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CUT&RUN of the kidneys from diabetic mice with MAT2A inhibitor II

ABSTRACT: H3K27me3 was enhanced in the gene loci related to the inflammatory pathway.

ORGANISM(S): Mus musculus

PROVIDER: GSE268596 | GEO | 2025/07/09

REPOSITORIES: GEO

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CUT&RUN of the kidneys from diabetic mice with MAT2A inhibitor I

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CUT&RUN of the kidneys from diabetic mice with MAT2A inhibitor II

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CRAMP1 CUT&RUN in K562 cells

Project description:The essential role of the core histones (H2A, H2B, H3 and H4) in DNA packaging has been well understood for decades, but the function of the linker histone (H1) remains enigmatic. H1 compacts nucleosome arrays in vitro, but the multiple H1 subtypes and variants encoded in mammalian genomes (11 in both mice and humans) hamper genetic studies to determine H1 function in vivo. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for linker histones in the function of Polycomb Repressive complex 2 (PRC2). Through a CRISPR/Cas9 genetic screen in a fluorescent reporter cell line responsive to PRC2 perturbation, we identified an essential requirement for the poorly characterised gene CRAMP1 in PRC2-mediated repression. CRAMP1 localises to the promoters of all expressed H1 genes where it acts as a positive transcriptional regulator. Ablation of CRAMP1 provides a unique tool to simultaneously deplete all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, in contrast to the broad genomic distribution previously ascribed to H1, we find that linker histones selectively localise to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, our data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.

2025-06-13 | GSE276852 | GEO

CRAMP1-dependent histone H1 biogenesis is essential for topoisomerase II inhibitor tolerance [CUT&RUN]

Project description:Topoisomerase II (TOP2) inhibitors (TOP2i) are mainstay chemotherapeutic agents that undermine genome integrity by stabilizing TOP2-DNA complexes accompanied by DNA damage formation. Here, we reveal the uncharacterized protein CRAMP1 and H1 linker histones as key effectors of TOP2i tolerance in human cells. We demonstrate that CRAMP1 defines a dedicated histone H1 biogenesis factor stimulating transcription of both replicative and non-replicative H1 genes, driven by its concurrent targeting to histone gene loci and H1-specific promoter motifs. CRAMP1 promotes TOP2i tolerance by maintaining H1 supply, involving a novel mechanism uncoupled from TOP2i-induced DNA damage whereby reducing the H1 pool triggers unscheduled TOP2 substrate formation in low accessibility chromatin states. This amplifies total demand for TOP2 activity, lowering the threshold for TOP2i-mediated exhaustion of TOP2. Our discoveries elucidate the mechanistic basis of histone H1 biogenesis in human cells, opening opportunities for selectively manipulating linker but not core histone supply and targeting cancer-associated H1 deficiency.

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Project description:Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

| S-EPMC6598765 | biostudies-literature

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