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Papers In Press, published online ahead of print October 18, 2000
Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030
Corresponding Author: Jeanie.B.McMillin{at}uth.tmc.edu
Transcriptional regulation of nuclear-encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear-encoded expression of cardiac-specific mitochondrial isoforms. The muscle CPT-I
J. Biol. Chem, 10.1074/jbc.M009352200
Submitted on October 13, 2000
Revised on October 17, 2000
Accepted on October 18, 2000
GATA-4 and Serum Response Factor Regulate Transcription of the Muscle-Specific Carnitine Palmitoyltransferase I
in Rat Heart
isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac Fatty Acid Response Element (FARE, -301/ -289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'flanking sequence of the carnitine palmitoyltransferase I[beta}(CPT-I) gene defines regulatory regions in the -391/ +80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/ luciferase reporter gene expression was significantly depressed at sites involving a putative MEF 2 sequence downstream from FARE and a cluster of cardiac-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, SRF and GATA-4 enhanced CPT-I reporter gene expression 4- to 36-fold in CV-1 cells. While cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-I. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4,drive robust gene transcription of a mitochondrial protein highly expressed in heart.
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