Purification and preliminary characterization of a cardiac KV1.5 repressor element binding factor

Abstract

We have previously demonstrated that the cell-specific expression of Kv1.5 promoter is regulated by a silencer (Kv1.5 repressor element; KRE) containing a dinucleotide-repetitive element, (GT)19(GA)1(CA) 15(GA)16. Electromobility gel shift assays (EMSAs) of KRE with GH3 nuclear extracts detected a unique DNA-protein complex, which was not detectable in Chinese hamster ovary or COS-7 cells. We further delineated KRE and determined that a 52-bp fragment that contained a (GT)10(GA)1(CA)10 dinucleotide-repetitive element was sufficient for silencer activity. EMSAs using nuclear extracts isolated from the heart and from GH3 cells demonstrated that the 52-bp element formed specific and identical gel shift effects. These complexes were not detectable in EMSA experiments with liver nuclear extracts. Magnetic DNA affinity purification and UV cross-linking experiments identified a 27-kDa KRE binding factor (KBF) in GH3 cell nuclear extracts. Purified KBF reacted specifically with double-stranded KRE, abolishing the formation of multimeric KRE-DNA complexes. Thus, the interaction between KRE and KBF may play an important role in regulating the GH3- and cardiac-specific expression of Kv1.5.