Purification and preliminary characterization of a cardiac Kv1.5 repressor element binding factor.
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.
Valverde
P
P
Cardiovascular Division, Brigham and Women's Hospital, Boston, Mass. 02115, USA.
Koren
G
G
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D051666:Kv1.5 Potassium Channel; D009206:Myocardium / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated; D011401:Promoter Regions, Genetic
D004268:DNA-Binding Proteins; D051666:Kv1.5 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D004247:DNA