A CT Repeat in the Promoter of the Chicken Malic Enzyme Gene Is Essential for Function at an Alternative Transcription Start Site

CT repeats are abundant in eukaryotic genomes and have been implicated in a number of biological events. The promoter of the chicken malic enzyme gene contains a long polypyrimidine/polypurine tract that includes seven tandem CTs. This CT repeat region together with 14 immediately downstream nucleotides functions as an active alternative promoter when linked to a reporter gene and may direct transcription initiation at a cluster of minor sites in the endogenous gene [G. Xu and A. G. Goodridge (1996)J. Biol. Chem.271, 16008–16019]. In the sequence required for promoter activity, −105 to −83 bp, there are two purines; only the A at −83 bp influences promoter activity. Mutation of different four-nucleotide stretches of the CT repeats to purines decreased promoter activity as a function of the increase in GC content. Increasing the number of CT repeats by changing pyrimidines downstream of (CT)7to CTs increased promoter activity. These sequences and other regions showed moderate sensitivity to S1 nuclease in supercoiled plasmids, suggesting the presence of non-B-DNA structures. Increasing the length of the CT repeats should increase the propensity to adopt non-B-DNA structures such as triplexes. Constructs with 10, 15, or 22 repeats had increased expression relative to wild type. Thus, the ability of CT repeats to form non-B-DNA structures may be functionally important.