TISSUE-SPECIFIC EXPRESSION OF PROMOTER REGIONS OF THE ALPHA-1(VI) COLLAGEN GENE IN CELL-CULTURES AND TRANSGENIC MICE

Cis-acting regions regulating transcription of the alpha 1/(VI) collag en chain have been Investigated ia vitro by transfection of promoter-C AT (where CAT is chloramphenicol acetyltransferase) constructs in diff erent types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomi c and cDNA sequences in which small deletions of the collagenous domai n had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transge nic lines, indicating that this fragment contains the basal promoter, but not activator sequences. Constructs with 0.6 and 1.4 kb of the 5'- flanking region produced significantly higher CAT activity in transfec ted cells and were expressed in tissues of about 30% of transgenic lin es. Although CAT activity was totally unrelated to the pattern of expr ession of the alpha(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start si te. When the promoter size was increased to 5.4 or 6.5 kb, CAT activit y was stimulated severalfold relative to the construct p1.4CAT and p4. 0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimul ation was, however, not observed in C2C12 myoblasts. Transgenic mice g enerated with 6.5CAT construct or minigenes, containing 6.2 kb of prom oter, exhibited very high levels of expression, which was similar to t he relative amount alpha 1(VI) mRNA in the majority of tissues, with t he exception of lung, adrenal gland and uterus. CAT activity in tissue s was 100-1000-fold higher than that measured in transgenic mite with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was de termined by RNase protection assay, the levels of mRNA per transgene c opy were compared to those of the chromosomal gene and found to be alw ays less than one quarter. These data suggest that the region -4.0/-5. 4 contains an important activator(s) sequence which induces transcript ion in several, but not all, type VI collagen-producing tissues. Final ly, analysis with the longest promoter fragment (7.5 kb) revealed a co mplex effect of the region -6.5/-7.5 on alpha 1(VI) chain transcriptio n. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblas ts and activating in chondroblasts in vitro, whereas transgenic animal s generated with 7.5CAT construct produced a pattern of expression com parable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreas ed. However, the ratio of transgene/chromosomal gene expression was no t constant, but varied in a way dependent on the tissue. This observat ion suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha 1(VI) gene. No phenotypic altera tions were induced by the presence of mutations in the minigenes.