EXPERIMENT_TITLE: GSM5629821: KD; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: STAG2 Knockdown or control cells were fixed in 1% of fresh formaldehyde for 10 minutes at room temperature, quenched with glycine and washed twice with ice-cold PBS. In situ Hi-C was then performed following the protocol by Rao et al (cell 2014). Briefly, nuclei were permeabilized, and DNA was digested overnight with 100 U MboI (New England BioLabs). The ends of the restriction fragments were labeled using biotin-14-dATP (Life Technologies) and then ligated in 1.2 mL final volume. After reversal of crosslinks, ligated DNA was purified and sheared to a length of ~400 bp with a Covaris E220 evolution instrument (Covaris, Woburn, MA). Ligation junctions were then pulled down with streptavidin beads. HiC libraries were constructed as described before (Rao et al, cell, 2014). Briefly,  DNA fragments were end-repaired, dA-tailed and Illumina adapters ligated. Libraries were produced by 8 cycles of PCR amplification with KAPA Hifi DNA polymerase (Roche). The HiC libraries were size selected with AMPure XP beads and sequenced at Quick Biology Inc. (Pasadena, CA)
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The cohesin complex participates in the organization of 3D genome through generating and maintaining DNA loops. Stromal antigen 2 (STAG2), a core subunit of the cohesin complex, is frequently mutated in various cancers. However, the impact of STAG2 inactivation on 3D genome organization and subsequent gene expression in cancer remain poorly understood. Here we show that depletion of STAG2 in melanoma cells leads to expansion of topologically associating domains (TADs) and enhances the formation of H3K27Ac-associated DNA loops at sites where binding of STAG2 is switched to its paralog STAG1. We further identify Interferon Regulatory Factor 9 (IRF9) as a major direct target of STAG2 in melanoma cells via integrated RNA-Seq, STAG2 ChIP-Seq and H3K27Ac HiChIP analyses. We demonstrate that loss of STAG2 activates IRF9 through modulating the 3D genome organization, which in turn enhances type I interferon signaling and increases the expression of PD-L1. Our findings not only establish a previously unknown role of the STAG2 to STAG1 switch in 3D genome organization, but also reveal a functional link between STAG2 and interferon signaling in cancer cells, which may contribute to malignant phenotype in STAG2-mutant cancer. Overall design: We investigated how STAG2 KD affact 3D genome structure and its related functional consequnece in malignant phenotype in STAG2-mutant melanomas.
SAMPLE_TITLE: KD
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "melanoma cell line"}, {"TAG": "tag", "VALUE": "STAG2 KD"}]
Pool_Member_@attributes_sample_title: KD
RUN_SET_RUN_Pool_Member_@attributes_sample_title: KD
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR16465802.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-zq-24/SRR016/16465/SRR16465802/SRR16465802.lite.1", "size": "17588329818", "date": "2022-08-28 23:48:31", "md5": "8b20d23d251dd270ac3f01a4c3d73f5b", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR16465802", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR16465802/SRR16465802", "size": "29087460646", "date": "2021-10-20 02:39:06", "md5": "93baccfc94bb5312fbb1f0562b45941c", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "kd_R2.fastq.gz", "size": "22103714055", "date": "2021-10-20 02:06:13", "md5": "d2f1acc048e123bc1798ffb16a40de24", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "kd_R1.fastq.gz", "size": "20326609041", "date": "2021-10-20 01:52:37", "md5": "ce48c8f09d520aabefe2567bfaca6991", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]