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Identification of Kel1 partners during the mating response in yeast

ABSTRACT: To understand the role of Kel1 during the mating response we immunoprecipitated native Kel1 in presence or absence of alpha-factor followed by mass spectrometry to find Kel1 interactors in presence and absence of pheromone.

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD020833 | Pride | 2021-11-17

REPOSITORIES: Pride

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Reciprocal regulation of cardiac chromatin by the chromatin structural proteins HMGB and CTCF

Project description:Transcriptome remodeling in heart disease occurs through the coordinated actions of transcription factors, histone modifications and other chromatin features at pathology-associated genes. It remains unknown the extent to which genome-wide chromatin reorganization also contributes to the pathologic gene expression. We examined the roles of two chromatin structural proteins, CTCF (CCCTC-binding factor) and HMGB2 (high mobility group protein B2), in regulating pathologic transcription and chromatin remodeling. Our data demonstrate a reciprocal relationship between HMGB2 and CTCF in controlling aspects of chromatin structure and gene expression. Both proteins regulate each otherâs expression as well as transcription in cardiac myocytes: however, only HMGB2 does so in a manner that involves global reprogramming of chromatin accessibility. We demonstrate that the actions of HMGB2 on local chromatin accessibility are conserved across genomic loci, whereas the effects on transcription are loci-dependent and emerge in concert with histone modification and other chromatin features. Lastly, while both proteins share gene targets, HMGB2 and CTCF neither bind these genes simultaneously nor do they physically co-localize in myocyte nuclei. Our study uncovers a previously unknown relationship between these two ubiquitous chromatin proteins and provides a mechanistic explanation for how HMGB2 regulates gene expression and cellular phenotype. Furthermore, we demonstrate direct evidence for hierarchical remodeling of chromatin on a genome-wide scale in the setting of cardiac disease. Examination of a chromatin structural protein, HMGB2 in basal and agonist-treated cells.

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Anti-Pol II pS5 co-immunoprecipitation in DIDO WT, KO and ΔSPOC HEK293T cells

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Proteomics of MCF7 cells (LC-MS/MS Orbitrap Fusion Tribrid)

Project description:We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. We compared riboLace translation estimates with protein levels determined by proteomics in MCF7 cells. RiboLace can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems.

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ChIP-seq of SMRT in CD8α+ DCs at 0hr and 6hr CpG stimulation

Project description:SMRT ChIP-seq were performed to identify binding site of SMRT in CD8α+ DCs at 0hr and 6hr CpG stimulation.

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K562 cell line LC-MSMS,Quantitative measurement of hydrophobicity dependent LLPS property of chromatin condensate

Project description:K562 cell line treated with 2,5-HD and proteins were captured via cross-linking. We quantified chromatin structure changes and chromatin binding proteins in K562 cell before and after 2,5-hexanediol treatment by Hi-C and Hi-MS, respectively.

2021-07-26 | PXD027565 | Pride

K562 cell lime LC-MSMS,Quantitative measurement of hydrophobicity dependent LLPS property of chromatin condensate

Project description:K562 cell line treated with 1,6-HD and proteins were captured via cross-linking. We quantified chromatin structure changes and chromatin binding proteins in K562 cell before and after 1,6-hexanediol treatment by Hi-C and Hi-MS, respectively.

2021-07-26 | PXD021434 | Pride

ChIP-seq of definitive endoderm differentiation of human embryonic stem cells in early G1 phase of the cell cycle

Project description:The experiment was designed to investigate histone modification changes during cell cycle progression of human embryonic stem cells (hESCs) during definitive endoderm differentiation. For this, FUCCI hESCs were sorted in Early G1 (EG1), and differentiation into endoderm was performed for up to 72 hours with a combination of cytokines as described in Pauklin and Vallier (2013) and Pauklin et al. (2016). ChIP-seq was generated at 12-hour and 36-hour time-points for H3K4me3, H3K27ac, H3K4me1, H3K36me3 and H3K27me3. Library preparation and sequencing were performed at the Wellcome Sanger Institute next-generation sequencing facility on Illumina HiSeq 2000, 2 x 75bp paired-end reads. ChIP-seq data from other time-points (0h, 24h, 48h and 72h) of the same experimental set-up is available elsewhere (GEO DataSet, accession PRJNA593217). All processed ChIP-seq data is publicly available at http://ngs.sanger.ac.uk/production/endoderm

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Genetic and proteomic screens suggest that signaling by the mechanosensitive ion channel MSL10 is influenced by its association with a synaptotagmin complex

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2022-10-26 | PXD018747 | Pride

Mapping ERβ genomic binding sites reveals unique genomic features and identifies EBF1 as an ERβ interactor [ChIP-Seq]

Project description:The C4-12/Flag.ERβ cell line which stably expressed Flag.ERβ is used to study ERβ genomic functions without ERα interference. Mapping ERβ binding sites in these cells reveals ERβ unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERβ target genes. Gene ontology analysis reveals that ERβ targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERβ binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERβ binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERβ binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERβ genomic functions in our cell model, we confirm the anti-proliferative role of ERβ and discover the novel cross talk of ERβ with EBF1 which has various implications in normal physiology. C4-12/Flag.ERβ cells were treated with 10nM E2 for 1 hour then crosslinked with 1% formaldehyde; EtOH treatment was used as control. Crosslinked samples were processed for ChIP-seq and sequenced with Illumina Genome Analyzer II and aligned to hg18. QuEST was used as the peak-calling software, using default parameters recommended to analyze transcription factor ChIP-seq data. The entire ChIP-seq process was performed once on each sample (Vehicle or E2-treated).

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