Dataset Information

The genomic structure of the human UBE1L gene.

ABSTRACT: The human UBE1L gene, for which the product may well play a role in the ubiquitin system because of its high degree of identity to the ubiquitin activating enzyme, is located at 3p21, a chromosomal region consistently showing loss of heterozygosity in lung cancer. The finding that UBE1L is well expressed in normal lung tissue, but hardly or not in lung cancer-derived cell lines, prompted us to investigate its genomic structure to find an explanation for the lack of expression in lung cancer. The gene has 22 exons distributed over 8.4 kb. Both anchored PCR experiments and mapping of DNase I-hypersensitive sites point to the region immediately upstream of exon 1 as the promoter site. Three moderately to well-informative polymorphisms were found, of which one is easily directly detectable. Cancer-specific mutations were not detected. The lack of expression in lung cancer cell lines correlated with a highly decreased sensitivity towards DNAse I of the promoter region and with an almost complete methylation of the HhaI site in the first exon. 5'-Azacytidine-induced demethylation did not result in a marked increase of the UBE1L mRNA level in the tumor cell lines. This leaves the possibility that mutation or absence of yet unknown transcription factors causes a regulatory block of the UBE1L gene.

SUBMITTER: Kok K

PROVIDER: S-EPMC6134375 | biostudies-literature | 1995

REPOSITORIES: biostudies-literature

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The genomic structure of the human UBE1L gene.

Kok K K Van den Berg A A Veldhuis P M PM Franke M M Terpstra P P Buys C H CH

Gene expression 19950101 3

The human UBE1L gene, for which the product may well play a role in the ubiquitin system because of its high degree of identity to the ubiquitin activating enzyme, is located at 3p21, a chromosomal region consistently showing loss of heterozygosity in lung cancer. The finding that UBE1L is well expressed in normal lung tissue, but hardly or not in lung cancer-derived cell lines, prompted us to investigate its genomic structure to find an explanation for the lack of expression in lung cancer. The ...[more]

PMID: 7734949

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Project description:The inducible IkappaB kinase (IKKi/IKKepsilon) is a recently described serine-threonine kinase that activates the transcription factors NFkappaB, interferon regulatory factor-3 (IRF3) and CCAAA/enhancer-binding protein (C/EBPdelta). Several inflammatory agents have been shown to induce the expression of the IKKi gene in macrophages and other cell types but the mechanism is unknown. We have found that the IKKi expression was constitutive in human chondrocytes from OA cartilage and a human chondrocytic cell line C28/I2 but was up-regulated by the inflammatory cytokines TNFalpha or IL-1betain an NFkappaB-dependent manner. To understand the constitutive and inducible expression of the IKKi gene we localized the transcription start site (TSS), cloned and sequenced a 2 kb genomic DNA fragment 5' of the TSS and characterized the putative promoter region (PPR), and identified the motifs therein that are required for basal and cytokine-induced IKKi gene promoter activity. We found that IKKi core promoter was TATA-less and by using PCR generated deletion mutants of the PPR we found that the cis-elements responsible for basal transcriptional activity were located between -51 and -100 bp upstream of the TSS while the cytokine response elements were located distally between -501 and -1000 bp upstream of the TSS. The DNA region containing the cytokine response elements had two kappaB sites as the most relevant regulatory motifs. The results of site-directed mutagenesis revealed that the kappaB site located between -833 and -847 bp upstream of the TSS was biologically functional and required for cytokine-induced IKKi promoter activity in human chondrocytes and HeLa cells. The silence of the other kappaB site (-816/-802) was positional, rather than sequence-specific. Over-expression of NFkappaB p65 mimics the TNFalpha-induced activation of the IKKi promoter. Also the gel shift assay suggested that NFkappaB p65 is responsible for activation of the IKKi promoter. These data for the first time characterize the promoter region and provide further insights into the transcriptional regulation of IKKi in human chondrocytes and other cell types.

Dataset Information

The genomic structure of the human UBE1L gene.

Publications

The genomic structure of the human UBE1L gene.

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