MOLECULAR-CLONING AND CHROMATIN STRUCTURE-ANALYSIS OF THE MURINE ALPHA-1(I) COLLAGEN GENE DOMAIN

We have isolated molecular clones of genomic mouse DNA spanning 55 kb, including the entire coding region of the murine alpha 1(I) collagen (Colla1) gene and 24 kb of 5' and 13 kb of 3'-flanking sequences, and have performed a detailed chromatin structure analysis of these sequen ces. Several new DNase-I-hypersensitive sites were identified. The dis tal 5'-flanking region contains two clusters of DNase-I-hypersensitive sites located between 7 and 8 kb and between 15 and 20 kb upstream of the start site of transcription, respectively. Several of these sites were shown to be present in collagen-producing, but not in non-produc ing cells, indicating that they are associated with transcription of t he gene and may function in its regulation. One strong constitutive DN ase-I-hypersensitive site at -18.5 kb was also cleaved by endogenous n ucleases. The 3'-flanking region of the gene contains a DNase-I-hypers ensitive site located 6 kb downstream of the end of the gene, as well as sequences that can induce a non-B DNA structure. Because these latt er sequences coincide with DNase-I-hypersensitive sites in the homolog ous human gene, our results suggest that some regulatory elements may play a role in gene regulation, not by specific protein-DNA interactio ns but by virtue of their ability to induce a non-B DNA structure and/ or an alternate chromatin conformation. A comparison of the murine and human Colla1 domains shows a similar, although not identical, distrib ution of DNase-I-hypersensitive sites, indicating a conserved arrangem ent of regulatory elements. Our results strongly suggest that these ne w sites constitute regulatory elements which are involved in the trans criptional regulation and/or chromatin loop organization of the Colla1 gene, and they are now amenable for functional analyses. (C) 1997 Els evier Science B.V.