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 re gulated. In the present study, we report the presence of a silencer region in the human ANTS promoter and the purification of a two-component factor t hat recognizes a specific hexanucleotide element, GTCCTG, of the silencer. Transfection of deletion constructs shows that ANTS silencer activity exten ds over a region of at least 310 nts, However, mutating the GTCCTG element completely relieves silencing activity in the context of the human ANTS pro moter. The data suggest that the GTCCTG element might be required for maint aining silencer activity of the extended silencer region. The ANTS silencer region cloned in front of the herpes simplex virus thymidine kinase promot er confers nearly complete inhibition to the heterologous promoter, However , unlike the ANTS promoter, mutating the GTCCTG element restores only parti al activity to the herpes simplex virus thymidine kinase promoter. A protei n complex consisting of two major polypeptides of 37 and 49 kDa was isolate d from HeLa nuclear extracts by affinity chromatography using the GTCCTG el ement as the affinity resin. Cross-linking studies and Southwestern analysi s indicate that p37 binds DNA. p49 appears to be loosely associated with th e p37/DNA complex but is necessary for strong binding of p37, Our data impl icating the GTCCTG element directly in silencing of the ANT2 promoter, toge ther with data from the literature reporting the presence of this element w ithin the silencer region of several additional promoters, suggest a genera l role of the GTCCTG element in transcriptional silencing.