Genomic DNA Sequence, Promoter Expression, and Chromosomal Mapping of Rat Muscle Carnitine Palmitoyltransferase I

Self Cite

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 heart-specific mitochondrial isoforms. The muscle CPT-I␤ isoform in heart is a TATAless gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (؊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␤ (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 MEF2 sequence downstream from the fatty acid response element and a cluster of heart-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, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4 -36-fold in CV-1 cells. Although 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. Expression of nuclear and mitochondrial encoded expression of respiratory chain subunits occurs despite physical separation of transcriptional events within separate genomes. Stimulation and coordination of mitochondrial gene expression from these two sites is accomplished by the nuclear respiratory factors, NRF-1 and NRF-2 (1, 2). Using electrical stimulation to produce hypertrophic growth of neonatal cardiac myocytes, the transcriptional activation of cytochrome c is preceded by induction of NRF-1 mRNA (3). This observation is consistent with NRF-1 induction as a prerequisite for synthesis of respiratory chain components. These basic insights into cellular factors that link nuclear events to mitochondrial gene activation are critical for adaptation to environmental stresses and the necessity for enhanced energy production.In contrast to subcellular coordination of mitochondrial biogenesis and respiratory chain synthesis, less is known concerning tissue-specific transcriptional regulation of nuclear encoded genes involved in energy metabolism. These genes are particularly important in cardiac...

Section: Resultsmentioning

confidence: 99%

“…Plasmids and Constructs-Construction of the rat CPT-I␤ promoter fragment Ϫ391/ϩ80 has been reported previously (18). The expression vector pCMV-Nkx 2.5 was a gift from Dr. Janet Mar.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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GATA-4 and Serum Response Factor Regulate Transcription of the Muscle-specific Carnitine Palmitoyltransferase I β in Rat Heart

Moore¹,

Wang²,

Belaguli³

et al. 2001

Self Cite

“…Alignment of these alternative ends with the complete gene sequence that has since been published has now confirmed this. As in the case of the mammalian CPT1A (35,36) and CPT1B promoters (37)(38)(39)(40), those for DmCPT1 are TATAless. Both promoter regions of the D. melanogaster CPT1 contain a number of sequence motifs with a high degree of similarity to known transcription factor-binding sites.…”

Section: Tablementioning

confidence: 99%

Alternative Exon Usage in the Single CPT1 Gene of Drosophila Generates Functional Diversity in the Kinetic Properties of the Enzyme

Price

Jackson

Müller

et al. 2010

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A. We performed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1 mRNA were found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5 ) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects.Lipid metabolism in insects has largely been investigated from the perspective of energy supply to flight muscle, particularly in migratory flight. Because their lipid metabolism is less complex than in vertebrates, insects are considered a good model system in which to study the fundamental aspects of fat metabolism. The increasing availability of insect gene sequence data and completely sequenced genomes allows comparative analyses with mammalian data and suggests that many of the fundamental molecular mechanisms involved in fatty acid metabolism and their regulation are conserved. Although in most insects there are major differences in the glycerolipids used to transport lipid between tissues (diacylglycerol in insects versus triacylglycerol in vertebrates), the transfer of the long chain acyl moiety across the mitochondrial inner membrane is likely to be substantially similar to that in vertebrates and to involve the carnitine shuttle (1). We have previously shown that the fruit fly Drosophila melanogaster has only a single CPT1 (carnitine palmitoyltransferase 1) gene (2). When we expressed the full-length cDNA (Berkeley Dro...

“…such that either exon 1A (7), also called U (8), or exon 1B, also called M, is transcribed. In the rat, only a single first exon has been found (9), equivalent to human exon 1B(M). The human CPT1B gene maps to the telomeric region of the long arm of chromosome 22 (6,10).…”

mentioning

confidence: 99%

Structural and Functional Genomics of the CPT1B Gene for Muscle-type Carnitine Palmitoyltransferase I in Mammals

Leij¹,

Cox²,

Jackson³

et al. 2002

Muscle-type carnitine palmitoyltransferase I (M-CPT I)is a key enzyme in the control of ␤-oxidation of long-chain fatty acids in the heart and skeletal muscle. Because knowledge of the mammalian genes encoding M-CPT I may aid in studies of disturbed energy metabolism, we obtained new genomic and cDNA data for M-CPT I for the human, mouse, rat, and sheep. The introns of these compact genes are 80% (mouse versus rat) and 60% (mouse versus human) identical. Sheep and goat, but not cow, pig, rodent, or human promoter sequences contain a short interspersed repeated sequence (SINE) upstream of highly conserved regulatory elements. These elements constitute two promoters in humans, sheep, and mice, and, contrary to previous reports, there is a second promoter in rats as well. Thus, the transcriptional organization of these genes is more uniform than previously supposed, with interspecies differences in the 5-ends of the mRNAs reflecting differences in splicing; only in humans extensive splicing and splice variation is found in the 5-and 3-untranslated regions. In the mouse, intron retention was detected in heart, muscle, and testes and may indicate an additional mechanism of regulation of M-CPT I expression. Splice variation in the coding region was previously proposed to lead to expression of CPT I enzymes with altered malonyl-CoA sensitivity (Yu, G. S., Lu, Y. C., and Gulick, T. (1998) Biochem. J. 334, 225-231). However, when expressed in the yeast Pichia pastoris, none of three earlier described splice variants had CPT I activity. Therefore, the involvement of splice variation of M-CPT