Project description:The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator – histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis, that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization.
Project description:The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator – histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis, that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. Med8-TAP strain ChIPed with IgG beads vs. Input in Saccharomyces cerevisiae
Project description:Transcription of mRNA products by RNA polymerase II (Pol II) is a multi-stage event subject to a multitude of regulatory processes. Transcription, RNA processing, and chromatin related factors all interact with Pol II to ensure proper timing and coordination of transcription and co-transcriptional processes. Many regulators must function simultaneously to coordinate these processes, yet few strategies exist to explore the full complement of factors regulating specific stages of transcription. To this end we developed a strategy to purify Pol II elongation complexes from specific loci of a single gene, namely the 5′ and 3′ regions, using sequences in the nascent RNA. Applying this strategy to Saccharomyces cerevisiae we determined the specific set of factors that interact with Pol II at precise stages during transcription. We identify many known region-specific factors as well as determine a role for the transcription termination factor Rai1 in regulating the early stages of transcription genome-wide. We also demonstrate a role for the ubiquitin ligase Bre1 in regulating Pol II dynamics during the latter stages of transcription. This strategy for gene and loci-specific isolation of transcription complexes will provide a useful tool to explore the host of factors that regulate the different stages of transcription and coordinate co-transcriptional processes.
Project description:To address the mechanisms of suppression, we analyzed time course of mRNA expression of four suppressed smc2-8 mutant strains. We addressed the question of genomic robustness by systematically screening genomic open reading frames, when induced for high-level expression, for their ability to suppress 55 conditional lethal mutations in yeast, and have discovered 636 suppressor genes participating in 822 novel dosage suppressor interactions. The suppressor genes are functionally broad and are enriched for overlapping open reading frames where mutually overlapping genes tend to be co-suppressors. Studies on suppressors of defects in chromosome condensation, telomere stability, and RNA polymerase II function suggest that adding interactions, by making significant connections where only weak or undetectable interactions were present (rewiring of gene regulatory pathways, and interaction within and between protein complexes) are frequent mechanisms of dosage suppression.
Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.
Project description:To address the mechanisms of suppression, we analyzed time course of mRNA expression of four suppressed smc2-8 mutant strains. We addressed the question of genomic robustness by systematically screening genomic open reading frames, when induced for high-level expression, for their ability to suppress 55 conditional lethal mutations in yeast, and have discovered 636 suppressor genes participating in 822 novel dosage suppressor interactions. The suppressor genes are functionally broad and are enriched for overlapping open reading frames where mutually overlapping genes tend to be co-suppressors. Studies on suppressors of defects in chromosome condensation, telomere stability, and RNA polymerase II function suggest that adding interactions, by making significant connections where only weak or undetectable interactions were present (rewiring of gene regulatory pathways, and interaction within and between protein complexes) are frequent mechanisms of dosage suppression. RNA samples isolated from two individual biological replicates, four time points each (0, 45, 90 and 180 min), of smc2-8 mutant strains harboring pGAL:SMC2, pGAL:UME1, pGAL:MEK1, pGAL:HTA2, pGAL:SNU66, and the negative control MORF plasmid (pGAL:negative, BG1766) and hybridized Affymetrix microarrays.