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Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

ABSTRACT: The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.

SUBMITTER: Erno H

PROVIDER: S-EPMC6081634 | biostudies-literature | 1993

REPOSITORIES: biostudies-literature

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Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

Ernø H H Monard D D

Gene expression 19930101 2

The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GD ...[more]

PMID: 8268720

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Project description:BACKGROUND:Urokinase-type Plasminogen Activator (uPA), a serine protease, plays a pivotal role in human breast cancer metastasis by mediating the degradation of extracellular matrix proteins and promoting cell motility. In more advanced breast cancers, uPA activity is significantly up regulated and serves as a prognostic indicator of poor patient outcome. Classically, regulation of uPA activity, especially in breast cancers, is thought to be mediated by Type 1 Plasminogen Activator Inhibitor (PAI-1). However, we have recently found that a lesser known natural inhibitor of uPA, Protease Nexin 1 (PN-1), is expressed in normal human mammary tissue. Based on this observation, we investigated if PN-1 is also expressed in human breast cancers where it may contribute to the regulation of uPA and participate in the development of a metastatic phenotype. RESULTS:Using quantitative real-time PCR analysis, we measured PN-1 mRNA expression in tissues obtained from 26 human breast tumor biopsies and compared these values with those obtained from 10 normal breast tissue samples. Since both PAI-1 and uPA expression levels are known to be elevated in metastatic breast cancer, we also measured their levels in our 26 tumor samples for direct comparison with PN-1 expression. We found that PN-1 expression was elevated over that found in normal mammary tissue; an increase of 1.5- to 3.5-fold in 21 of 26 human breast tumors examined. As anticipated, both PAI-1 and uPA mRNA levels were significantly higher in the majority of breast tumors; 19 of 26 tumors for PAI-1 and 22 of 26 tumors for uPA. A quantile box plot of these data demonstrates that the elevated PN-1 expression in breast tumor tissues directly correlates with the increased expression levels found for PAI-1 and uPA. CONCLUSION:The fact that PN-1 expression is elevated in human breast cancer, and that its increased expression is directly correlated with increases measured for PAI-1 and uPA, suggests that PN-1 may contribute to the regulation of uPA-mediate tumor cell motility and metastatic spread.

Project description:BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat ?1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a ?1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

Dataset Information

Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

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Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter.

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