Spt4 helps RNAPII pass the +2 nucleosome barrier in vivo
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ABSTRACT:
PROVIDER: PRJNA668299 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12800700.fastq.gz | Fastqsanger.gz | |||
| SRR12800800_1.fastq.gz | Fastqsanger.gz | |||
| SRR12800800_2.fastq.gz | Fastqsanger.gz | |||
| SRR12800701.fastq.gz | Fastqsanger.gz | |||
| SRR12800801_1.fastq.gz | Fastqsanger.gz |
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Project description:Aim and Methods: To understand the precise role of Spt4 in budding yeast, various high throughput sequencing techniques were used. Nucleosome positions were studied using MNase-seq in WT and spt4∆ cells. The position of RNAPII was examined using NET-seq in spt4∆ cells or cells in which Spt4 has been anchored away to the cytoplasm (Spt4 AA). RNAPII was also mapped in cells in which Spt5 has been anchored away to the cytoplasm (Spt5 AA). Steady-state transcript levels were assessed using RNA-seq in samples coupled with NET-seq. The position of the Spt4 on RNAPII was mapped at single nucleotide resolution using TEF-seq. With the same technique, the position of Spt5 on RNAPII was investigated in wild type (WT) and spt4 knock-out (spt4∆) cells. The position and levels of the pre-initiation complex were assessed using ChIP-seq on Sua7(TFIIB) in WT and spt4∆ cells. Results: The interaction between the Spt4/5 complex and RNAPII periodically changes as RNAPII transitions through nucleosomes. The dynamic interaction of Spt5 with RNAPII is dependent on Spt4. In spt4∆ and Spt4 AA cells, RNAPII distribution is altered in a similar way compared to their respective controls. After transcribing into nucleosomes, RNAPII accumulates upstream of nucleosome dyads, especially around the +2 nucleosome. In Spt5 AA cells, the distribution and amount of RNAPII on genes are severely affected. Nucleosome positions are altered in spt4∆ cells. Although the position of the +1 nucleosome remains unchanged, nucleosome spacing from this point is increased, and the level of increase in nucleosome spacing correlates with the level of RNAPII accumulation. Conclusion: This work suggests that Spt4 regulates nucleosome positioning by a mechanism related to transcription and promotes RNAPII movement through nucleosome barriers, especially the barrier set by the +2 nucleosome.
2021-09-27 | GSE159291 | GEO
Project description:Promoter-proximal pausing of RNAPII is a critical step in early transcription elongation to precisely regulate gene expression. Here, we observed evidence of promoter-proximal pausing like distributions of RNAPII in S. cerevisiae. We find that Ino80p loss caused the transition of RNAPII pausing to the alternative pausing site at which is tightly associated with the +1 nucleosome. These Ino80p dependent genes showed better nucleosome positioning around the main pausing site which suggested the regulation of RNAPII pausing by Ino80p in a nucleosome context-dependent manner. Furthermore, we examined the similar INO80 dependent RNAPII pausing site determination in mESCs. Altogether, we hypothesize a highly conserved role of the chromatin remodeling Ino80 complex in controlling the early RNAPII elongation to establish intact pausing in various organisms, from budding yeast to mouse.
2021-08-20 | GSE158622 | GEO
Project description:Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 interacts with elongating RNA polymerase II (RNAPII) via a tandem Src2 homology (tSH2) domain, but it is not known whether this particular interaction is required for the nucleosome reassembly activity of Spt6. Here, we show that Spt6 recruitment to genes and its nucleosome reassembly functions are largely independent of association with RNAPII. Instead, the Spt6-RNAPII association is required for post-transcriptional mRNA turnover. Mechanistically, association of Spt6 with RNAPII couples the Ccr4-Not complex to the transcribed regions of genes, which we show regulates the timely deadenylation and degradation of a broad range of mRNAs including those required for cell cycle progression. Thus, our findings reveal an unexpected control mechanism for mRNA turnover facilitated by a histone chaperone during transcription.
2018-04-18 | GSE111815 | GEO
Project description:All histone-DNA contacts in a nucleosome must be disrupted for RNA polymerase II (RNAPII) to transcribe through chromatin. However, the structural transitions of the nucleosome during transcription in vivo are unknown. To identify nucleosomal intermediates formed during transcription in vivo, we mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by deep sequencing. At the first nucleosome position downstream of the transcription start site, we identified hexasomes lacking an H2A/H2B dimer. Inhibiting topoisomerases or depleting histone chaperones increased dimer loss, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased dimer loss. Our results suggest that positive torsion generated by elongating RNAPII evicts the distal H2A/H2B dimer in vivo. We also identified diagnostic subnucleosomal particle remnants in cell-free human DNA data as a relic of transcribed genes from the tissue-of-origin. Thus the identification of subnucleosomal particle fragments from nuclease protection data represents a general strategy for structural epigenomics.
2017-12-22 | GSE98351 | GEO
Project description:The carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the heptapeptide consensus Y1-S2-P3-T4-S5-P6-S7. RNAPII CTD is intrinsically disordered and has been shown to promote liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the precise role of the conserved heptad residues in LLPS has been hampered by the lack of direct characterization of the biochemical properties of the CTD. Here, we generated a systematic array of RNAPII CTD variants to unravel the sequence-encoded molecular grammar underlying LLPS of the human CTD.
2024-09-19 | PXD049700 | Pride
Project description:Purpose: The ultimate goal of this study is the identification of transcription elongation-related chromatin remodeler and its in vivo mechanism in fission yeast through Next-generation sequencing (NGS) based ChIP-seq technique. Methods: Every ChIP-seq cells were grown up to mid-log phase and harvested after final 1% formaldehyde fixation and 125mM glycine quencing. Cells were harvested by centrifugation at 4 Celcius degrees and the cell pellets were washed by 15mL of pH7.5 TBS buffer per a conical tube. ChIP cells were disrupted by glass bead vortexing in ChIP lysis buffer and sonicated by Sonic Dismembrator Model 500, Fisher Scientific. Debris of the sonicate was excluded by centrifugation by 14,800rpm at 4 Celcius degree. IP samples were aliqouted from the sonicate and immunoprecipiated by proper amount of antibody and protein A/G beads, washed by ChIP lysis buffer for over-night at 4 Celcius degrees. IP samples were washed and eluted by proper buffers in Sigmaprep spin column. Proteins in both IP and input samples were degraded by 2hr 30ug proteinase K rxn and de-crosslinked at 65 Celcius degrees for at least 8hrs. The IP and input DNA were eluted by Quiagen PCR purification kit. Results: RNAPII and factor ChIP-seq in various mutants of chromatin remodelers showed that FUN30 family promotes transcription at gene coding regions. RNAPII ChIP-seq in fft3Δ, spt16-1 and fft3Δspt16-1 demonstrated that RNAPII regulation function of Fft3 is largely overlapped with that of Spt16. Histone H3 ChIP-seq results in fft3Δ and spt16-18 demonstrated that Fft3 is major chromatin remodeler to promote nucleosome turnover through nucleosome disassembly. Finally, histone H3 and RNAPII ChIP-seq in fft3Δ, H3/H4DD and fft3Δ H3/H4DD were showed that Fft3 facilitates RNAPII elongation at ORF regions through nucleosome barrier regulation. Conclusions: Fun30-Fft3 is major chromatin remodeler to facilitate RNAPII elongation by nucleosome disassembly-driven nucleosome barrier alleviation in fission yeast.
2016-12-18 | GSE83600 | GEO
Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches.
2015-07-31 | GSE59956 | GEO
Project description:RNA Polymerase II (RNAPII) termination for transcripts containing a polyadenylation signal (PAS) is thought to differ mechanistically from termination for PAS-independent RNAPII transcripts such as sn(o)RNAs. In a screen for factors required for PAS-dependent termination, we identified Sen1, a putative helicase known primarily for its role in PAS-independent termination. We show that Sen1 is required for termination on hundreds of protein-coding genes and suppresses cryptic transcription from nucleosome-free regions on a genomic scale. These effects often overlap with but are also often distinct from those caused by Nrd1 depletion, which also impacts termination of protein-coding and cryptic transcripts, including many genic antisense transcripts. Sen1 controls termination through its helicase activity and stimulates recruitment of factors previously implicated in both PAS-dependent (Rna14, Rat1) and PAS-independent (Nrd1) termination. Thus, RNAPII termination for both protein-coding genes and cryptic transcripts is dependent on multiple pathways. The 2 RNAPII datasets were produced in duplicates and the Sen1 and Nrd1 datasets in triplicates (all IP/Input).
2011-06-01 | E-GEOD-19015 | biostudies-arrayexpress
Project description:We present a strategy termed PASS-DIA (Precursor ion And Small Slice-DIA) in which MS/MS spectra are acquired with small isolation window (slices) and MS/MS spectra are interpreted with accurately measured precursor ion masses. This method enables direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was superior to both DDA and conventional DIA experiment with regard to identification of peptides. Application of PASS-DIA for analysis of samples with post-translationally modified peptides such as phosphorylation and N-glycosylation again revealed its superior performance. Finally, use of PASS-DIA to characterize a rare proteome of human fallopian tube organoid samples revealed biologically relevant and low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool which outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that PASS-DIA method could become an important strategy for discovery type studies when deeper proteome characterization is required.
2024-08-02 | PXD018180 | Pride
Project description:Deregulation of RNA Polymerase II (RNAPII) by oncogenic signaling leads to collisions of RNAPII with DNA synthesis machinery (transcription-replication conflicts, TRCs). TRCs can result in DNA damage and underlie genomic instability in tumor cells. Here we provide evidence that elongating RNAPII promotes activation of the ATM kinase at TRCs to drive DNA repair. We show the ATPase Wrnip1 that binds and protects stalled replication forks, associates with RNAPII and limits ATM activation. Wrnip1 binding to elongating RNAPII requires catalytic activity of the ubiquitin ligase Huwe1. Mutation of Huwe1 promotes the transfer of Wrnip1 onto replisome and induces TRCs stimulating ATM activation on RNAPII. This mechanism is evoked early upon replicative stress to induce Wrnip1 translocation and ATM signaling at TRCs. Thus, although primarily considered as genotoxic events, TRCs can provide a mechanism to maintain genome stability under replicative stress.
2023-07-31 | PXD037680 | Pride