A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

Abstract

The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-Int⌬). The Col-Int⌬ allele contains a 1.3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two-to threefold decrease in Col1A1 mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies of cis-acting elements in gene regulation.

Insertional Mutation in the Intron 1 of Unc5h3 Gene Induces Ataxic, Lean and Hyperactive Phenotype in mice.

“…Regulation of the cardiac homeobox gene CSX1 is mediated by an enhancer element in the first intron [26]. There is substantial evidence for the involvement of the first intron in the tissue-specific expression of the alpha 1 (I) collagen gene [6,15]. A negative regulatory element was identified and characterized within the first intron of the epidermal growth factor receptor gene [33].…”

UV Irradiation Activates JNK and Increases αI(I) Collagen Gene Expression in Rat Hepatic Stellate Cells

“…A previous study found that an AP-1 binding site in the first intron of the ␣I(I) collagen gene played a critical role in the stimulation of the ␣I(I) collagen gene by transforming growth factor (TGF␤) (33). Another recent report indicated that the first intron of the ␣I(I) collagen gene had a tissue-specific and developmentally regulated function in transcription of the gene (34). In the present study, HSCs transfected with plasmid p1.7/1.6 or p1.7 (GC box mut)/1.6 showed significant differences in the CAT activity in their responses to dominant negative JUN (Fig.…”

δEF1 Binds to a Far Upstream Sequence of the Mouse Pro-α1(I) Collagen Gene and Represses Its Expression in Osteoblasts

“…It had a repressor effect in transient transfection experiments, but sequences adjacent to this element completely abolished its repressor effect (11). Moreover, deletion of most of the first intron did not increase the levels of expression of the pro-␣1(I) collagen gene in vivo (8). A member of the Krü ppel-like family of transcription factors named cKrox is the only transcription factor that has been reported as being able to down-regulate the level of expression of the pro-␣1(I) collagen gene (12).…”

“…The first intron of the pro-␣1(I) collagen gene also contains positive regulatory elements such as an AP-1 binding site (6) or an Sp1-binding site that is involved in maintaining bone density (7). The role of this site in maintaining normal levels of expression of the pro-␣1(I) collagen gene throughout life has been shown by knock-in experiments (8). A cis-acting element located in the 3Ј-flanking region of the pro-␣1(I) collagen gene has been shown to drive high levels of reporter gene expression in transiently transfected fibroblastic cells (9).…”