Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A Universal RNA Polymerase II CTD Cycle Is Orchestrated by Complex Interplays Between Kinase, Phosphatase and Isomerase Enzymes Along Genes

ABSTRACT: Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is non-uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phospho-isoforms in wild type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. We took a systematic approach to examine the genome-wide distribution of the various CTD modifications using a panel of RNAPII CTD phospho-specific antibodies; both in wild type cells and in mutants for most of the CTD kinases, phosphatases and the isomerase. Immunoprecipitation of CTD phospho-isoforms were done using the following antibodies: H14 and 3E8 for Ser5, H5 and 3E10 for Ser2, 4E12 for Ser7, 8WG16 (anti-Rpb1-CTD) and W0012 (anti-Rpb3) for RNAPII (global localization). A list of the mutant strains and their genotypes can be found in the supplemental files of the related publication. Most ChIPs (in Cy5) were hybridyzed against a non-immunoprecipitated (whole cell extract, WCE) in Cy3. Ssu72, Pti1 and Rpb1 were immunoprecipitated using tagged proteins (3myc-Ssu72, Pti1-3myc, Rpb1-9myc) and the ChIP DNA hybridized in competition with a control ChIP DNA prepared from an isogenic untagged strain (NoTag). ChIP from wild type strains yFR116 (W303) and yFR117 (S288C) were used to obtains wild type profiles that can be compared to mutants strains of the same background. All ChIP-chip experiments were done at least in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Francois Robert

PROVIDER: E-GEOD-29403 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. We examined the genome-wide distribution (using ChIP-chip) of Spt6. Spt6 occupancy was also assayed in mutants for CTD Serine 2 and Serine 5 kinases and in mutants for histone deacetylases. ChIPs were performed with a Myc-tagged version of Spt6. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In the ChIP experiments with the spt6-202del mutant, non immunoprecipitated DNA (input) was used as the control. In addition to Spt6 ChIPs, the project includes RNAPII (Rpb3) ChIP-chip datasets, where an anti-Rpb3 antibody was used to ChIP RNAPII and non immunoprecipitated DNA (input) was used as the control. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

Project description:The C-terminal domain (CTD) of the largest subunit in DNA-dependent RNA polymerase II (RNAP II) is essential for mRNA syntheses, coordinating an astounding array of protein-protein interactions. In yeast, mutations that separate established functional units of the CTD result in pleiotrophic effects, producing slow growth phenotypes and the accumulation of abnormally large cells. In general, the downstream pathways for such CTD related phenotypes are uncharacterized and probably highly complex. We also observed that a sizeable fraction of CTD mutants retain multiple buds on parent cells and are elongated relative to wild-type yeast. FACScan analyses revealed a trend toward increased DNA content over multiple rounds of growth, indicating that insertions into the normal tandemly repeated CTD result in aneuploidy. To begin to address the role of the CTD in such complex changes in cell structure and behavior, we applied an integrated approach using the Saccharomyces cerevisiae Genome Database (SGD) and microarray data. We were able to identify candidate genetic networks that could explain chromosome missegregation and related phenotypic traits in CTD mutants. These networks provide links between CTD-associated proteins and kinetochore function, control of cell cycle checkpoint mechanisms, and expression of cell wall and membrane components. The essential elements required for CTD function have been determined in yeast though substitution and insertion mutations (West and Corden 1995; Stiller and Cook 2004; Liu et al. 2008); the core CTD functional unit lies within tandem heptapeptides or â??diheptadsâ??. In addition, CTD mutants with progressively longer polyalanine insertions between diheptads show a continuous decline in growth rates, and the induction of conditional phenotypes; this has been demonstrated out to five alanine (5A) insertions (Stiller and Cook, 2004). Attempts to restore the global amino acid register via extending insertions to seven alanines between diheptad units proved to be lethal, leading to the conclusion that too great a separation between functional units puts undo stress on at least some essential CTD-protein interactions. (Liu et al, 2008). During these prior investigations, we observed that mutants containing 5A insertions are, on average, twice as large as normal yeast, prompting us to investigate these cells in more detail. Yeast cells were transformed with mutated CTD sequences containing 5 alanine insertions between diheptad units via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004). For comparison yeast cells were transformed with YSPTSPS sequences via the plasmid shuffle, as described in detail previously (West and Corden 1995; Stiller and Cook 2004). RNA was extracted from fresh pellets of yeast cultures at log phase, grown to an optical density of 0.8 at 600nm in 100 ml of YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) at 30 ÂºC in a shaker at 225 rpm. Total RNA was extracted using Qiagenâ??s RNeasy Kit (Valencia, CA). Four replicates for each sample yeast strain (5A and wild-type CTD control) were prepared, and 10ug total RNA for every replicates were sent to Duke university DNA microarray center for further analyses. Array ID YO06N from Operon Yeast Genome Oligo Set version 1.1.2 was used for the hybridization. The direct labeling protocol was performed for sample RNA, which included steps of first strand synthesis, slide preparation, hydrolysis, cDNA purification, hybridization and array washing. Cy3 and Cy5 were used for labeling the samples. Maui hybridization and TIGR washing system were used in this protocol for array hybridization and washing respectively. The protocol can be found in Duke University microarray center website as http://www.genome.duke.edu/cores/microarray/services/spotted-arrays/protocols/. Fluorescent DNA bound to the microarray was detected with a GenePix 4000B scanner (Axon Instruments, Foster city, CA), using the GenePix 4000 software package to locate signal from spots. Data processing was performed using Duke Univerisity BASE web server (https://base-server.duhs.duke.edu/). GO annotation was used for gene ontology.

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A Universal RNA Polymerase II CTD Cycle Is Orchestrated by Complex Interplays Between Kinase, Phosphatase and Isomerase Enzymes Along Genes

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