Tissue‐Specific Expression of Promoter Regions of the αr1(VI) Collagen Gene in Cell Cultures and Transgenic Mice

Abstract

Cis-acting regions regulating transcription of the alpha1(VI) collagen chain have been investigated in vitro by transfection of promoter-CAT (where CAT is chloramphenicol acetyltransferase) constructs in different types of cultured cells and in vivo in transgenic mice carrying the same CAT constructs or minigenes derived from the fusion of genomic and cDNA sequences in which small deletions of the collagenous domain had been engineered. 215 bp of 5'-flanking sequence showed promoter activity in vitro, yet were not expressed in any tissue of six transgenic 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 transfected cells and were expressed in tissues of about 30% of transgenic lines. Although CAT activity was totally unrelated to the pattern of expression of the alpha1(VI) mRNA, these results suggest the presence of an activator(s) between -0.2 and -0.6 kb from the transcription start site. When the promoter size was increased to 5.4 or 6.5 kb, CAT activity was stimulated severalfold relative to the construct p1.4CAT and p4.0CAT in NIH3T3 fibroblasts and chick embryo chondroblasts. This stimulation was, however, not observed in C2C12 myoblasts. Transgenic mice generated with 6.5CAT construct or minigenes, containing 6.2 kb of promoter, exhibited very high levels of expression, which was similar to the relative amount alpha1(VI) mRNA in the majority of tissues, with the exception of lung, adrenal gland and uterus. CAT activity in tissues was 100-1000-fold higher than that measured in transgenic mice with shorter promoter (0.6 or 1.4 kb). Since expression of minigenes was determined by RNase protection assay, the levels of mRNA per transgene copy were compared to those of the chromosomal gene and found to be always less than one quarter. These data suggest that the region -4.0/-5.4 contains an important activator(s) sequence which induces transcription in several, but not all, type VI collagen-producing tissues. Finally, analysis with the longest promoter fragment (7.5 kb) revealed a complex effect of the region -6.5/-7.5 on alpha1(VI) chain transcription. The sequence was inhibitory in NIH3T3 cells, indifferent in myoblasts and activating in chondroblasts in vitro, whereas transgenic animals generated with 7.5CAT construct produced a pattern of expression comparable to that of 6.5CAT and minigenes. During postnatal development transcription from both the endogenous gene and the transgenes decreased. However, the ratio of transgene/chromosomal gene expression was not constant, but varied in a way dependent on the tissue. This observation suggests that the fragment studied contains key sequences for the age-dependent regulation of the alpha1(VI) gene. No phenotypic alterations were induced by the presence of mutations in the minigenes.

Cell Type-specific Transcription of the α1(VI) Collagen Gene

“…Although, as stated above, no locus control regions have been defined yet, a correspondence between hypersensitive sites and actual transcription has been found also for collagen genes, in particular ␣2(I) and ␣1(I) (5,11). As for the manner in which the different cis-acting regulatory elements contribute to the transcriptional regulation of a collagen gene, the available data suggest that they act in a modular way (4,5,12,13). As proposed recently by Arnone and Davidson (14), this means that each region contributes "a particular regulatory function that is a subfraction of the overall combined regulatory function executed by the complete system" independently from the other regions.…”

Analysis of Transcription of the Col6a1 Gene in a Specific Set of Tissues Suggests a New Variant of Enhancer Region

“…Finally, the sequence between about Ϫ5.4 to Ϫ3.9 kb from the transcription start site contains information for expression in articular cartilage, intervertebral disks, nerves, vibrissae, and subepidermal mesenchyme. The strong activating capacity of the Ϫ5.4-to Ϫ3.9-kb sequence has been confirmed by experiments with transgenic mice, where promoter-CAT fusions including this region are expressed in several tissues over 100 times more efficiently than constructs lacking it (9). One interesting feature of the Ϫ5.4/Ϫ3.9 region is that information controlling transcription in cells with different embryological origin and function, including articular chondrocytes, Schwann cells, 2 and fibroblasts, is enclosed in a relatively small DNA fragment, 1.5 kb, a size compatible with that of a single enhancer (11).…”

“…C2C12 myoblasts were not used in this experiments because we had previously demonstrated that there is no significant difference of CAT expression between p1.4(A-P)CAT and p1.4CAT (9). On the contrary, the whole enhancer region increased transcription considerably in NIH3T3, MC615, and sternal chondroblasts from chick embryos (Fig.…”

“…These include p5.4⌬(4.0 -1.4)lacZ and p5.4⌬(4.0 -1.4)CAT (8,9), renamed p1.4(A-P)lacZ and p1.4(A-P)CAT.…”

“…Our group has undertaken the study of regulation of collagen VI in the mouse and has identified several sequences of the 5Ј-flanking region of the Col6a1 gene active in transcriptional control (7)(8)(9)(10). In particular, analyses in transgenic mice have located three regions responsible for tissue-specific transcription at high frequency (8).…”