EXPERIMENT_TITLE: GSM8396149: HeLa HP1 knockdown biol + HP1αI165E overexpression biological rep 1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Genomic DNA was extracted from 500,000 cells Crosslinking was carried out with 1% formaldehyde for 10min at room temperature. Nuclei were permeabilized. DNA was digested with 100 units of MboI. After reversal of crosslinks, ligated DNA was size selected between 200-1000 bp and subsequently prepped for Illumina sequencing.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Fo¨rster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1a). Intriguingly, this super- resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1a protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1a oligomeric state. Specifically, we found HP1a monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1a dimerisation. Collectively, these results demonstrate HP1a to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function. Overall design: To investigate our CV-based quantitation of nuclear wide chromatin density, and identify, on what spatial scales the HP1a monomers and dimers are in opposition, we applied high-throughput chromosome conformation capture (Hi-C) to the chromatin network of HeLa, HeLaKD (H (HP1KD) and HeLaHP1aI165E+KD (KDOE), and quantified the frequency of short to long range chromatin interactions. These three conditions were carried out in duplicate.
SAMPLE_TITLE: HeLa HP1 knockdown biol + HP1αI165E overexpression biological rep 1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HeLa"}, {"TAG": "cell line", "VALUE": "HeLa"}, {"TAG": "cell type", "VALUE": "Cervical Cancer"}, {"TAG": "genotype", "VALUE": "HP1 knockdown + HP1alphaI165E overexpression"}, {"TAG": "hic digestion_enzyme", "VALUE": "MboI"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HeLa HP1 knockdown biol + HP1αI165E overexpression biological rep 1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HeLa HP1 knockdown biol + HP1αI165E overexpression biological rep 1
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