Major Transcription Start Site Research Articles

Targeting the Human CD3γ Gene Promoter By HIV-1 and HTLV-1: Two Distinct Mechanisms Involving A Transcriptional Regulatory Element and Chromatin Remodeling

Our studies show that HIV-1, HIV-2, and HTLV-I infection all provoke a progressive defect in surface T cell receptor expression. A specific loss of CD3γ transcripts is responsible for the defect after HIV-1 or HIV-2 infection. Alternatively, while CD3γ transcripts are lost first after HTLV-I infection, their reduction is followed several months later by a loss of CD3δ and subsequently CD3e mRNA. Studies of CD3γ transcriptional control revealed parallels with elements regulating HIV-1 gene expression, including a downstream element reminiscent of HIV TAR. Mutant and deletion CD3γ promoter constructs delimited a 53 bp region downstream from the major transcription start site as critical for positive gene expression. EMSA experiments demonstrate that this sequence functions through an RNA rather than a DNA intermediate, which can bind three specific nuclear protein complexes. Deletion of U at +9 and +37 kills promoter activity. Alternatively, progressive silencing of the CD3 gene locus by HTLV-I functions via chromatin remodeling, characterized by increased binding of Ikaros to the CD3 γ promoter and the CD3δ enhancer. Expression of the CD3 genes can be reactivated in HTLV-I infected cells by the synergistic action of the histone deactylase inhibitor trichostatin A and the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. The importance of viral targeting of the CD3 genes will be discussed. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The IL-7 Receptor alpha Promoter Is Activated at Distinct Stages during B Cell Development by the Ets Transcription Factors PU.1 and GABP.

The cytokine interleukin-7 (IL-7) is required for B and T lymphocyte development, and for the survival and maintenance of both naive and memory T lymphocytes. The receptor for IL-7 (IL-7R) is heterodimeric, consisting of a common gamma chain (γc) and an alpha subunit (IL-7Rα). The γc is expressed in most hematopoietic cell types, but the IL-7Rα gene is regulated in a cell type and developmental stage-specific manner. We have previously shown that the Ets-family transcription factor PU.1 is required to activate transcription of the IL-7Rα gene during fetal lymphocyte development. However, several questions remain unanswered. First, the IL-7Rα promoter is poorly characterized. Second, the IL-7Rα is expressed at high levels in the T cell lineage where PU.1 is not expressed. Third, the transcription factor early B cell factor (EBF) can activate IL-7Rα transcription in developing B cells in the absence of PU.1. To address these questions, we have characterized the IL-7Rα promoter region in detail. First, we determined that the major transcription start sites in the IL-7Rα gene are downstream of an Ets/PU.1 binding site. We found that the intact Ets site is required for IL-7Rα promoter activity, as well as to mediate enhancer action from a distance. IL-7Rα promoter activity depends on the proper orientation of the Ets site relative to functional initiator sequences. We found, using gel shift analysis, that both PU.1 and the Ets transcription factor GA binding protein (GABP) are expressed in developing B cells, and can interact with the Ets binding site in the IL-7Rα promoter. However, the function of PU.1 is distinct from GABP during B cell development. Retroviral transduction of PU.1 mutant progenitor cells with a PU.1 retrovirus robustly rescues IL-7Rα transcription and IL-7-dependent B cell development. In contrast, transduction with GABPα and GABPβ1 subunits fails to activate IL-7Rα transcription in PU.1 mutant progenitor cells. We conclude that activation of the IL-7Rα gene requires PU.1 during the earliest stages of lymphocyte development, but is alternatively utilized by PU.1 and GABP after commitment to the B cell lineage.

Short Nucleotide Insertions in the MCL-1 Promoter Affect Its Gene Expression.

We have recently reported a short nucleotide (six and eighteen) insertion (6- and 18-nt) in the MCL-1 promoter (−190bp upstream of the major transcription start site, GenBank, AN: AF198614) in B-cell chronic lymphocytic leukemia patients. These insertions were found to be polymorphisms. CD38-negative CLL patients with these promoter insertions had higher mRNA levels, poor response to standard chemotherapy and shorter survival. The aim of the present study was to test the hypothesis that these insertions directly effect MCL-1 gene expression. K562 and HeLa cells were transfected with pGL3-Basic, pGL3-Control and four reporter plasmids were constructed with a +0/0, +6/6, +18/18, and −23/23 MCL-1 promoter linked to Luciferase. Firefly activity in each transfection was normalized to Renilla Luciferase activity. At 24-hours post-transfection cells were induced with phorbol 12-myristate 13-acetate (PMA), granulocyte-macrophage colony stimulating factor (GM-CSF), both PMA and GM-CSF, or with fresh media only. At 48-hours post-transfection cells were harvested and Dual-Luciferase Assay was performed. Compared to transfection with pGL3-(+0/0) plasmid, the Luciferase PMA-stimulated activity for K562 cells transfected with pGL3-(+6/6) and pGL3-(+18/18) was 1.7 (P=0.0277) and 1.5 times higher (P=0.0067) respectively, and for pGL3-(−23/23) was 3.2 times lower (P=0.0021). The Luciferase PMA-stimulated activity for HeLa cells transfected with pGL3-(+6/6) was 3.3 times higher (P=0.0038), pGL-3-(+18/18) was 2.3 times higher (P=0.0245), and did not differ significantly for pGL3-(−23/23), compared to pGL3-(+0/0). Similar results were observed for GM-CSF-stimulated activity for HeLa cells, where activity was 3.9 times (P=0.0068) for pGL3-(+6/6) and 2.5 times (P=0.0258) higher for pGL3-(+18/18). Unstimulated Luciferase activity was also 2.5 times higher (P=0.0115) for pGL3-(+18/18) compared to pGL3-(+0/0) plasmid. These results provide experimental evidence for the positive effect of these nucleotide insertions (6- and 18-nt) on MCL-1 gene expression in both hemopoietic (K562) and epithelial (HeLa) cells.

Regulatory elements of microfibril-associated glycoprotein-1 gene expression in muscle cells

The Mfap2 gene encodes the microfibril-associated glycoprotein-1 (MAGP1), an extracellular matrix protein of microfibrillar structures. The gene is transcribed from a major transcription start site embedded in a CpG island. Mapping of transcriptionally active regions in the 5′ flanking sequence identified a region, located between nucleotides −339 and −109 as the Mfap2 basal promoter. Site-directed and random mutagenesis demonstrated that a KLF sequence motif at −256/−270, an E-box at −222/−229, and a GC-box at −117/−125, are critical for the promoter function. Using electrophoresis mobility shift assays, we find that the KLF motif mediates the binding of GKLF/KLF4, whereas the E-box is a target for both Upstream Stimulatory Factors 1 and 2, and the GC box at −117/−125 forms complexes with Sp1 and Sp3, but not with Sp4 or AP2α. A sequence element spanning position −150 may represent the binding motif of an uncharacterized transcription factor. The basal transcriptional regulation of Mfap2 in muscle cells is discussed.

Placental cathepsin M is alternatively spliced and exclusively expressed in the spongiotrophoblast layer

Cathepsin M and cathepsin-3 are cysteine peptidases expressed exclusively in the murine placenta. Their expression increases continuously from 11.5 dpc until the end of gestation. The cathepsin M gene consists of 8 exons and 7 introns covering 6 kb of genomic DNA on mouse chromosome 13. Multiple variants of CTSM were identified which display alternative splicing of exon 2 or exon 7. Alternative splicing of exon 2 does not affect the translated region of CTSM whereas aberrant splicing of exon 7 will results in enzymatically inactive versions of CTSM which still might retain inhibitory activity towards cysteine peptidases. Besides two defined major transcription start sites the putative promoter region comprises of a TATA-box and a relatively low (41%) G + C content reflecting its highly specific spatial and temporal expression pattern. Similar features are found within the promoter region of CTS3 which is highly homologous to CTSM. Both cathepsin M and -3 expression are confined to the spongiotrophoblast layer of the mouse placenta an expression pattern which is unique among cysteine peptidases located within the cluster of cathepsin J-like peptidases on mouse chromosome 13.

PHOX2B Regulates Its Own Expression by a Transcriptional Auto-regulatory Mechanism

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomain transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5'-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription.

Transforming Growth Factor-β1 Upregulates Transcription of α3 Integrin Gene in Hepatocellular Carcinoma Cells Via Ets-transcription Factor-binding Motif in the Promoter Region

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Characterization of the promoter for the α3 integrin gene in various tumor cell lines: Roles of the Ets‐ and Sp‐family of transcription factors

The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.

ATG deserts define a novel core promoter subclass

The MHC class I gene, PD1, has neither functional TATAA nor Initiator (Inr) elements in its core promoter and initiates transcription at multiple, dispersed sites over an extended region in vitro. Here, we define a novel core promoter feature that supports regulated transcription through selective transcription start site (TSS) usage. We demonstrate that TSS selection is actively regulated and context dependent. Basal and activated transcriptions initiate from largely nonoverlapping TSS regions. Transcripts derived from multiple TSS encode a single protein, due to the absence of any ATG triplets within approximately 430 bp upstream of the major transcription start site. Thus, the PD1 core promoter is embedded within an "ATG desert". Remarkably, extending this analysis genome-wide, we find that ATG deserts define a novel promoter subclass. They occur nonrandomly, are significantly associated with non-TATAA promoters that use multiple TSS, independent of the presence of CpG islands (CGI). We speculate that ATG deserts may provide a core promoter platform upon which complex upstream regulatory signals can be integrated, targeting multiple TSS whose products encode a single protein.

Structure and function of the upstream promotor of the human Mafbx gene: The proximal upstream promotor modulates tissue‐specificity

Muscle loss has been linked to increased expression of an ubiquitin ligase termed muscle atrophy F-box (MAFbx), a nuclear protein involved in degradation of MyoD. To gain insights into mechanisms by which the human MAFbx gene is controlled, the structure of its upstream promotor were studied, and its expression in cultured cells was characterized. Expression of MAFbx was found only in cells of muscle lineage. A reporter gene controlled by 948 bases of human MAFbx upstream promotor displayed similar cell-type selectivity. MAFbx levels were greatly enhanced upon myogenic differentiation of C2C12 myoblasts, and differentiation markedly increased activity of a reporter gene constructed with 400 bp of upstream promotor from the MAFbx gene. The core promotor spanned approximately 160 bases beginning at -241 bp upstream of the first codon, included potential binding sites for MyoD and myogenin, and was highly conserved among mouse, rat, and humanMAFbx genes. The major transcription start site for the human MAFbx gene was 340 bases upstream of the ATG and was localized the highly conserved region of 140 bp. The findings indicate an important role for the immediate upstream promotor of the human MAFbx gene in mediating its developmental expression and tissue specificity.

Functional studies of the 5'-untranslated region of human 5-HT4 receptor mRNA.

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.

Chromatin Immunoprecipitation (ChIP) on Chip Experiments Uncover a Widespread Distribution of NF-Y Binding CCAAT Sites Outside of Core Promoters

The CCAAT box is a prototypical promoter element, almost invariably found between -60 and -100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40-50% of NF-Y sites are not in core promoters but are in introns or at distant 3' or 5' locations. The abundance of "unorthodox" CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network.

Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter

Multiple lines of evidence indicate that the P-type Mg 2+-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon–intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position −143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5′-flanking sequence (−1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5′ end of this sequence up to nucleotide −291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to −217 caused a drastic decrease in activity in all three cell lines, but a −148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFκB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein.

Transcription of macrophage IGF-I exon 1 is positively regulated by the 5′-untranslated region and negatively regulated by the 5′-flanking region

Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3'-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from -1711 to -855 and -855 to -337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5'-untranslated region and negatively regulated by elements in the 5'-flanking region of the IGF-I gene.

The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism.

The PerC protein of enteropathogenic Escherichia coli (EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the LEE1 promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic E. coli (EHEC) lacks PerC-dependent activation due to utilization of an alternative LEE1 promoter and lack of a perC gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC LEE1 promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The perC1 genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual perC1 copies appear to be poorly expressed, the total perC1 expression level from a strain encoding multiple copies approaches that of perC in EPEC and may therefore contribute significantly to LEE1 activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the LEE1 promoter, suggesting that other core E. coli proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.

TAL1 Regulates Expression of the Band 3 Gene in Erythroid Progenitors through Both Upstream Regulatory and Proximal Promoter Elements.

The TAL1 (also known as SCL and TCL-5) protein is a class II basic helix-loop-helix (bHLH) transcription factor that plays an important role during embryonic and adult hematopoiesis. We previously established that the Protein 4.2 (P4.2) gene is a physiologic target of TAL1 in erythroid cells and showed that tandem E box-GATA elements in its proximal promoter mediated TAL1-directed gene activation. Through database searches, we identified two E box-GATA consensus sequences in the proximal promoter of another erythroid membrane skeleton gene, that encoding the Band 3 protein. Identical to what was observed for P4.2, overexpression of wild-type TAL1 slightly increased while enforced expression of a DNA binding-defective TAL1 mutant severely reduced endogenous Band 3 gene expression in murine erythroleukemia (MEL) cells induced to differentiate with dimethyl sulfoxide (DMSO). Overexpression of Ldb1 significantly inhibited and a LIM protein interaction-defective Ldb1 mutant virtually ablated Band 3 mRNA accumulation in DMSO-induced MEL cells. Quantitative chromatin immunoprecipitation (ChIP) analysis with Tal1 antibody confirmed that Tal1 was associated with the proximal promoter of the Band 3 gene in MEL cells, while analysis of approximately 28 kb of genomic sequence spanning the Band 3 gene, including 5 kb 5′ and 3′ of the gene, revealed another consensus E box-GATA element in intron 16; however, no significant TAL1 binding was detected in this region by ChIP analysis. To identify potential non-conventional TAL1 binding site(s), scanning ChIP analysis was applied to the entire 28 kb Band 3 genomic region, and a region ~2.5 kb upstream of the major transcriptional start site was found to bind significantly more TAL1 protein than the proximal promoter. Fine mapping of TAL1 binding to this region by ChIP analysis using more highly sheared DNA and smaller sized amplicons narrowed TAL1 binding to a region of ~100 bp, which we designated as the Band 3 upstream regulatory region (B3URE). Luciferase reporter assays in transiently transfected MEL cells with vectors containing genomic sequence revealed the presence of an orientation- and position- independent repressor in this upstream region, with a 107 bp fragment retaining nearly all the repressing activity of a larger, 700 bp fragment. In contrast, coexpression of TAL1, E47, GATA-1, LMO2, and Ldb1 in COS cells led to transactivation of a reporter gene linked to the promoter-proximal E box-GATA element. These data suggest that TAL1 acts as a repressor on the upstream B3URE in undifferentiated cells and then, as differentiation proceeds, as an activator on the E box-GATA elements in the proximal promoter.

The Transcription Factor CCAAT-binding Factor CBF/NF-Y and Two Repressors Regulate the Core Promoter of the Human Pro-α3(V) Collagen Gene (COL5A3)

To elucidate the mechanisms underlining alpha3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the -129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to -122 to -117, the -101 to -96, the -83 to -78, and the -68 to -57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity.

Functionally significant SNP MMP8 promoter haplotypes and preterm premature rupture of membranes (PPROM).

Matrix metalloproteinase 8 (MMP8), an enzyme that degrades fibrillar collagens imparting strength to the fetal membranes, is expressed by leukocytes and chorionic cytotrophoblast cells. We identified three single nucleotide polymorphisms (SNPs) at -799C/T, -381A/G and +17C/G from the major transcription start site in the MMP8 gene, and determined the functional significance of these SNPs by analyzing their impact upon MMP8 promoter activity and their association with preterm premature rupture of membranes (PPROM). The minor alleles +17 (G) and -381 (G) were in complete linkage disequilibrium. A promoter fragment containing the three minor alleles had 3-fold greater activity in chorion-like trophoblast cells (BeWo, JEG-3 and HTR-8/SVneo) compared with the major allele promoter construct. Electrophoretic mobility shift assays revealed differences in BeWo nuclear protein binding to oligonucleotides representing the -381 and -799 SNPs, suggesting that the minor alleles have reduced transcription factor binding. A case-control study of African-American neonates using allele-specific primers revealed a statistically significant association between the three minor allele haplotype, which displays the highest MMP8 promoter activity in trophoblast cells, with PPROM with an odds ratio (OR) of 4.63 (P < 0.0001), whereas the major allele promoter appeared to be protective (OR = 0.52, P < 0.0002). None of the minor alleles were individually associated with PPROM. These findings demonstrate the functional significance of SNP haplotypes in the MMP8 gene and associations with obstetrical outcomes.

Cloning, gene organization and identification of an alternative splicing process in lecithin:retinol acyltransferase cDNA from human liver

Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters in many tissues and is crucial for the transport and intracellular storage of vitamin A. LRAT expression is highly regulated in the liver. In this study, we have cloned and sequenced the full-length LRAT mRNA from human liver and identified its 5′- and 3′-ends. Full-length LRAT mRNA comprises 5023 nt with a predicted ORF of 230 amino acids, a short 5′UTR, and a relatively long 3′UTR of 4 kb containing several polyadenylation signals and AU-rich regions. Based on alignment of this mRNA with human genomic DNA in the GenBank database, the human LRAT gene spans about 9.1 kbp and consists of two exons and a relatively long 4-kbp intron. Further analysis of normal liver revealed a minor alternative splicing variant which lacks a 103 nt polynucleotide contained in the 5′UTR of the full-length LRAT transcript. This variant predicts that the LRAT gene is organized into three exons and two introns, as reported for LRAT cloned from retinal pigment epithelium (RPE) cells. These two LRAT mRNA variants are also present in testis, which is known to express LRAT and contain retinyl esters. Major and minor transcription start sites for human liver LRAT mRNA were identified and the sequence of the upstream proximal promoter region was retrieved from the GenBank database and physically analyzed for the presence of putative cis-acting elements essential for basal transcription. This region contains a TATA box, CCAAT box and Sp1 site, which are apparently conserved in mouse and rat LRAT genes. Our results provide evidence that multiple LRAT mRNA transcripts, which are expressed in a tissue-specific manner, may result from several mechanisms including differential splicing of the 5′UTR region and the use of multiple polyadenylation signals in the 3′UTR.

Transcriptional activation of c-myc proto-oncogene by WT1 protein.

The Wilms' tumor 1 gene (WT1) plays an essential role in urogenital development and malignancy. Through DNA binding, WT1 can either enhance or repress transcription depending on the context of the DNA-binding sites or the cell type in which it is expressed. WT1 is overexpressed in a variety of human cancers, including leukemia and breast cancer; in these diseases, the expression of WT1 is associated with a poor prognosis. To determine how WT1 affects c-myc expression in the context of breast cancer cells, we have examined the ability of both endogenous and exogenous WT1 proteins in breast cancer cells to bind to the c-myc promoter in vivo. Using c-myc-promoter-driven luciferase constructs, we found that different forms of WT1 could enhance the expression of the reporter. Unlike other studies where WT1 is reported to be a negative regulator of c-myc, we found that both the - and + KTS forms of WT1 could act to enhance c-myc expression, depending on the cell type. The WT1-binding site near the second major transcription start site of the c-myc promoter was confirmed to be involved in upregulation of human c-myc by WT1. Finally, we demonstrated that overexpression of WT1 induced a significant increase in the abundance of endogenous c-myc protein in breast cancer cells, consistent with the upregulation of c-myc transcription following WT1 induction. These observations strongly argue that in the case of breast cancer WT1 is functioning as an oncogene in part by stimulating the expression of c-myc.