Rat Argininosuccinate Lyase Promoter: The Dyad-Symmetric CCAAT Box Sequence CCAATTGG in the Promoter Is Recognized by NF-Y

Abstract

The gene for rat argininosuccinate lyase (AL), which catalyzes the last step of arginine biosynthesis, is expressed in many tissues, though its expression is much higher in the liver where the enzyme is involved in the ornithine cycle (urea cycle), and moderately higher in the kidney. In transient transfection analysis using various cell lines, the AL promoter exhibited considerable levels of activity in all tested cell lines, with no apparent liver cell specificity, presumably reflecting the ubiquitous expression of this gene. Analysis of 5'-deletion mutants of the promoter region revealed several cis-regulatory elements, whose contribution to the promoter activity seemed to vary according to cell type. The most prominent positive regulatory region was detected around the position -80 base pairs, and it overlapped with a dyad-symmetric CCAAT box sequence CCAATTGG. A factor(s) interacting with this sequence element was found to be present ubiquitously. Gel shift competition analysis and antibody double shift analysis showed that this factor was identical or highly similar to a transcription factor, nuclear factor-Y (NF-Y), which consists of two different subunits and recognizes CCAAT motifs in various promoters. Point mutagenesis at each half site of the palindrome CCAATTGG and the introduction of a space between the half-site motifs abolished the binding with NF-Y. Thus, it seems that NF-Y recognizes the correctly spaced dyad-symmetric CCAAT box.