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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Nup170 with/without Brl1 degradation (DIA)

ABSTRACT: This project was aimed at investigating nuclear pore complex biogenesis upon inducible degradation of Brl1 using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, the nuclear pore complex was purified through endogenously tagged Nup170 at several time points following auxin inducible degradation of Brl1. Simultaneously with the degradation of Brl1, newly synthesized proteins were metabolically labeled using a SILAC approach. The metabolic labeling of all nuclear pore complex constituents was assessed. Proteins that depend on Brl1 for their assembly show delayed metabolic labeling.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Federico Uliana

LAB HEAD: Federico Uliana

PROVIDER: PXD032024 | Pride | 2022-08-17

REPOSITORIES: Pride

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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Brl1 (DIA)

Project description:This project was aimed at analyzing the binding of Brl1 to the nuclear pore complex during the assembly process using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, we affinity purified Brl1 at several time points following SILAC metabolic labeling in growing S. cerevisiae cultures. Then, we analyzed the metabolic labeling for all co-purified nucleoporins and used these labeling kinetics to infer the timing of Brl1 binding during the native nuclear pore complex assembly.

2022-08-17 | PXD032017 | Pride

An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - Lysate intermixing text (DIA)

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The histone acetyltransferase Mst2 prevents epigenetic silencing via specific acetylation of the ubiquitin ligase Brl1 [microarray data]

Project description:Faithful propagation of distinct chromatin states is crucial to maintain cellular identity. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report the discovery of a self-reinforcing feedback loop that preserves the transcription-competent state of RNA polymerase II transcribed genes. We found that the chromatin reader Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3 marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides histone H3K14, Mst2 specifically acetylates Brl1, a component of the histone H2B ubiquitin ligase complex (HULC). Brl1 acetylation results in increased histone H2B ubiquitination, which together with H3K14ac positively feeds back on transcription and prevents assembly of ectopic heterochromatin. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

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The histone acetyltransferase Mst2 prevents epigenetic silencing via specific acetylation of the ubiquitin ligase Brl1 [NGS data]

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The histone acetyltransferase Mst2 protects active chromatin from epigenetic silencing by acetylating the ubiquitin ligase Brl1

Project description:Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we discover a self-reinforcing feedback loop that preserves the transcription-competent state of RNA polymerase II transcribed genes. We found that the chromatin reader Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3 marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation results in increased histone H2B ubiquitination, which together with H3K14ac positively feeds back on transcription and prevents assembly of ectopic heterochromatin. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

2017-06-28 | PXD005714 | Pride

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The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome [RNA-Seq]

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Association of NUP153 on genomic DNA in TE5 cells [ChIP-seq]

Project description:To understand the regulatory regions of genomic DNA by nuclear pore, the genomic region associated by NUP153, one of nuclear pore protein, was determined by Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) .

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The WNT-dependent gating of MYC requires non-canonical functions of a distal CTCF binding site

Project description:Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer (OSE)-mediated gating of MYC to the nuclear pore. This process enables MYC transcripts to escape the higher degradation rate in the nucleus compared to the cytoplasm. By targeted mutation, we show here that a single OSE-specific CTCF binding site (CTCFBS) mediates the WNT-regulated increased nuclear export rate of mRNAs derived from its interacting MYC and FAM49B loci, located >0.5 and >2.6 mbp distal of the OSE. The CTCFBS coordinated OSE-MYC proximity with their peripheral localisation, and mediated the WNT-dependent recruitment of the nuclear pore anchor AHCTF1 to the OSE. Furthermore, the CTCFBS up-regulated CCAT1 eRNA transcription, implicated in OSE-MYC interactions, correlating with an enhanced ability of the OSE to reach the nuclear periphery. We submit that WNT-dependent gating of MYC requires CTCF complexed with the OSE to coordinate multiple steps of the gating process.

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