Identification and analysis of the human neural polypyrimidine tract binding protein (nPTB) gene promoter region.

Neural polypyrimidine tract-binding protein nPTB, originally identified as the neuronal counterpart of the hnRNPI/PTB protein, is an RNA binding protein involved into tissue-specific alternative splicing regulation. Here we describe the functional characterization of the promoter sequence of nPTB in HeLa and neuroblastoma cells. By means of genomic sequence analysis we have isolated and cloned a 1587-base pair region upstream the human nPTB coding region. The nPTB proximal promoter, although rich in G+C content and presenting putative binding sites for the transcription factors Sp1, NF-1, NF-kB and Oct-1, lacks a typical TATA box. Luciferase transient expression assays using deletion mutants have identified the proximal promoter region at -125 relative to the transcription start site. Alignment of human, murine and chimpanzee genomic sequences upstream the nPTB exon 1 has provided evidences for the evolutionary conservation of specific transcription factor binding sites.

Romanelli MG ，Lorenzi P ，Morandi C 《GENE》

Characterization of the infection-responsive bovine lactoferrin promoter.

The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5' flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a non-canonical TATA box, multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4 kb of 5' flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basal promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS) significantly stimulated its activity up to 2.3-fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to -543 was sufficient for basal activity, whereas the sequence up to -1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative regulatory region (-1407/-1029). LPS-responsive regions of the promoter were localized to the region from -1029 to -543 containing one STAT3 site and two NF-kappaB sites, and the region from -4355 to -2462 containing three AP1 sites and six NF-kappaB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-kappaB pathway.

Zheng J ，Ather JL ，Sonstegard TS ，Kerr DE ... -  《GENE》

Transcriptional regulation of the mouse PNRC2 promoter by the nuclear factor Y (NFY) and E2F1.

PNRC2 (Proline-rich Nuclear Receptor Coactivator 2) was previously identified through its interaction with SF1 (steroidogenic factor 1) and has been demonstrated to be a novel coactivator for multiple nuclear receptors. In this study, PNRC2 was found to be widely expressed in mouse tissues with a strong expression in lung, spleen, ovary, thymus, and colon. Alignment of mouse genomic sequence with mouse cDNA sequence (BC006598), using mouse genome browser, defines that PNRC2 gene, located on chromosome 4, contains 3 exons: 166 bp-exon I, 205 bp-exon II, and 1526 bp-exon III. The translational start site is located in exon III. The first two exons are not translated. The 420 bp coding sequence in exon III encodes a 140 amino acid protein. To understand the molecular mechanisms that regulate the expression of PNRC2 gene, we have cloned and characterized the 5'-flanking region of the gene. Potential transcriptional start sites were determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the mPNRC2 gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -67/+53 region is the minimal promoter of the mouse PNRC2 gene in HeLa cells. Within this sequence we identified two putative binding sites (inverted CCAAT box) for the transcription factor NFY (nuclear factor Y), a factor mediating cell type-specific and cell-cycle regulated expression of genes, and one binding site for E2F1, a founding member of the E2F family that displays the properties of both an oncogene and a tumor suppressor gene. Mutating each individual CCAAT site or changing the orientation of the CAATT box led to a 5-fold decrease in PNRC2 promoter activity in transient transfection experiments. Gel shift, supershift assay, and ChIP analysis demonstrated the specific binding of NFY and E2F1 proteins to the mouse PNRC2 promoter. Transient transfections and luciferase assays further revealed that overexpression of NFY enhanced-promoter activity of PNRC2 gene in a dose-dependent manner while overexpression of E2F1 strongly repressed the activity of the PNRC2 promoter. Since most genes regulated by E2F1 or NFY play a regulatory role in the cell cycle, the finding that the PNRC2 promoter is activated by NFY and repressed by E2F1 indicates that in addition to functioning as nuclear receptor coactivator, PNRC2 may also play a role in the cell cycle.

Zhou D ，Masri S ，Ye JJ ，Chen S ... -  《GENE》

Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein.

In a study of the role of transcription factor Sp1 in the formation of tumors by human fibrosarcoma cell lines that overexpress it [Cancer Res., 65 (2005) 1007], we found that expression of an Sp1-specific ribozyme, not only reduced the level of Sp1 protein, but also that of Sp3 protein, and that when the protein levels of these two transcription factors in the fibrosarcoma cell lines were reduced to near that found in normal human fibroblasts, the cell lines could no longer form tumors. An Sp1-specific ribozyme could reduce the level of expression of both Sp1 protein and Sp3 protein if the promoter of the Sp1 gene and that of the Sp3 gene both have Sp1/Sp3 transcription factor binding sites and if such sites are critically responsible for the level of expression of both Sp1 and Sp3 protein in the cells. The Sp1 minimal promoter has been identified and it has two Sp1/Sp3 sites [J. Biol. Chem. 276 (2001) 22126]. To characterize the Sp3 promoter, we isolated 2.1 kb of the 5'-flanking region of the Sp3 gene, which contains Sp1/Sp3 binding sites, and using an expression reporter assay, showed that it has promoter activity. We then systematically reduced the size of the 5' flanking region, and determined that the nt-339 to nt-39 fragment, which contains an Sp1/Sp3 binding site at nt-181 and another at nt-168, retained the same promoter activity as the 2.1 kb region. Electrophoretic mobility shift assays indicated that both Sp3 protein and Sp1protein bind to these two sites. By mutating either or both of these binding sites, we showed using the reporter assay that each site is required for full promoter activity. We then designed an Sp3-specific ribozyme, expressed it in a human fibrosarcoma cell line in which Sp1 protein and Sp3 protein are expressed at high levels, and found that, indeed, the level of expression of both proteins was significantly reduced.

Lou Z ，Maher VM ，McCormick JJ 《GENE》

The mouse chemerin receptor gene, mcmklr1, utilizes alternative promoters for transcription and is regulated by all-trans retinoic acid.

CMKLR1 (chemoattractant-like receptor 1) is a G-protein-coupled receptor implicated in cartilage and bone development and is expressed in organs like the parathyroid gland, brain, and lung. The receptor is also expressed in dendritic cells and in macrophages where it acts as a co-receptor for entry of HIV/SIV isolates into human CD4(+) cells. Recently, a protein named "chemerin" (also known as TIG2) was isolated from human inflammatory fluids and hemofiltrate and found to be the endogenous ligand for CMKLR1. We have previously described the genomic organization of the cmklr1 gene and characterized its promoter in mouse neuroblastoma NB4 1A3 cells. In the present study we identify a second transcript, cmklr1b, in mouse microglia BV2 cells. Cmklr1b is transcribed from an alternative promoter with a transcription start site located 6780 bp downstream of the previously identified exon 1 (cmklr1a). The cmklr1b promoter lacks a TATA box but contains two CCAAT boxes in opposite directions. 5' Deletion analysis of the promoter region in BV2 cells using a luciferase reporter gene assay indicates two regions, between 623-755 bp and 56-125 bp upstream of transcription start site, to be important for promoter function. The proximal promoter region includes both CCAAT boxes, and site-directed mutagenesis separately within these elements revealed that only the forward CCAAT element was important for transcription. Although the forward CCAAT element is essential for transcription electrophoretic mobility shift and super-shift assays demonstrated that both CCAAT elements actually bind nuclear proteins from BV2 cells and identified the binding factor as NFY. Real-time reverse transcriptase-PCR experiments of cmklr1b expression in all-trans retinoic acid (ATRA)- stimulated BV2 cells showed strong up-regulation of receptor transcript. Luciferase reporter gene assay of the promoter in ATRA-stimulated BV2 cells confirmed that transcriptional activity of the cmklr1b promoter is increased by ATRA. However, deletion analysis could not identify an ATRA-responsive element within the promoter region suggesting that gene activation is likely to occur through alternative mechanisms. The results emphasise a possible role of cmklr1 in bone modelling.

Mårtensson UE ，Bristulf J ，Owman C ，Olde B ... -  《GENE》