Characterization of a Silencer Element and Purification of a Silencer Protein That Negatively Regulates the Human Adenine Nucleotide Translocator 2 Promoter

Expression of adenine nucleotide translocator isoform 2 (ANT2) is growth regulated. In the present study, we report the presence of a silencer region in the human ANT2 promoter and the purification of a two-component factor that recognizes a specific hexanucleotide element, GTCCTG, of the silencer. Transfection of deletion constructs shows that ANT2 silencer activity extends over a region of at least 310 nts. However, mutating the GTCCTG element completely relieves silencing activity in the context of the human ANT2 promoter. The data suggest that the GTCCTG element might be required for maintaining silencer activity of the extended silencer region. The ANT2 silencer region cloned in front of the herpes simplex virus thymidine kinase promoter confers nearly complete inhibition to the heterologous promoter. However, unlike the ANT2 promoter, mutating the GTCCTG element restores only partial activity to the herpes simplex virus thymidine kinase promoter. A protein complex consisting of two major polypeptides of 37 and 49 kDa was isolated from HeLa nuclear extracts by affinity chromatography using the GTCCTG element as the affinity resin. Cross-linking studies and Southwestern analysis indicate that p37 binds DNA. p49 appears to be loosely associated with the p37/DNA complex but is necessary for strong binding of p37. Our data implicating the GTCCTG element directly in silencing of the ANT2 promoter, together with data from the literature reporting the presence of this element within the silencer region of several additional promoters, suggest a general role of the GTCCTG element in transcriptional silencing.