Function of a C-Rich Sequence in the Polypyrimidine/Polypurine Tract of the Promoter of the Chicken Malic Enzyme Gene Depends on Promoter Context

Abstract The promoters of many genes contain C-rich polypyrimidine/polypurine (PPY/PPU) sequences that are important for gene expression. The promoter of the chicken malic enzyme gene contains a long PPY/PPU tract that can act as an alternative promoter. This tract can be separated functionally into a C-rich and (CT) 7 sequences. The (CT) 7 region together with some 3′ nucleotides is essential for function of the alternative transcription start site and the C-rich sequence as a regulatory element. In constructs that contained the PPY/PPU tract or the −147/+31-bp promoter of the malic enzyme gene connected to a reporter gene, deletion of the C-rich region increased gene expression. In constructs containing 5.8-kb 5′-flanking DNA of the gene, deletion of the same C-rich region decreased expression of the reporter gene. Positive function of the C-rich sequence required two upstream DNA regions, −237 to −147 bp and −3474 to −2715 bp. To understand the mechanism(s) by which the same sequence exerts different effects, we examined the transcription start sites in the construct where the C-rich region was deleted. We directly visualized transcription start sites by performing 5′-rapid amplification of cDNA ends and a subsequent primer extension on a single-stranded template. Deletion of the C-rich region from constructs containing 5.8 kb of 5′-flanking DNA almost completely abolished transcription initiation from the PPY/PPU promoter and reduced transcription from the major endogenous start site. DEAE fractionation of hepatic nuclear extract revealed more than 10 proteins that bound specifically to C-rich DNA. These results suggest that interactions between upstream DNA elements and the C-rich sequence and the selective use of DNA-binding activities may bestow different functions on the same nucleotide sequence.