Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (À78/ þ 59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFjB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between À194 and À78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

High expression of the peroxisome proliferator-activated receptor ␣ (PPAR␣) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPAR␣ activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPAR␣ increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (؊4551)UCP1-CAT construct, containing the 5-regulatory region of the rat ucp-1 gene, was activated by PPAR␣ co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor ␣. The coactivators CBP and PPAR␥-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPAR␣-dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPAR␣-responsive element was located in the upstream enhancer region of the ucp-1 gene. This ؊2485/؊2458 element bound PPAR␣ and PPAR␥ from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPAR␣ or PPAR␥ activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPAR␥) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPAR␣).

SummaryUnicellular eukaryotes that lack mitochondria typically contain related organelles such as hydrogenosomes or mitosomes. To characterize the evolutionary diversity of these organelles, we conducted an expressed sequence tag (EST) survey on the freeliving amoeba Mastigamoeba balamuthi, a relative of the human parasite Entamoeba histolytica. From 19 182 ESTs, we identified 21 putative mitochondrial proteins implicated in protein import, amino acid interconversion and carbohydrate metabolism, two components of the iron-sulphur cluster (Fe-S) assembly apparatus as well as two enzymes characteristic of hydrogenosomes. By immunofluorescence microscopy and subcellular fractionation, we show that mitochondrial chaperonin 60 is targeted to small abundant organelles within Mastigamoeba. In transmission electron micrographs, we identified doublemembraned compartments that likely correspond to these mitochondrion-derived organelles, The predicted organellar proteome of the Mastigamoeba organelle indicates a unique spectrum of functions that collectively have never been observed in mitochondrion-related organelles. However, like Entamoeba, the Fe-S cluster assembly proteins in Mastigamoeba were acquired by lateral gene transfer from e-proteobacteria and do not possess obvious organellar targeting peptides. These data indicate that the loss of classical aerobic mitochondrial functions and acquisition of anaerobic enzymes and Fe-S cluster assembly proteins occurred in a free-living member of the eukaryote super-kingdom Amoebozoa.