EXPERIMENT_TITLE: GSM5018573: U2OS cells - LowMYC_rep1_HiC; Homo sapiens; Mus musculus; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Human U2OS cells and mouse 3T3 cells were fixed with 2% formaldehyde using the Arima Hi-C Kit (Arima). 1 million fixed 3T3 mouse cells were added to each sample of 4 million human U2OS cells before subsequent steps of restriction enzyme digest, biotin end filling and ligation according to the Arima Hi-C protocol. Libraries were prepared using the KAPA Hyper-Prep Kit (KAPA), according to manufacturers' protocols.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The MYC oncogene encodes for the MYC protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. MYC overexpression leads to MYC binding at active enhancers, resulting in a global transcriptional amplification of active genes. Since superenhancers are frequently dysregulated in cancer, we hypothesized that MYC preferentially invades into superenhancers and alters the cancer genome organization. To that end, we performed ChIP-seq, RNA-seq, 4C-seq and SIQHiC (Spike-in Quantitative Hi-C) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC. MYC overexpression in U2OS cells modulated histone acetylation and increased MYC binding at superenhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting MYC binding sites at promoters and enhancers. Immunofluorescence staining showed that MYC molecules formed punctate foci at these transcriptionally active domains after MYC overexpression. These results demonstrate the accumulation of overexpressed MYC at promoter-enhancer hubs and suggest that MYC invades into enhancers through spatial proximity. At the same time, the increased protein-protein interactions may strengthen these chromatin interactions to increase chromatin contact frequency. CTCF siRNA knockdown in MYC overexpressed U2OS cells demonstrated that removal of architectural proteins can disperse MYC and abrogate the increase in chromatin contacts. By elucidating the chromatin landscape of MYC driven cancers, we can potentially target MYC associated chromatin interactions for cancer therapy. Overall design: Spike-In Quantitative Hi-C (SIQHiC), ChIP-Seq, RNA-Seq and 4C-Seq performed on 2 biological replicates of U2OS cells treated with doxycycline or vehicle (H2O) for 30h, with NIH3T3 mouse cells spiked in.
SAMPLE_TITLE: U2OS cells - LowMYC_rep1_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "U2OS human cells; spike in NIH3T3"}, {"TAG": "cell line", "VALUE": "U2OS; NIH3T3"}, {"TAG": "doxycyline treatment", "VALUE": "-"}, {"TAG": "restriction enzyme", "VALUE": "Arima (multi-enzyme)"}, {"TAG": "chip antibody", "VALUE": "Not Applicable"}]
Pool_Member_@attributes_sample_title: U2OS cells - LowMYC_rep1_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: U2OS cells - LowMYC_rep1_HiC
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