putative Sp-1, AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites significantly diminished, whereas mutation of the Ets sites abolished, the inducibility of the promoter construct. Chromatin immunoprecipitation assays showed that histone acetylation correlates with mRNA expression and that Ets-1/2 binds this region. Thus, transcription factors that bind these sites, especially Ets family members, are likely to be important regulators of Jak3 expression.

Transcriptional regulation of the human Sp1 gene promoter by the specificity protein (Sp) family members nuclear factor Y (NF-Y) and E2F.

We analysed in detail the minimal promoter of transcription factor Sp1, which extends 217 bp from the initiation of transcription. Within this sequence we identified putative binding sites for Sp1, nuclear factor Y (NF-Y), activator protein 2 ('AP-2'), CCAAT/enhancer-binding protein ('C/EBP') and E2F transcription factors. In one case, the boxes for Sp1 and NF-Y are overlapping. Gel-shift and supershift assays demonstrated specific binding of Sp1, Sp3 and NF-Y proteins. Transient transfections and luciferase assays revealed activation of the Sp1 minimal promoter upon overexpression of Sp1 itself, NF-Y and E2F. Whereas overexpression of NF-Y or E2F had an additive effect on Sp1 overexpression, the activation of Sp1 transcription due to Sp1 was counteracted by Sp3 overexpression. Mutagenesis analysis of the NFY/Sp1-overlapping box revealed that both factors compete for this box, and that when the NF-Y site of this overlapping box is specifically mutated there is an increase in Sp1 binding, thus increasing transcriptional activity. These results help to explain the complex regulation of the Sp1 gene, which depends on the relative amounts of Sp1, Sp3, E2F and NF-Y proteins in the cell.

Assessment by Molecular Dynamics Simulations of the Structural Determinants of DNA-binding Specificity for Transcription Factor Sp1

The DNA-binding domain (DBD) of the ubiquituous transcription factor Sp1 consists of three consecutive zinc fingers that recognize a number of nucleotide sequences different from, but related to and sometimes overlapping, those recognized by the structurally better characterized early growth response protein 1 (EGR1, also known as Zif268, Krox-24, and NGFI-A). The accepted consensus binding sequence for Sp1 is usually defined by the asymmetric hexanucleotide core GGGCGG but this sequence does not include, among others, the GAG (=CTC) repeat that constitutes a high-affinity site for Sp1 binding to the wt1 promoter. Since no 3D structure of the whole DBD of Sp1 is available, either alone or in complex with DNA, a homology-based model was built and its interaction with two DNA 14-mers was studied using nanosecond molecular dynamics simulations in the presence of explicit water molecules. These oligonucleotides represent Sp1 target sites that are present in the promoters of the mdr1 and wt1 genes. For comparative purposes and validation of the protocol, the complex between the DBD of EGR1 and its DNA target site within the proximal mdr1 promoter was simulated under the same conditions. Some water molecules were seen to play an important role in recognition and stabilization of the protein–DNA complexes. Our results, which are supported by the available experimental evidence, suggest that the accuracy in the prediction of putative Sp1-binding sites can be improved by interpreting a set of rules, which are a blend of both stringency and tolerance, for the juxtaposed triplet subsites to which each zinc finger binds. Our approach can be extrapolated to WT1 and other related natural or artificial zinc-finger-containing DNA-binding proteins and may aid in the assignment of particular DNA stretches as allowed or disallowed-binding sites.

Genomic structure and promoter analysis of human NeuAc α2,3Gal β1,3GalNAc α2,6-sialyltransferase (hST6GalNAc IV) gene

We have cloned the genomic DNA encoding the human NeuAc α2,3Gal β1,3GalNAc α2,6-sialyltransferase (hST6GalNAc IV) and analysed its structure. The hST6GalNAc IV gene was found to span about 9 kb and to be composed of six exons. The 5′-RACE (rapid amplification of cDNA ends) results indicated that mRNA isoform of the hST6GalNAc IV was generated by alternative splicing in the 5′-untranslated region. The expression of this gene was highly restricted in human fetal tissues. The potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5′-flanking region of this gene lacked canonical TATA and CAAT boxes, but contained several putative binding sites for transcription factors SP1, MZF1, GATA1, LMO2COM, NFAT, HFH8 and USF, etc. Functional analysis of the 5′-flanking region by transient expression method revealed a high transcriptional activity in both HepG2 cells and Molt4 cells in a cell type-dependent manner, but not in SK-N-MC cells. These results suggest cell type-specific regulation of the basal hST6GalNAc IV promoter activity.

Genomic organization and promoter characterization of the murine dopamine receptor regulating factor (DRRF) gene

To study the transcriptional mechanisms by which expression of the dopamine receptor regulating factor (DRRF) gene is regulated, a murine genomic clone was isolated using a DRRF cDNA as probe. A 24 kb genomic fragment which comprises 13 kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Sp1. The DRRF gene also has consensus sequences for AP1 and AP2 binding sites. The transcriptional activity of five deletion mutants of a 1.5 kb fragment was analyzed by modulating transcription of the heterologous chloramphenicol acetyltransferase (CAT) gene in the promoterless plasmid pCAT-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3, except the construct stretching from −901 to +17. These transient expression assays suggested the presence of positive regulators between −1153 and −901 and between −118 and −93 while a negative regulator was found in the region between −901 and −118. Comparison among cell types revealed strong transcriptional activity of the DRRF promoter in neuronal NB41A3 cells and moderate activity in hepatic HepG2 and renal OK cells, but none in skeletal muscle C2C12 or glial C6 cells. These findings confirm the tissue-specific activity of the DRRF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

Betaglycan Expression Is Transcriptionally Up-regulated during Skeletal Muscle Differentiation: CLONING OF MURINE BETAGLYCAN GENE PROMOTER AND ITS MODULATION BY MyoD, RETINOIC ACID, AND TRANSFORMING GROWTH FACTOR-β

Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.

Sp1 Transactivation of the TCL1 Oncogene

Cis-regions and trans-factors controlling TCL1 oncogene expression are not known. We identified the functional TCL1 promoter by mapping four transcriptional start sites 24-30 bp downstream of a TATA box. A 424-bp fragment upstream of the major start site showed robust promoter activity comparable with SV40 in both TCL1 expressing and non-expressing cell lines. Additional constructs spanning 10 kb upstream and 20 kb downstream of the start site showed only modest increases in reporter activity indicating that TCL1 expression is primarily controlled by the promoter. Ten putative Sp1-binding sites were identified within 300 bp of the start site, and three of these specifically bound Sp1. A dose-dependent transactivation of the TCL1 promoter with Sp1 addition in Sp1-negative Drosophila SL2 cells was observed, and mutation of the three identified Sp1-binding sites significantly repressed reporter gene expression in 293T cells, confirming a key role for Sp1 in activating the TCL1 promoter in vivo. In TCL1 silent cell lines, CpG DNA methylation was rarely observed at functional Sp1 sites, and methylation of a previously reported NotI restriction site was associated with dense CpG methylation rather than endogenous TCL1 gene silencing. Together, these results indicate that Sp1 mediates transactivation of the TCL1 core promoter and that TCL1 gene silencing is not dependent on mechanisms involving Sp1 and NotI site methylation.

Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a

UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene ( Ube4b) for UFD2a. A full-length (∼5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5 ′ flanking region of Ube4b revealed that nucleotides −1018 to −943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin–proteasome pathway.