Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5⌬ and med15⌬ deletions. The med5⌬ and med16⌬ deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.The multiprotein Mediator complex is required for basal and regulated expression of nearly all RNA polymerase II (pol II) 3 -dependent genes in the Saccharomyces cerevisiae genome. Mediator conveys regulatory information from enhancers and other control elements to the promoter (1). The functional activities identified for Mediator include stimulation of basal transcription, support of activated transcription, and enhancement of phosphorylation of the C-terminal domain of pol II by the transcription factor IIH kinase (2, 3). S. cerevisiae Mediator also contains a histone acetyltransferase (HAT) activity, which is not found in other eukaryotic Mediator complexes (4). The HAT activity was localized to Med5 (Nut1), a S. cerevisiae-specific protein, which lacks homologues in higher eukaryotes (4, 5). The MED5 gene was originally isolated in a screen for mutants that would suppress the Swi4/Swi6 dependence of a synthetic reporter gene containing part of the HO promoter (6). Several other genes encoding Mediator proteins were identified in the same screen, including MED10 (NUT2), MED16 (SIN4), MED19 (ROX3), MED12 (SRB8), MED13 (SRB9), CDK8 (SRB10), and CYCC (SRB11). The MED5 gene is nonessential in yeast. A deletion of MED5 relieves repression at the URS2 element in the HO promoter but only in combination with a mutant allele of either MED10 or CCR4 (6). These effects on the HO promoter were seen with a lacZ reporter gene but not at the endogenous HO gene locus. The in vivo role of Med5 in Mediator-dependent gene expression therefore remains an open question.In the presence of RNA pol II, Mediator adopts an extended conformation that embraces the globular pol II core complex (7). The extended structure reveals three distinct submodules of Mediator. Direct contacts are formed between pol II and the head and middle region (7,8). The largest part of Mediator is made up of an elongated tail region, which does not appear to contact pol II. Structural analysis of mutant Mediator complex has demonstrated that the tail region contains the Med2, Med3, Med15 (Gal11), and Med16, proteins, which are involved in interactions with a number of different activators,...

PPARδ is involved in the inflammatory response and its expression is induced by cytokines, however, limited knowledge has been produced regarding its regulation. Since recent findings have shown that microRNAs, which are small non-coding RNAs that regulate gene expression, are involved in the immune response, we set out to investigate whether PPARδ can be regulated by microRNAs expressed in monocytes. Bioinformatic analysis identified a putative miR-9 target site within the 3′-UTR of PPARδ that was subsequently verified to be functional using reporter constructs. Primary human monocytes stimulated with LPS showed a downregulation of PPARδ and its target genes after 4 h while the expression of miR-9 was induced. Analysis of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages showed that human PPARδ mRNA as well as miR-9 expression was higher in M1 compared to M2 macrophages. Furthermore, treatment with the PPARδ agonist, GW501516, induced the expression of PPARδ target genes in the pro-inflammatory M1 macrophages while no change was observed in the anti-inflammatory M2 macrophages. Taken together, these data suggest that PPARδ is regulated by miR-9 in monocytes and that activation of PPARδ may be of importance in M1 pro-inflammatory but not in M2 anti-inflammatory macrophages in humans.

Background:The amount of intra-thoracic fat, of which mediastinal adipose tissue comprises the major depot, is related to various cardiometabolic risk factors. Autopsy and imaging studies indicate that the mediastinal depot in adult humans could contain brown adipose tissue (BAT). To gain a better understanding of this intra-thoracic fat depot, we examined possible BAT characteristics of human mediastinal in comparison with subcutaneous adipose tissue.Materials and methods:Adipose tissue biopsies from thoracic subcutaneous and mediastinal depots were obtained during open-heart surgery from 33 subjects (26 male, 63.7±13.8 years, body mass index 29.3±5.1 kg m−2). Microarray analysis was performed on 10 patients and genes of interest confirmed by quantitative PCR (qPCR) in samples from another group of 23 patients. Adipocyte size was determined and uncoupling protein 1 (UCP1) protein expression investigated with immunohistochemistry.Results:The microarray data showed that a number of BAT-specific genes had significantly higher expression in the mediastinal depot than in the subcutaneous depot. Higher expression of UCP1 (24-fold, P<0.001) and PPARGC1A (1.7-fold, P=0.0047), and lower expression of SHOX2 (0.12-fold, P<0.001) and HOXC8 (0.14-fold, P<0.001) in the mediastinal depot was confirmed by qPCR. Gene set enrichment analysis identified two gene sets related to mitochondria, which were significantly more highly expressed in the mediastinal than in the subcutaneous depot (P<0.01). No significant changes in UCP1 gene expression were observed in the subcutaneous or mediastinal depots following lowering of body temperature during surgery. UCP1 messenger RNA levels in the mediastinal depot were lower than those in murine BAT and white adipose tissue. In some mediastinal adipose tissue biopsies, a small number of multilocular adipocytes that stained positively for UCP1 were observed. Adipocytes were significantly smaller in the mediastinal than the subcutaneous depot (cross-sectional area 2400±810 versus 3260±980 μm2, P<0.001).Conclusions:Human mediastinal adipose tissue displays some characteristics of BAT when compared with the subcutaneous depot at microscopic and molecular levels.