RESUMEN
BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. RESULTS: Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. CONCLUSIONS: In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.
RESUMEN
Sterol regulatory element binding protein-1a (SREBP-1a) is a transcription factor that is a major player in lipid metabolism and insulin action. We have generated human liver cells (HepG2) overexpressing active SREBP-1a constitutively called SREBP-1a (+). These cells show massive intracellular lipid accumulation. To elucidate the effect of SREBP-1a on lipid metabolism at the level of the cellular protein network, we have analyzed the protein pattern of mitochondria using the novel technique two-dimensional difference gel electrophoresis (2D-DIGE). Mitochondria were enriched by subcellular fractionation using differential and isopyknic centrifugation. Proteins of isolated organelles were labeled with Cy dyes and separated on 2D gels. These gels revealed more than 100 protein spots, which were significantly different in their abundance between wild-type and SREBP-1a (+) cells. MALDI mass spectrometry showed that 68% of identified proteins belong to mitochondria. In SREBP-1a (+) cells, several enzymes involved in lipid metabolism were significantly altered, suggesting that cellular lipid metabolism is triggered by accumulation of fatty acids rather than by its degradation. To test the possible functional relevance of this finding, intracellular fatty acid (FA) patterns were analyzed by gas chromatography. The results showed a significant increase in total fatty acid content with a shift in composition to long-chain unsaturated FAs. Therefore, the detected protein differences might be an explanation for the observed intracellular lipid accumulation and might link SREBP-1a to features like steatosis hepatis.