A regulatory element within a coding exon modulates keratin 18 gene expression in transgenic mice.
Multiple tissue-specific, DNase-hypersensitive sites are correlated with known or potential regulatory regions of the human keratin 18 (K18) gene. One of these sites is found within exon 6, close to a potential AP-1 binding site. Footprint analysis confirmed that this site is capable of binding c-Jun and c-Fos in vitro. However, exon 6 can stimulate expression of a reporter gene driven by the K18 proximal promoter independent of AP-1 in F9 cells and additionally modulates AP-1 responsiveness when in combination with an intron enhancer. Analysis in transgenic mice and by transient transfections of mutant forms of the K18 gene showed that exon 6 contributes to the expression of the K18 gene. However, substitution of part of exon 6 with the corresponding part of the keratin 19 gene which lacks an AP-1 site decreased but did not destroy the regulatory activity of the exon. Furthermore, this mutation did not alter either the tissue specificity or the position-independent and copy number-dependent behavior of the K18 gene. In contrast, a frameshift mutation within exon 6 dramatically decreased the expression of the gene. K18 RNA expression from the frameshift mutation was less than 10% of the wild type K18 transgene. This decline in expression was the result of a combination of decreased stability of mutant K18 RNA and the creation of a negative regulatory element that can interact with the first intron regulatory elements and actively suppress K18 expression. These results demonstrate that a protein-coding portion of the K18 gene also has a regulatory function.
Neznanov
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N
Burnham Institute, La Jolla, California 92037, USA.
Umezawa
A
A
Oshima
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins; D005091:Exons; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D007633:Keratins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D015534:Trans-Activators / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; C494070:Ets2 protein, mouse; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D012313:RNA; D007633:Keratins