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(Circulation Research. 1999;84:937-944.)
© 1999 American Heart Association, Inc.

Original Contribution

Purification and Preliminary Characterization of a Cardiac Kv1.5 Repressor Element Binding Factor

Paloma Valverde, Gideon Koren

From the Cardiovascular Division, Brigham and Women's Hospital, Boston, Mass.

Correspondence to Dr Gideon Koren, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail koren{at}calvin.bwh.harvard.edu

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.

Key Words: Kv1.5 • transcription • silencer • heart • K⁺ channel

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