Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE60181 | GEO | 2014/08/08
SECONDARY ACCESSION(S): PRJNA257690
REPOSITORIES: GEO
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Project description:The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3'-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphate-regulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD. RNA-seq of wild type and T4V mutant of budding yeast RNAP II CTD in duplicates
2014-08-08 | E-GEOD-60181 | biostudies-arrayexpress
Project description:The primary structure and phosphorylation pattern of the tandem YSPTSPS repeats of the RNA polymerase II CTD comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding “letters” to gene expression, we analyzed the poly(A)+ transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n=227) >> Y1F (n=71) > S7A (n=58) >> T4A (n=7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code “word”. Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, while S7A increased pho1+ expression. Meiotic genes were enriched among those up-regulated in S7A cells. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.
2014-03-13 | GSE52370 | GEO
Project description:The primary structure and phosphorylation pattern of the tandem YSPTSPS repeats of the RNA polymerase II CTD comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding M-bM-^@M-^\lettersM-bM-^@M-^] to gene expression, we analyzed the poly(A)+ transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n=227) >> Y1F (n=71) > S7A (n=58) >> T4A (n=7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code M-bM-^@M-^\wordM-bM-^@M-^]. Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, while S7A increased pho1+ expression. Meiotic genes were enriched among those up-regulated in S7A cells. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues. Interrogation of the S. pombe transcriptome using polyA+ strand specific RNA sequencing (Illumina HiSeq 2000) in cultures. A total of 16 samples were analysed: two biological repeates of each WT, Y1F, S2A, T4A, S7A,Y1F-S7A, S2A-S7A and T4A-S7A strains
2014-03-13 | E-GEOD-52370 | biostudies-arrayexpress
Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the genome-wide occupancy of Pol II, phospho-Thr4, and key reader Rtt103 in WT and CTD-mutant strains of S. cerevisiae.
2017-05-02 | GSE95418 | GEO
Project description:RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While the significance of phosphorylation on Ser2/Ser5 is well-established, the role of Thr4 remains enigmatic. Thr4 phosphorylation has been implicated in elongation, termination, and mRNA processing, yet it has only been detected after transcription end sites, presenting a paradox. Our investigation revealed that Thr4 phosphorylation is initially obscured by flanking Ser5 phosphorylation at the onset of transcription. By selectively removing this masking effect using a phosphatase, we unveiled the true genomic location of this evolutionarily conserved phosphorylation mark, shedding light on Thr4 phosphorylation cellular functions. Subsequent proteomic analyses unearthed that many proteins previously associated with Ser2 are actually recruited to transcription via Thr4. Crucially, Thr4 phosphorylation primes Ser2 phosphorylation, leading to lethal defects in 3'-end processing. Our findings delineate the true genomic location and function of this "phantom" mark, prompting a reassessment of functional assignments of the CTD heptad.
2024-03-13 | MSV000094312 | MassIVE
Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5M-bM-^@M-^Y and 3M-bM-^@M-^Y regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3M-bM-^@M-^Y region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells. In this study, we investigated the function and ChIPseq genome-wide profiling of Thr4P residue (using the 6D7 antibody) of the Pol II CTD in Raji human B cells in comparison with either total Pol II profiling (N20 antibody, santa-cruz sc-899x), Ser5P CTD (3E8) or Ser2P (3E10) profiling in WT Raji cells. In another set of experiments, we also analysed total Pol II profiling (using an HA tag at the N-terminus of RPB1 and HA antibody Abcam ab9110) when endogenous enzyme is shut down by alpha-amanitin and replaced by either a recominant Pol II with 48 consensus repeats of the CTD (con48) or a mutated version where Thr4 residues were replaced by Ala (Thr4-Ala).In total 6 experimental sets (Pol IIt, Ser5P, Ser2P, Thr4P, con48, Thr4-Ala) were generated for our analysis and for each a biological replicate was performed. Biological replicates were merged when the data showed comparable signal noise ratio. Otherwise a unique replicate, showing the best noise ratio, was chosen for further analysis although the second replicate (for Ser2P and Thr4-Ala experiments). An input control (genomic DNA extracted after reverse crosslinking of the nuclear chip extracts) was performred and used for substraction to the ChIP experiments. One specific input material was used for wt cells, one for con48 and one for Thr4-Ala. Our data were processed to generate final wig files using our in house analysis pipeline essentially as described in Koch et al, (2011) NSMB 18 (8) p956.In brief, after alignment, sequence tags are: (i) artefact removed, (ii) elongated to an in silico optimized actual size of the initial fragments , (iii) input substracted, (iv) merged if applicable, (v) scaled for all experiments to correct for variation of tag number in between experiments. Several of the raw data files were no longer available.
2012-06-04 | E-GEOD-37519 | biostudies-arrayexpress
Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5’ and 3’ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3’ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.
2012-06-04 | GSE37519 | GEO
Project description:Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here we show phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3'-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of Asp1 and Aps1 pyrophosphatases (i.e., too much IP8) is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1∆ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3'-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.
2019-06-16 | GSE127550 | GEO
Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcrption. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific readers. While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the genome-wide occupancy of Pol II, phospho-Thr4, Hrr25, and key reader Rtt103 in WT and irreversibly sensitive Hrr25 (hrr25is) mutant strains of S. cerevisiae.
2018-12-02 | GSE107164 | GEO
Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the RNA expression profile in WT and CTD-mutant strains of S. cerevisiae.
2017-05-02 | GSE95417 | GEO