Genomic organization and promoter analysis of the mouse ADP-ribosylarginine hydrolase gene.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases (ADPRH) catalyzing the opposing arms of an ADP-ribosylation cycle. The ADPRH cDNA had been cloned from human, rat, and mouse tissues and high levels of mRNA were found in brain, spleen, and testis. To begin to understand the molecular mechanisms that regulate ADPRH gene expression, we cloned the full-length cDNA, determined the genomic structure of mouse ADPRH, and investigated promoter function. Northern analysis using different regions of the ADPRH cDNA as probes identified two mRNAs of 1.7 and 3.0 kb, which resulted from the use of alternative polyadenylation signals, CATAAC and ATTAAA, beginning at positions 1501 and 2885, respectively, of the nucleotide sequence (A of ATG = 1). The ADPRH gene, represented in two overlapping genomic clones, spans approximately 9 kilobases with four exons and three introns. The 5'-flanking region contains the features of a housekeeping gene; it has neither a TATA nor a CAAT box, but is, instead, highly GC-rich with multiple transcription initiation sites. Promoter analysis, assessed using transient transfection of PC12, NB41A3, NIH/3T3, and Hepa 1-6 cells with truncated constructs, revealed potent stimulatory (-119 to -89) and inhibitory (-161 to -119) elements, which were utilized similarly in the different cell lines. Further mutational analysis of the promoter and electrophoretic mobility-shift assays identified a positive GC-box element (-107 to -95); Sp1 and Sp3, which bound to this motif, were also detected by supershift assays. In co-transfection experiments using Drosophila SL2 cells that lack endogenous Sp1, Sp1 trans-activated the ADPRH promoter in a manner dependent on the presence of an Sp1-binding motif. The promoter activity pattern and involvement of Sp transcription factors are consistent with prior observations of widespread hydrolase expression in mammalian tissues.

Aoki K ，Kato J ，Shoemaker MT ，Moss J ... -  《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》

Sp1 and AP2 enhance promoter activity of the mouse GM3-synthase gene.

Promoters of the glycosyltransferase genes for ganglioside synthesis are TATA-less and often have multiple binding sites for transcription factors Sp1 and AP2 in their proximal regions. However, the function of Sp1 and AP2 in the promoters has not yet been defined. Here, we cloned 5'-flanking fragments of the mouse GM3-synthase gene and assessed the promoter activity of these fragments in mouse Neuro-2a cells. This promoter is TATA-less and contains a number of potential transcription factor-binding sites. Multiple putative transcriptional initiation sites for this gene were identified, including several downstream initiation sites. We then set out to dissect the regulatory elements important for GM3-synthase promoter function. We found that a 5'-flanking 254-bp DNA fragment of the gene contained regulatory elements including two Sp1-binding and six AP2-binding sites that were essential for the basal activity of the promoter in mouse Neuro-2a cells. The effects of the individual Sp1- and AP2-binding sites on basal activity of the GM3-synthase gene were investigated. Mutations in the juxtaposed Sp1/AP2-binding site and in an AP2-binding site decreased the activity of the proximal promoter to approximately 50%. In vitro and in vivo interactions between transcription factors Sp1 and AP2 and these regulatory elements were confirmed by EMSA and the chromatin immunoprecipitation approach, respectively. Therefore, our results demonstrate that Sp1 and AP2 enhance the basal activity of the TATA-less mouse GM3-synthase promoter.

Xia T ，Zeng G ，Gao L ，Yu RK ... -  《GENE》

Genomic organization and transcriptional analysis of gonadotropin-regulated testicular RNA helicase--GRTH/DDX25 gene.

The gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a new member of the DEAD-box protein family. Phylogenetic analysis revealed that GRTH is distantly related to other members of the family. GRTH is transcriptionally up-regulated by gonadotropin, displays ATPase and RNA helicase activities, and participates in germ cell development. To understand the regulation of GRTH gene expression, we investigated its structural organization and aspects of basal transcriptional regulation at the promoter domain. The 20-kb mouse GRTH gene contains 12 coding exons and all but one of its conserved helicase motifs are contained within single exons. GRTH is a TATA-less gene with multiple transcriptional start sites (TSS), GC-rich sequences and a promoter located within -205/+63 bp of the gene. Sequences -852/-354 and -501/-354 bp caused 40-60% and >80% inhibition of transcription in expressing and non-expressing cells, respectively. Transcriptional activity was recovered only in expressing cells by the addition of upstream sequences (-1085/-852 bp). Sp1/Sp3 supported basal transcriptional activity in all cell types, while E-box was an activator-binding site only in non-expressing cells. These findings indicate that a differential pattern of transcriptional regulation may be involved in the control of GRTH gene expression in a cell-specific manner.

Tsai-Morris CH ，Lei S ，Jiang Q ，Sheng Y ，Dufau ML ... -  《GENE》