Mediator is a general coactivator of RNA polymerase II (Pol II) transcription. Genomic location analyses of different Mediator subunits indicate a uniformly composed core complex upstream of active genes but unexpectedly also upstream of inactive genes and on the coding regions of some highly active genes. The repressive Cdk8 submodule is associated with core Mediator at all sites but with a lower degree of occupancy, indicating transient interaction, regardless of promoter activity. This suggests gene-specific regulation of Cdk8 activity, rather than regulated Cdk8 recruitment. Mediator presence is not necessarily linked to transcription. This goes beyond Cdk8-repressed genes, indicating that Mediator can mark some regulatory regions ahead of additional signals. Overlap with intergenic Pol II location in stationary phase points to a role as a binding platform for inactive Pol II during quiescence. These results shed light on Cdk8 repression, suggest additional roles for Mediator, and query models of recruitment-coupled regulation.

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.Maintenance of adequate ATP supply is of crucial importance for the mechanisms of structural remodeling in cells with high shape plasticity, especially under high energy-demanding circumstances (1). During processes like cell motility or phagocytosis, the cell movement and cellular shape changes require active restructuring of the actin cytoskeleton. By spatially controlled polymerization of ATP-bound G-actin monomers to the plus end of growing actin filaments and formation of multiple branches, a dense network is built, which is called the actin cortex, based on its specific localization within the cell (2). In this network, ATP hydrolysis and release of P i drive actin filament dynamics by modulating filament stability and determine nucleotide-dependent filament conformation and interaction(s) with regulatory actin-binding proteins (3). Furthermore, force generation needed for contraction and cell movement is controlled by myosin and nonmuscle myosin ATPases (4), and also upstream signaling, involving small GTPases, is contingent upon nucleotide exchange (5). Taken together, actomyosin dynamics is overall an energy-demanding process, directly coupled to ATP availability.Indeed, actomyosin-based processes may consume a major fraction of cellular energy (6). Moreover, the coupling between global ATP suppl...

Many different transcriptional activators and repressors regulate RNA polymerase (Pol) II transcription in eukaryotic cells. These regulatory factors generally bind to upstream DNA sites and recruit large coregulatory complexes such as the Mediator (Malik and Roeder 2000;Naar et al. 2001;Bjorklund and Gustafsson 2005;Kornberg 2005). Mediator complexes were isolated from fungi, metazoans, and a plant (Boube et al. 2002;Backstrom et al. 2007). Mediator from the yeast Saccharomyces cerevisiae (Sc) is a 1-MDa complex that comprises 25 subunits, of which 11 are essential for viability and 22 are at least partially conserved among eukaryotes. Mediator promotes initiation complex assembly through contacts with activators, Pol II, and general transcription factors.Mediator subunits reside in different modules named head, middle, tail, and kinase modules (Dotson et al. 2000;Kang et al. 2001). Apparently the Mediator modules are required for the regulation of different subsets of genes. Gene deletion studies implicated the middle module in regulating HSP genes and low-iron response genes, the tail module in regulating HSP and OXPHOS genes, and the kinase module in regulating genes required during nutrient starvation (Holstege et al. 1998;Beve et al. 2005;van de Peppel et al. 2005;Singh et al. 2006).The Mediator head module is important for initiation complex assembly, stimulates basal transcription, and is necessary for activated transcription (Ranish et al. 1999;. The head module contains subunits Med6, Med8, Med11, Med17, Med18, Med20, and Med22, which are conserved from yeast to human. Head subunits are essential for yeast viability, except for Med18 and Med20 (Koleske et al. 1992;Thompson et al. 1993;Lariviere et al. 2006). In vitro, Med18 and Med20 are required for formation of a stable initiation complex, for efficient basal transcription, and for activated transcription (Thompson et al. 1993;Lee et al. 1999;Ranish et al. 1999). In vivo, Med18 and Med20 regulate transcription of the same subset of genes and have a mainly positive function (van de Peppel et al. 2005).Based on structural analysis, we proposed previously that the trimeric subcomplex of the C-terminal domain of Med8 (Med8C), Med18, and Med20 (the Med8C/18/20 subcomplex) forms a conserved functional submodule of the Mediator head (Lariviere et al. 2006). Here, we confirm this proposal with a combination of X-ray analysis, yeast genetics, biochemistry, and transcriptomics. Our results indicate that Mediator contains functionally distinct submodules within its previously defined modules, and show how gene regulatory submodules can be identified by a combination of structural and functional studies on the molecular level and gene expression analysis on the systems level. Results and Discussion Med8C/18/20 is a subcomplex of the Mediator headOur previous analysis revealed that Sc Med8 contains an essential N-terminal domain (Med8N, residues 1-137), followed by a nonessential linker (residues 138-189) and a C-terminal region that includes a ␣-helix (Med8C, re...

BackgroundCellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays.ResultsIn general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system.ConclusionsThe tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of tps2Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.

Summary Behçet disease is a multi‐system disease associated with human leukocyte antigen (HLA) class I polymorphism. High‐resolution next‐generation sequencing (NGS) with haplotype analysis has not been performed previously for this disease. Sixty Egyptian patients diagnosed according to the International Study Group (ISG) criteria for Behçet disease and 160 healthy geographic and ethnic‐matched controls were genotyped for HLA class I loci (HLA‐A, B, C). For HLA class II loci (DRB1, DRB3/4/5, DQA1, DQB1, DPA1, DPB1), 40 control samples were genotyped. High‐resolution HLA genotyping was performed using NGS and the results were analyzed. Clinical manifestations were oral ulcers (100%), genital ulcers (100%), eye (55%) and neurological (28%) and vascular involvement (35%). HLA‐B*51:08 [odds ratio (OR) = 19·75, 95% confidence interval (CI) = 6·5–79; P < 0·0001], HLA‐B*15:03 (OR = 12·15, 95% CI = 3·7–50·7; P < 0·0001), HLA‐C*16:02 (OR = 6·53, 95% CI = 3–14; P < 0·0001), HLA‐A*68:02 (OR = 3·14, 95% CI = 1·1–8·9; P < 0·01) were found to be associated with Behçet disease, as were HLA‐DRB1*13:01 and HLA‐DQB1*06:03 (OR = 3·39, 95% CI = 0·9–18·9; P = 0·04 for both). By contrast, HLA‐A*03:01 (OR = 0·13, 95% CI = 0–0·8; P = 0·01) and HLA‐DPB1*17:01 were found to be protective (OR = 0·27, 95% CI = 0·06–1·03; P = 0·02). We identified strong linkage disequilibrium between HLA‐B*51:08 and C*16:02 and A*02:01 in a haplotype associated with Behçet disease. HLA‐B*51:08 was significantly associated with legal blindness (OR = 2·98, 95% CI = 1·06–8·3; P = 0·01). In Egyptian Behçet patients, HLA‐B*51:08 is the most common susceptibility allele and holds poor prognosis for eye involvement.