In the present study, through deletion analysis of the ?1785 to ?336 bp region, we have identified an activation domain (?1285/?1017) in the human CIC gene promoter. The activation of gene expression by this domain is due to the presence of an active FOXA site (from ?1098 to ?1088 bp) that acts as a strong enhancer of the CIC gene transcription. This conclusion is directly demonstrated by the observation that (a) the wild-type (but not the mutated) FOXA site markedly enhances gene reporter expression activity, and (b) the wild-type FOXA-driven gene reporter activity was further enhanced in
TAK-632 expressing the transcription factor FOXA1. The role of FOXA1 in the regulation of CIC gene transcription is also shown by the increase in the levels of both CIC transcript and protein caused by overexpression of FOXA1. Vice versa, silencing not only of FOXA1 but also of the other FOXA subfamily transcription factors (FOXA2 and FOXA3) strongly reduces CIC gene expression as measured by transcript and protein levels. It is noteworthy that the FOXA sequence is present in the promoters of other mitochondrial CIC genes sequenced. For example, Rattus norvegicus, Canis familiaris, Danio rerio, and Gallus gallus CIC gene promoters exhibit FOXA sites at ?1011/?1001, ?527/?517, ?560/?550, and ?3141/?3131 bp, respectively, which are 85–90% identical to the canonical FOXA [22]. Interestingly, transgenic mice overexpressing FOXA2 in their liver exhibit increased mRNA levels of fatty
acid synthase, which converts malonyl-CoA into fatty acids, and glyceraldehyde-3-phosphate acyltransferase, which is the first step in triglyceride synthesis [23]. The CIC transcriptional activation by FOXA, reported here, is therefore another mechanism by which FOXA regulates lipogenesis. Like FOXA, also SREBP-1, which is upregulated by insulin [24] and downregulated by polyunsaturated fatty acids [25], has been shown to be a positive transcriptional regulator not only of CIC [15] but also of some cytosolic lipogenic enzymes [25] which work in sequence to CIC.
