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Binding Changes in Chromatin Factors Upon Targeted XPO1 Inhibition in T Cells

ABSTRACT: We assessed the chromatin localization of XPO1, different histone markers, and transcription factors associated with T cell activation with and without SP100030 treatment We used CUT&RUN sequencing as a lower-input (500,000 cells per sample), higher-sensitivity alternative to ChIP-Seq to assess chromatin binding of different factors

ORGANISM(S): Homo sapiens

PROVIDER: GSE228211 | GEO | 2024/02/01

REPOSITORIES: GEO

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Binding Changes in Chromatin Factors Upon Targeted XPO1 Inhibition in T Cells

Project description:We assessed the chromatin localization of XPO1 and p300 in response to C646 (10 uM), S109 (1 uM), and SP100030 (1 uM). We used CUT&RUN sequencing as a lower-input (500,000 cells per sample), higher-sensitivity alternative to ChIP-Seq to assess chromatin binding of different factors

2024-05-07 | GSE244396 | GEO

Binding Changes in Chromatin Factors Upon Targeted XPO1 Inhibition in T Cells

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Binding Changes in Chromatin Factors Upon Targeted XPO1 Inhibition in T Cells

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NPM1-fusion proteins promote myeloid leukemogenesis through XPO1-dependent HOX activation [Cut & Run]

Project description:NPM1-fusion proteins promote myeloid leukemogenesis through XPO1-dependent HOX activation [Cut & Run]

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Targeted in situ genome-wide profiling with high efficiency for low cell numbers

Project description:Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely- used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench and requires no special skills.

2018-03-21 | GSE104550 | GEO

XPO1 amplification abrogates the effects of E571K XPO1 mutation in B-cell lymphoma

Project description:XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphomas (PMBL) and classical Hodgkin lymphomas (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K modification within the hydrophobic groove of the protein, the site of cargo proteins binding. We investigated the functional impact of XPO1E571K mutation using cell lines having various XPO1 status. We first confirmed that the mutation was present both within the XPO1 mRNA and the corresponding protein. In the U2940 cell line having a wild-type (wt) XPO1 gene, we introduced the E571K mutation using several CRISPR-Cas9 strategies. Using a barcoding method to trace the mutation, we observed that the mutation gave no advantage in vitro. Indeed, the effects of XPO1E571K mutation were hidden by the presence of the XPO1 wt allele. Strikingly, XPO1E571K mutation never occurred as homozygous or hemizygous in our patients�� cohort. We further showed that XPO1E571K mutation modified selinexor binding to XPO1 and in turn, its sensitivity to this drug. We concluded that the balance between the wild-type and the mutated allele of XPO1 is a key element in defining XPO1 functions and oncogenic properties.

2020-10-01 | PXD016916 | Pride

CUT&RUN LoV-U of beta-catenin, TCF/LEF transcription factors and histone modifications in HEK293T and mouse embryonic hindlimb tissue at 11.5 days post coitum

Project description:In our attempts to profile different regulators of the WNT/b-catenin transcriptional complex, CUT&RUN failed to produce consistent binding patterns of the non-DNA-binding b-catenin. We developed a modified CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible b-catenin binding profiles. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested, as shown by datasets targeting the TCF/LEF transcription factors and various histone modifications. CUT&RUN-LoV-U uncovers direct WNT/β-catenin target genes in human cells, as well as in ex vivo cells isolated from developing mouse tissue.

2022-07-16 | E-MTAB-11951 | biostudies-arrayexpress

High-throughput mRNA sequencing of CLL cells containing an E571K XPO1 mutations

Project description:Purpose: Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. Thus, we aimed to identify disrupted cellular pathways in response to the E571K XPO1 mutation, the mutation most frequently cited in CLL patients, via high-throughput RNA-sequencing Results & Conclusion: Multidimensional scaling (MDS) analysis of the top 1000 most variable genes demonstrated distinctly unique clustering along the first dimension for XPO1-E571K patient and XPO1-wt patient samples. This clustering pattern was driven by a number of genes that were either over- or under-expressed in XPO1-E571K patient cells, contributing to a unique gene expression pattern in patients with this mutation signature.

2021-01-21 | GSE163370 | GEO

Prolonged XPO1 inhibition is essential to achieve optimal antileukemic activity in NPM1-mutated acute myeloid leukemia

Project description:NPM1-mutated acute myeloid leukemia (AML) accounts for one third of AML in adults. NPM1-mutated AML maintenance depends on the interaction between mutated NPM1 (NPM1c) and the nuclear exporter Exportin 1 (XPO1). In this work, we show that continous XPO1 inhibition is necessary to achieve stable disruption of the NPM1c-XPO1 interaction and to induce HOX downregulation and differentiation of AML cells with mutated NPM1. In contrast, intermittent XPO1 inhibition only results in minimal transcriptional perturbation and limited antileukemic activity.

2022-09-21 | GSE181176 | GEO

Demonstration of CUT&RUN motif footprint analysis using key blood progenitor transcription factors

Project description:We introduce CUT&RUNTools (https://bitbucket.org/qzhudfci/cutruntools/) as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. We illustrate the functionality of CUT&RUNTools through analysis of CUT&RUN data acquired for GATA1, a master regulator in erythroid lineage cells. Results were compared initially to published GATA1 ChIP-seq data for cells under the same conditions. We performed de novo analysis of CUT&RUN peaks to retrieve not only GATA1’s primary motif, but also the GATA1-TAL1 composite motif, and co-factor motifs GCCCCGCCTC, CMCDCCC, and RTGASTCA that correspond to SP1, KLF1, and NFE2 co-factors. Cofactor binding was verified by independent TAL1 and KLF1 CUT&RUN, and other ChIP-seq experiments. CUT&RUNTools also generated base-pair resolution motif footprint for sequence-specific binding factors, and located likely direct binding sites by quantifying log-odds of binding scores. Overall, CUT&RUNTools should enable biologists to realize advantages of cleavage data provided by CUT&RUN, and make high-quality footprinting analysis accessible to a broad audience.

2019-08-24 | GSE136251 | GEO

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