No abstract

Mediator is a general coactivator of RNA polymerase II (RNA pol II) bridging enhancer-bound transcriptional factors with RNA pol II. Mediator is organized in three distinct subcomplexes: head, middle, and tail modules. The head and middle modules interact with RNA pol II and the tail module interacts with transcriptional activators. Deletion of one of the tail subunits SIN4 results in derepression of subset of genes, including FLR1, by a largely unknown mechanism. Here we show that derepression of FLR1 transcription in sin4Δ cells occurs by enhanced recruitment of the mediator, as well as Swi/Snf and SAGA complexes. The tail and head/middle modules of the mediator behave as separate complexes at the induced FLR1 promoter. While the tail module remains anchored to the promoter, the head/middle modules are also found in the coding region. The separation of the tail and head/middle modules in sin4Δ cells is also supported by the altered stoichiometry of the tail and head/middle modules at several tested promoters. Deletion of another subunit of the tail module MED2 in sin4Δ cells results in significantly decreased transcription of FLR1, pointing to the importance of the integrity of the separated tail module in derepression. All tested genes exhibited increased recruitment of the tail domain; however, only genes with increased occupancy of the head/middle modules displayed also increased transcription. The separated tail module thus represents a promiscuous transcriptional factor that binds to many different promoters and is necessary for derepression of FLR1 in sin4Δ cells.

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in benomyl sensitivity, alterations in chromatin structure of centromeres, mitotic delay, and a higher frequency of chromosome loss. Here we intended to utilize benomyl sensitivity as a phenotype that would allow us to identify genes that are important for kinetochore function and are downstream of Plc1p. However, our screen identified SIN4, encoding a component of the Mediator complex of RNA polymerase II. Deletion of SIN4 gene (sin4⌬) does not suppress benomyl sensitivity of plc1⌬ cells by improving the function of kinetochores. Instead, benomyl sensitivity of plc1⌬ cells is caused by a defect in expression of FLR1, and the suppression of benomyl sensitivity in plc1⌬ sin4⌬ cells occurs by derepression of FLR1 transcription. FLR1 encodes a plasma membrane transporter that mediates resistance to benomyl. Several other mutations in the Mediator complex also result in significant derepression of FLR1 and greatly increased resistance to benomyl. Thus, benomyl sensitivity is not a phenotype exclusively associated with mitotic spindle defect. These results demonstrate that in addition to promoter-specific transcription factors that are components of the pleiotropic drug resistance network, expression of the membrane transporters can be regulated by Plc1p, a component of a signal transduction pathway, and by Mediator, a general transcription factor. The results thus suggest another layer of complexity in regulation of pleiotropic drug resistance.The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC) 3 yields two prominent eukaryotic second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ). In higher eukaryotes, the hydrophilic IP 3 triggers the release of calcium from internal stores and thus modulates Ca 2ϩ /calmodulin-regulated pathways, whereas the hydrophobic 1,2-diacylglycerol activates the phospholipid-and Ca 2ϩ -dependent protein kinase C (1-3).In yeast cells, PLC (Plc1p encoded by PLC1) and three inositol polyphosphate (InsPs) kinases (Ipk2p/Arg82p, Ipk1p, and Kcs1p) constitute a nuclear signaling pathway that affects transcriptional control (4) and export of mRNA from the nucleus (5). Recently, InsPs produced by a Plc1p-dependent pathway have been shown to regulate the activity of chromatin remodeling complexes in vivo and in vitro (6, 7). The induction of the phosphate-responsive PHO5 gene, chromatin remodeling of its promoter, and recruitment of Swi/Snf and Ino80 chromatin remodeling complexes are impaired in the ipk2/arg82 mutant strain (7). In vitro, nucleosome mobilization by the yeast Swi/Snf complex is stimulated by IP 4 and IP 5 , whereas IP 6 inhibits nucleosome mobilization by yeast Isw2 and Ino80 complexes and Drosophila NURF complex (6). A possible mechanism by which InsPs affect chromatin remodeling may involve effects on protein conformation of the chromatin remodeling complexes (7). Alternativ...