Directional regulatory activity of cis-acting elements in the bidirectional alpha 1(IV) and alpha 2(IV) collagen gene...

The genes for the type IV collagen alpha 1 and alpha 2 chains are regulated by a common alpha 130-base pair (bp) bidirectional promoter. In an attempt to explore the regulation of expression of both genes, we have constructed a minilocus plasmid containing minigenes for the human alpha 1 (IV) and alpha 2(IV) collagen genes located head-to-head like the endogenous genes. The directional regulation of this promoter was studied by introducing site-directed mutations into the promoter of the minigenes. In transient transfections the levels of transcripts of both minigenes were measured, and the influence of mutations on the direction of transcription was studied. The mutational analysis showed that a CCCTCCC motif and a GC box located in the center of the common promoter as well as GC boxes in exon 1 of the alpha 2(IV) gene are potent activating elements for both genes. The promoter of the alpha 1(IV) and alpha 2(IV) collagen genes contains no classical TATA boxes. Even so, A+T-rich sequence motifs, which have no obvious homology to the TATA box located 25-30 bp upstream of exon 1 in each gene, direct the initiation of transcription predominantly about 30 bp downstream of their location. Disruption of the A+T-rich sequence decreased the activity of the promoter considerably, and in addition, the generated classical TATA boxes hindered maintenance of normal bidirectional transcription and changed the ratio of the alpha 1(IV) and alpha 2(IV) transcripts. The CCAAT box in the alpha 2(IV) chain coding strand activated the promoter in the alpha 2(IV) gene direction, but not in the alpha 1(IV) gene direction.