Abstract
Carnitine palmitoyltransferase-I (CPT-I) catalyzes the rate-controlling step of fatty acid oxidation. CPT-I converts long-chain fatty acyl-CoAs to acylcarnitines for translocation across the mitochondrial membrane. The mRNA levels and enzyme activity of the liver isoform, CPT-Ialpha, are greatly increased in the liver of hyperthyroid animals. Thyroid hormone (T3) stimulates CPT-Ialpha transcription far more robustly in the liver than in non-hepatic tissues. We have shown that the thyroid hormone receptor (TR) binds to a thyroid hormone response element (TRE) located in the CPT-Ialpha promoter. In addition, elements in the first intron participate in the T3 induction of CPT-Ialpha gene expression, but the CPT-Ialpha intron alone cannot confer a T3 response. We found that deletion of sequences in the first intron between +653 and +744 decreased the T3 induction of CPT-Ialpha. Upstream stimulatory factor (USF) and CCAAT enhancer binding proteins (C/EBPs) bind to elements within this region, and these factors are required for the T3 response. The binding of TR and C/EBP to the CPT-Ialpha gene in vivo was shown by the chromatin immunoprecipitation assay. We determined that TR can physically interact with USF-1, USF-2, and C/EBPalpha. Transgenic mice were created that carry CPT-Ialpha-luciferase transgenes with or without the first intron of the CPT-Ialpha gene. In these mouse lines, the first intron is required for T3 induction as well as high levels of hepatic expression. Our data indicate that the T3 stimulates CPT-Ialpha gene expression in the liver through a T3 response unit consisting of the TRE in the promoter and additional factors, C/EBP and USF, bound in the first intron.
Highlights
Thyroid hormone (T3)1 has profound effects on various aspects of metabolism and development [1]
Our findings show that the thyroid hormone receptor (TR) in the promoter and CCAAT enhancer binding proteins (C/EBPs) and Upstream stimulatory factor (USF) bound in the first intron comprise a T3 response unit that mediates the liver-selective T3 induction of Carnitine palmitoyltransferase-I (CPT-I)␣
To define smaller regions in the intron that contribute to the T3 stimulation, additional internal deletions were made in the context of the Ϫ4495/ϩ1240 CPT-I␣-Luc vector
Summary
Thyroid hormone (T3) has profound effects on various aspects of metabolism and development [1]. The TRE is located in the promoter of the CPT-I␣ gene between nucleotides Ϫ2938 and Ϫ2923 Mutation of this DR4 motif results in the complete loss of T3 responsiveferase; COUP-TF, chicken ovalbumin upstream promoter transcription factor; CPT, carnitine palmitoyltransferase; CPT-I, mitochondrial outer membrane CPT; CPT-II, mitochondrial inner membrane CPT; CPT-I␣, liver isoform of CPT-I; DR, direct repeat; C/EBP, CCAAT enhancer binding protein; USF, upstream stimulatory factor; CREB, cAMP-response element-binding protein; luc, luciferase; DMEM, Dulbecco’s modified Eagle’s medium; ChIP, chromatin immunoprecipitation; PEPCK, phosphoenolpyruvate carboxykinase. We identified regions within the first intron that are necessary to achieve the full T3 induction Elements within these regions bind proteins that participate in the T3 response, including CCAAT enhancer-binding proteins (C/ EBP) and upstream stimulatory factor (USF-1 and USF-2). Our findings show that the TR in the promoter and C/EBP and USF bound in the first intron comprise a T3 response unit that mediates the liver-selective T3 induction of CPT-I␣
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