TRANSCRIPTIONAL REGULATION OF THE HUMAN MITOCHONDRIAL CITRATE TRANSPORTER GENE

The mitochondrial citrate carrier (CIC) is a nuclear-encoded protein located in the inner mitochondrial membrane. It exports citrate from mitochondria to the cytosol where it produces acetyl-CoA and NADPH, which are both necessary for fatty acid and sterol biosynthesis. CIC mRNA and CIC protein levels are high in liver, pancreas and kidney. Recently, we have functionally characterized the promoter of the human CIC gene. Firstly, we found that insulin upregulates and polyunsaturated fatty acids downregulate CIC gene transcription through the SRE/SREBP-1 regulatory system. Then, we assayed the gene reporter activity of CIC promoter deletion fragments and identified three important regions: the proximal promoter from -335 to -20 bp, an activation domain from -1285 to -1017 bp and an inhibitory domain from -742 to -499 bp. In the proximal promoter five active wild-type Sp1-binding elements were present. The DNA demethylating agent 5-aza-2-deoxycytidine (AzaC) and/or trichostatin A (TSA) increased CIC transcript and protein levels as well as Sp1 and acetylated histone binding to the CIC proximal promoter in SK-N-SH cells. Furthermore, Sp1 silencing decreased proximal promoter-driven gene reporter activity as well as CIC expression levels in AzaC- and TSA-treated SK-N-SH cells. In the activation domain a FOXA site was present. The wild-type CIC FOXA site cloned in front of the luciferase promoter conferred gene reporter transcriptional activation, and FOXA overexpression and silencing, respectively, increased and reduced CIC expression. In addition, FOXA1 silencing in INS-1 cells decreased not only CIC mRNA and protein but also the amount of cytosolic citrate and glucose-stimulated insulin secretion. Finally, in the CIC inhibitory domain, we characterized a silencer element of 26 bp that binds the transcriptional factor ZNF224. ZNF224 overexpression decreased LUC activity in cells transfected with a construct containing the CIC silencer, whereas ZNF224 silencing activated reporter transcription in cells transfected with the same construct. Remarkably, ZNF224 and CIC displayed an opposite pattern of expression in fetal tissues. In conclusion, our functional analysis of the CIC gene promoter has led to identify some interesting mechanisms regulating the expression of this gene in different tissues and under various physiological conditions