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Genome-wide mapping of m6A sites in cells expressing normal- versus slow-elongating RNA polymerase II (RNAPII)

ABSTRACT: We report enhanced deposition of m6A in cells expressing slow-elongated version of RNAPII

ORGANISM(S): Homo sapiens

PROVIDER: GSE145421 | GEO | 2020/03/10

REPOSITORIES: GEO

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Genome-wide mapping of m6A sites in cells expressing normal- versus slow-elongating RNA polymerase II (RNAPII)

Project description:Genome-wide mapping of m6A sites in cells expressing normal- versus slow-elongating RNA polymerase II (RNAPII)

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Slow RNAPII transcription elongation rate, low levels of RNAPII pausing, and elevated histone H1 content at promoters associate with higher m6A deposition on nascent mRNAs

Project description:N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

2022-09-14 | GSE213270 | GEO

Slow RNAPII transcription elongation rate, low levels of RNAPII pausing, and elevated histone H1 content at promoters associate with higher m6A deposition on nascent mRNAs

Project description:Slow RNAPII transcription elongation rate, low levels of RNAPII pausing, and elevated histone H1 content at promoters associate with higher m6A deposition on nascent mRNAs

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A dual role for H2A.Z.1 in modulating Initiating and Elongating RNAPII dynamics

Project description:RNA Polymerase II (RNAPII) pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step for the induction of most metazoan genes. During pause-release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells (ESCs). Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts Negative Elongation Factor (NELF) dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the Pre-Initiation Complex (PIC) components TFIIB and TBP. Together, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs by regulating RNAPII dynamics and pause release.

2021-04-03 | GSE143737 | GEO

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

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Phase separation of CTD of RNAPII

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.

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Project description:Endogenous aldehydes induce inter-strand crosslinks (ICL) and DNA-protein crosslinks (DPC). While DNA-repair and aldehyde-clearance systems cope with cellular toxicity, deficiencies in these mechanisms cause genome-instability disorders. The FA-pathway, defective in Fanconi anemia (FA), specifically removes ICL. In contrast, SPRTN, compromised in Ruijs-Aalfs syndrome, eliminates DPC during replication. However, AMeDS patients lacking aldehyde-detoxification display combined features of FA and Cockayne syndrome, associated with transcription-coupled repair (TCR) deficiency, suggesting a novel repair mechanism for aldehyde-induced DNA lesions in active genes. In this report, we demonstrate efficient resolution of aldehyde-induced transcription-blocking lesions by TCR. Mass-spectrometry and DPC-seq identify the TCR complex and additional factors involved in DPC removal and formaldehyde-induced damage tolerance. Notably, TFIIS-dependent cleavage of stalled-RNAPII transcripts exclusively protects against formaldehyde-induced damage. A mouse-model lacking both aldehyde-clearance and TCR pathways confirms endogenous DPC accumulation in transcribed regions. These findings highlight the importance of DPC removal in preventing transcription-roadblocks and contribute to understanding disorders related to aldehyde clearance and TCR deficiencies.

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pSer2-RNAPII interaction partners in MEF cells (label free)

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

Fresh versus slow-frozen ATACseq samples for salmon tissues

Project description:Fresh versus slow-frozen ATACseq samples for salmon tissues

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Fresh versus slow-frozen ATACseq samples for salmon tissues

Project description:When sampling many samples at locations with poor laboratory facilities, the ability of freezing samples for later processising is crucial. ATAC-seq on fresh tissue is still considered the star method. Freezing has shown to affect chromatin architecture and nucleosome pattern. However, the freezing method might determine the chromatin integrity. We therefore ask whether or not slow frozen samples give the same results as fresh samples when assaying tissues for transposase accessible chromatin?

2020-05-08 | E-MTAB-9001 | biostudies-arrayexpress

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