Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARα) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARα ligands. Here, we characterized a binding site for PPARα in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARα binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1α) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1α did not enhance CPT-1A transcription through the PPARα binding site in the second intron. Following knockdown of PGC-1α with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARα and PGC-1α stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.
Objective The goal of this study was to examine the effects of thyroid hormone status on the ability of serum to accept cellular cholesterol. Methods and Results Sera from hypophysectomized rats treated ± T3 was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T3 treated rats had increased ApoA-I, ApoA-IV and fetuin A levels with decreased Apo E levels. Microarray and real-time RT-PCR analysis of rat liver revealed large increases in ApoA-I, ApoA-IV, ABCG5, and ABCG8 in response to T3. J774 macrophages, BHK cells, and Fu5AH rat hepatoma cells were used to measure cholesterol efflux mediated by ABCA1, ABCG1 transporters or SR-BI. Sera from T3-treated rats stimulated efflux via ABCA1 but not by ABCG1 or SR-BI. Gel filtration chromatography revealed that T3 treatment caused a decrease in HDL particle size accompanied by higher levels of lipid-poor ApoA-I. Conclusions Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter. This effect is most likely attributable to increases in small HDL and lipid poor ApoA-I in response to T3.
The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation.Methylation of DNA cytosine residues is strongly associated with higher order chromatin formation, heterochromatin, and repression of RNA transcription. Several molecular mechanisms leading to transcriptional repression, as a result of DNA methylation, have been elucidated. For example, MeCP2, one of a family of proteins with a methyl-DNA binding domain (MBD), 2 binds to 5-methylcytosines recruiting a histone deacetylase, which in turn leads to nucleosome stabilization, recruitment of proteins that mediate chromatin condensation, and the repression of transcription (1-8).In several rare cases, DNA methylation has been shown to enhance transcription. DNA methylation of the far upstream region of the INTERLEUKIN-8 gene and methylation of the EARLY GROWTH RESPONSE-2 intron are associated with increased promoter activity (9, 10). The mechanism of these effects is unknown, and no DNA-binding proteins involved in these processes have been identified. Other studies have indicated that methylated DNA can be transcribed, presumably when there is an absence of proteins that directly mediate transcriptional repression (11). However, none of these studies involve a transition from repressed to activated methylated DNA. Recently, Lembo et al. (12) identified a protein that facilitates a transition from MBD-mediated repression to MBD-mediated activation. However, the molecular mechanism that specifies a transition from repressed methylated DNA to methylated DNA that becomes or remains transcriptionally active, for a given promoter, remains unknown.Here we report that the sequence-specific DNA-binding protein, RFX, can mediate the transcriptional activation of a methylated major histocompatibility (MHC) gene promoter that was repressed by the methylation. These results indicate that DNA demethylation is not necessary for promoter de-repression, i.e. for a loss of proteins that block the binding of positive transcriptional regulatory proteins; and that DNA demethylation is not necessary for promoter activation, defined as an in...
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