Identification of a novel Sp1 splice variant as a strong transcriptional activator.

The transcription factor Sp1 regulates expression of numerous genes involved in many cellular processes. Different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, alternative splicing isoforms may also be the basis of its distinct functional activities. In this study, we identified a novel alternative splice isoform of Sp1 named Sp1c. This variant is generated by exclusion of a short domain, which we designate a, through alternative splice acceptor site usage in the exon 3. The existence of this new isoform was confirmed in vivo by Western blotting analysis. Although at very low levels, Sp1c is ubiquitously expressed, as seen in its fulllength Sp1. A preliminary characterization of Sp1c shows that: (a) Sp1c works as stronger activator of transcription than full-length Sp1; (b) percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than percentage of HEK293 Sp1-overexpressing cells

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's Sindrome

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented

Transcriptional Regulation of the Mitochondrial Citrate and Carnitine/Acylcarnitine Transporters: Two Genes Involved in Fatty Acid Biosynthesis and E-oxidation

Transcriptional regulation of genes involved in fatty acid metabolism is considered the major long-term regulatory mechanism controlling lipid homeostasis. By means of this mechanism, transcription factors, nutrients, hormones and epigenetics control not only fatty acid metabolism, but also many metabolic pathways and cellular functions at the molecular level. The regulation of the expression of many genes at the level of their transcription has already been analyzed. This review focuses on the transcriptional control of two genes involved in fatty acid biosynthesis and oxidation: the citrate carrier (CIC) and the carnitine/ acylcarnitine/carrier (CAC), which are members of the mitochondrial carrier gene family, SLC25. The contribution of tissue-specific and less tissue-specific transcription factors in activating or repressing CIC and CAC gene expression is discussed. The interaction with drugs of some transcription factors, such as PPAR and FOXA1, and how this interaction can be an attractive therapeutic approach, has also been evaluated. Moreover, the mechanism by which the expression of the CIC and CAC genes is modulated by coordinated responses to hormonal and nutritional changes and to epigenetics is highlighte

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's Sindrome

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented

The sequences of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms.

The sequences of the human and bovine genes for the phosphate carrier from the inner membranes of mitochondria have been determined. The genes have similar structures and each is divided into nine exons. In both genes, two exons, named IIIA and IIIB, are closely related, and they appear to the alternatively spliced. The human exon IIIB sequence is found in a published human heart cDNA sequence, and bovine exon IIIA forms part of a published bovine heart cDNA sequence. By further examination of the human heart cDNA library, sequences arising from both alternatively spliced forms of the phosphate carrier have been characterized. Both forms were also found in several bovine tissues, but the ratios of expression of the two forms varied. The form containing exon IIIA was expressed most highly in bovine heart and liver, less highly in brain and kidney, and only in low amounts in lung. The opposite hierarchy was found for the form containing exon IIIB; it was most highly expressed in lung and least in heart and liver. The alternative splicing mechanism affects amino acids 4-45 of the mature phosphate carrier protein, which is believed to form one of six transmembrane segments of the phosphate carrier and to emerge into a large extramembranous loop. The alternative splicing mechanism changes 13 and 11 amino acids in the human and bovine carrier proteins, respectively. As the function of this region of the phosphate carrier is not known, the effects of the changes on carrier function are not understood at present

TRANSCRIPTION OF THE MITOCHONDRIAL CITRATE CARRIER GENE: ROLE OF SREBP-1, UPREGULATION BY INSULIN AND DOWNREGULATION BY PUFA

In this study we investigated the transcriptional role of the sterol regulatory element (SRE) present in the promoter of the mitochon- drial citrate carrier (CIC). We show that wild-type (but not mutated) CIC SRE cloned in front of the luciferase promoter confers tran- scriptional activation of the gene reporter. We also demonstrate that insulin activates, and polyunsaturated fatty acids (PUFA) inhibit, the gene reporter activity driven by the CIC promoter containing wild-type (but not mutated) SRE. Finally, both insulin treatment and overexpression of SRE binding protein (SREBP-1) increase the CIC transcript and protein levels, whereas PUFA have an opposite effect. These results show that SRE/SREBP-1 play a role in the transcriptional regulation of CIC by insulin and PUFA

Statins, fibrates and retinoic acid upregulate mitochondrial acylcarnitine carrier gene expression

In this study, we investigated the effects of statins, fibrates, 9-cis-retinoic acid and forskolin on the transcription of the mitochondrial carnitine/acylcarnitine carrier (CAC) gene. Statins, fibrates, retinoic acid and forskolin activate luciferase gene reporter activity driven by the -334/+3 bp region of the human CAC promoter containing wild-type (but not mutated) PPRE. These four agents also increase CAC transcript and protein levels. The combinations of statins and fibrates, retinoic acid and fibrates and fibrates and forskolin act synergistically. Mevalonate abolishes the activation of CAC gene expression by statins; the inhibitor of the PKA pathway H89 suppresses the stimulation of CAC gene expression by forskolin. Because CAC is essential for fatty acid beta-oxidation, the above results on the regulation of CAC gene expression provide a novel contribution to the understanding of the hypolipidemic action of statins, fibrates and retinoic acid

A key role of the mitochondrial citrate carrier (SLC25A1) in TNFα- and IFNγ-triggered inflammation

The chronic induction of inflammation underlies multiple pathological conditions, including metabolic, autoimmune disorders and cancer. The mitochondrial citrate carrier (CIC), encoded by the SLC25A1 gene, promotes the export of citrate from the mitochondria to the cytoplasm, a process that profoundly influences energy balance in the cells. We have previously shown that SLC25A1 is a target gene for lipopolysaccharide signaling and promotes the production of inflammatory mediators. We now demonstrate that SLC25A1 is induced at the transcriptional level by two key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), and such induction involves the activity of the nuclear factor kappa B and STAT1 transcription factors. By studying the down-stream events following SLC25A1 activation during signals that mimic inflammation, we demonstrate that CIC is required for regulating the levels of nitric oxide and of prostaglandins by TNFα or IFNγ. Importantly, we show that the citrate exported from mitochondria via CIC and its downstream metabolic intermediate, acetyl-coenzyme A, are necessary for TNFα or IFNγ to induce nitric oxide and prostaglandin production. These findings provide the first line of evidence that the citrate export pathway, via CIC, is central for cytokine-induced inflammatory signals and shed new light on the relationship between energy metabolism and inflammation

Transcription of the mitochondrial citrate carrier gene: identification of a silencer and its binding protein ZNF224

In the last few years, we have been functionally characterizing the promoter of the human mitochondrial citrate carrier (CIC). In this study we show that CIC silencer activity extends over 26 bp (-595/-569), which specifically bind a protein present in HepG2 cell nuclear extracts. This transcription factor was purified by DNA affinity and identified as ZNF224. Overexpression of ZNF224 decreases LUC transgene activity in cells transfected with a construct containing the CIC silencer region, whereas ZNF224 silencing activates reporter transcription in cells transfected with the same construct. Moreover, overexpression and silencing of ZNF224 diminishes and enhances, respectively, CIC transcript and protein levels. Finally, ZNF224 is abundantly expressed in fetal tissues contrary to CIC. It is suggested that CIC transcriptional repression by ZNF224 explains, at least in part, the low expression of CIC in fetal tissues in which fatty acid synthesis is low