Transcriptional Regulation of the Human α2(I) Collagen Gene

Abstract

This study identifies three regions of the human α2(I) collagen promoter involved in the binding of nuclear factors. These regions include sequences from −173 to −155 (footprint I), −133 to −119 (footprint II), and −101 to −72 (footprint III). A novel positive cis-element containing a TCCTCC motif was identified within footprint II. In addition, we demonstrated that a pyrimidine-rich region within footprint I is a binding site for a transcriptional repressor, and a CCAAT motif within footprint III is a binding site for a transcriptional activator. Comparative functional analysis of the cis-acting elements within the proximal 350 base pairs of this promoter, including previously characterized Sp1 binding sites at −300, indicates that constitutive activity of this promoter is regulated equivalently by the three positive cis-acting elements at −300, −125, and −80. Mutations in the repressor site at −160 increase constitutive activity by 4-6-fold. However, simultaneous mutations of the repressor site and the cis-regulatory element at either the −300 or −125 sites result in no increase in constitutive transcription activity suggesting interaction between the activators and repressor elements. In contrast, simultaneous mutation of the CCAAT motif and the repressor site results in about a 4-fold increase, suggesting that activation via the CCAAT motif may be independent of this repressor. This study identifies three regions of the human α2(I) collagen promoter involved in the binding of nuclear factors. These regions include sequences from −173 to −155 (footprint I), −133 to −119 (footprint II), and −101 to −72 (footprint III). A novel positive cis-element containing a TCCTCC motif was identified within footprint II. In addition, we demonstrated that a pyrimidine-rich region within footprint I is a binding site for a transcriptional repressor, and a CCAAT motif within footprint III is a binding site for a transcriptional activator. Comparative functional analysis of the cis-acting elements within the proximal 350 base pairs of this promoter, including previously characterized Sp1 binding sites at −300, indicates that constitutive activity of this promoter is regulated equivalently by the three positive cis-acting elements at −300, −125, and −80. Mutations in the repressor site at −160 increase constitutive activity by 4-6-fold. However, simultaneous mutations of the repressor site and the cis-regulatory element at either the −300 or −125 sites result in no increase in constitutive transcription activity suggesting interaction between the activators and repressor elements. In contrast, simultaneous mutation of the CCAAT motif and the repressor site results in about a 4-fold increase, suggesting that activation via the CCAAT motif may be independent of this repressor.