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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.

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Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5-flanking region of the ␣1(VI) collagen gene, mapping at ؊4.6, ؊4.4, ؊2.5, and ؊0.1 kilobase (kb) from the RNA start site. The site at ؊2.5 kb was independent from, whereas the other three sites could be related to, ␣1(VI) mRNA expression. The site at ؊0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides ؊104 and ؊73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at ؊0.

The region extending from ؊5.4 to ؊3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163-1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E-L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E-J, which spans the 5 245 base pairs of E-L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E-J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing ␣1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the ؊5.4/؊3.9 region of the Col6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity.Genes of the extracellular matrix are very often among targets of terminal differentiation programs. In most cases, expression of the genes is the result of transcriptional regulation attained by tissue-specific enhancers. Well characterized examples are genes such as osteocalcin, collagen I, osteopontin, and bone sialoprotein in osteoblasts, and collagen II and XI in chondroblasts. The exclusive transcription of osteocalcin and the high level expression of ␣1(I) collagen, osteopontin, and bone sialoprotein are controlled by sequences binding Osf2/ Cbfa1, a transcription factor necessary for the differentiation of osteoblasts (1), whereas transcription of ␣1(II) and ␣2(XI) genes requires sequences recognized by Sox9 and other members of the high mobility group class of transcription factors, which are involved in cartilage differentiation (2-6). Thus, the identification and analysis of enhancers responsible for tissuespecific expression of extracellular matrix components are important not only to understand the regulation of their genes but also to clarify the genetic control of differentiation programs.Our group has undertaken the study of regulation of collagen VI in the mouse and has identified several sequences...