Genome-Wide Occupancy Profile of Mediator and the Srb8-11 Module Reveals Interactions with Coding Regions

120

112

Wnt target gene transcription is mediated by nuclear translocation of stabilized ␤-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of ␤-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.Drosophila ͉ kinase module ͉ kohtalo ͉ skuld ͉ Wnt

Section: Discussionmentioning

confidence: 51%

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Carrera

Janody

Leeds

et al. 2008

120

112

“…In contrast, another study found Mediator to be associated with upstream regions of a large number (Ϸ1,200) of genes, and with coding sequences of many genes, in yeast grown in CSM (26). Similarly, Mediator association with a large number of both intergenic regions and coding sequences was reported in fission yeast (Schizosaccharomyces pombe) (43). More recently, comparison of Mediator association at promoters in yeast grown in YPD and in CSM suggested that although differences in growth conditions could account for a small proportion of the discrepant results published previously, the greater portion of the discrepancy could not be explained in this way (42).…”

Section: Discussionmentioning

confidence: 90%

Mediator complex association with constitutively transcribed genes in yeast

Ansari

Morse

2009

Mediator is a large, multisubunit complex that is essential for transcription of mRNA by RNA Pol II in eukaryotes and is believed to bridge transcriptional activators and the general transcription machinery. However, several recent studies suggest that the requirement for Mediator during transcriptional activation is not universal, but rather activator dependent, and may be indirect for some genes. Here we have investigated Mediator association with several constitutively transcribed genes in yeast by comparing a yeast strain that harbors a temperature-sensitive mutation in an essential Mediator subunit, Srb4, with its wild-type (WT) counterpart. We find modest association of Mediator with constitutively active genes and show that this association is strongly decreased in srb4 ts yeast, whereas association with a nontranscribed region or repressed gene promoters is lower and unaffected in the mutant yeast. The tail module of Mediator remains associated with ribosomal protein (RP) gene promoters in srb4 ts yeast, while subunits from the head and middle modules are lost. Tail module association at Rap1-dependent gene promoters is lost in rap1 ts yeast, indicating that Rap1 is required for Mediator recruitment at these gene promoters and that its recruitment occurs via the tail module. Pol II association is also rapidly and severely affected in srb4 ts yeast, indicating that Mediator is directly required for pol II association at constitutively transcribed genes. Our results are consistent with Mediator functioning as a general transcription factor in yeast. Rap1 ͉ transcription

“…Moreover, genomewide profiling indicates that Mediator can associate with gene-coding regions, suggesting that a portion of copurifying Mediator may even be in elongating pol II complexes (36,37). Purification of a tagged Mediator subunit under cross-linking conditions might distinguish between some of these possibilities.…”

Section: Discussionmentioning

confidence: 99%

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Tardiff

Abruzzi

Rosbash

2007

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.mass spectrometry ͉ nascent RNP ͉ RNA processing ͉ transcription ͉ affinity purification P urification and mass spectrometric analysis of protein complexes is a ubiquitous approach in modern molecular biology (1). The identification of interacting proteins and their subsequent characterization provides valuable insight into the complexes that carry out essential cellular functions. Purifications can use multiple biochemical fractionations (e.g., ion exchange, gradients, gel filtration, and affinity purification) or a streamlined tandem affinity purification (TAP) approach (2). Conventional purification methods, however, often preclude the accurate identification of in vivo-relevant complexes. Major complications include dissociation of protein complexes, as a function of off-rates and extract dilution. Moreover, strong affinities can promote in vitro associations that do not exist in vivo. These can occur as a consequence of protein exchange and the absence of subcellular localization. Finally, major complexes may be insoluble, or harsh methods required to effect solubility can perturb complex composition. Ribonucleoprotein particles (RNPs) and especially transcription complexes are particularly subject to these caveats.The synthesis of protein-encoding mRNAs is directed by RNA polymerase II (pol II), an extensively studied macromolecular machine. The composition of pol II is dynamic and changes with the stage of transcription, namely, initiation, elongation, and termination (3, 4). Difficulties with complex purification underlie controversies, for example, to what extent initiation occurs by stepwise assembly of a preinitiation complex on DNA. Moreover, elongating pol II is tightly associated with DNA, which makes its characterization challenging (5).ChIP is a major tool for characterizing DNA-associated proteins and complexes, including transcription and cotranscriptional ...