Characterization of an evolutionarily conserved far-upstream enhancer in the human alpha 2(I) collagen (COL1A2) gene

We have examined the chromatin structure around and upstream of the transcr iptional start site of the human alpha2(I) collagen (COL1A2) gene. Four str ong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. A nother hypersensitive site potentially involved in COL1A2 silencing was fou nd in intron 1 (HS(In)), HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped similar to 20 kilobases upstream of COL1A2 at about th e same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretch es nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to t he otherwise minimally active COL1A2 promoter. Characterization of the huma n element documented functional differences with the mouse counterpart. Enh ancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3' of HS3 augmented positio n-independent expression of the transgene. Hence, subtle differences may ch aracterize the regulation of mammalian alpha2(I) collagen genes by evolutio narily conserved sequences.