EXPERIMENT_TITLE: GSM4984004: BCBL-1_TREx-K-Rta_latent_CHi-C_Rep1; Homo sapiens; Human gammaherpesvirus 8; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Chimeric DNA ligation products were prepared for KSHV Capture Hi-C (CHi-C) using kitted reagents from Arima Genomics (San Diego, CA) based on methods described for in situ Hi-C and CHi-C. Briefly, cells were crosslinked with 2% formaldehyde, lysed, and the genomic DNA digested with a cocktail of 4-cutter restriction endonucleases by incubation for 30 minutes at 37°C. The 5'-overhangs were then filled in and labeled with biotinylated dATP (biotin-14-dATP) by incorporation with Klenow fragment of DNA polymerase I (incubation for 45 minutes at 25°C). Ligation of the spatially proximal blunt-ended fragments was then performed with T4 DNA ligase (incubation for 15 minutes at 25°C). The formaldehyde crosslinks were reversed and the proximally-ligated, chimeric DNA products were purified with Agencourt AMPure XP paramagnetic beads (Beckman Coulter). Hi-C and KSHV Capture Hi-C (CHi-C) libraries were prepared from the chimeric DNA ligation products using kitted reagents from Arima Genomics (San Diego, CA). The DNA was fragmented to an average size of 400 bp with a Covaris E220 Focused-ultrasonicator (Covaris, Inc.) and size-selected to have a fragment size distribution of 200-600 bp with AMPure XP beads. The biotin-labeled ligation products were then selectively enriched by affinity capture with streptavidin magnetic beads (Arima Enrichment Beads). Subsequently, sequencing libraries were prepared with the Kapa HyperPrep Kit with Library Amplification Module (Roche) using a modified protocol for on-bead end repair, dA-tailing, and ligation of Illumina TruSeq sequencing adaptors. The KSHV CHi-C library was then prepared from the above Hi-C libraries. Briefly, target enrichment for KSHV genomic content was performed by solution hybridization with a custom-designed KSHV genomic capture probe library (xGen Lockdown Probes; IDT) and subsequent capture of the hybridized targets with streptavidin beads (DynaBeads MyOne Streptavidin C1; Thermo Fisher Scientific) according to the manufacturer's standard protocol (IDT). The KSHV genome-enriched CHi-C library DNA was eluted and PCR enrichment (12 cycles) performed with high-fidelity KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: lncRNAs regulate protein functions via formation of protein-RNA complexes. Previous studies have shown that expression of viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we show an underlying molecular mechanism and regulation of KSHV latency by a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Knock-out of the viral RNA binding protein, ORF57 protein, leads to decreased inducible and static viral genomic loops in latent chromatin and a failure to form RNA polymerase II aggregates in the nucleus during reactivation. We identified that CHD4's enzymatic activity silences viral gene expression by preventing nuclear aggregate formation. Furthermore, integrated genomic and proteomic studies together show that KSHV episomes frequently tether near the host cell centromeres and colocalize with a CHD4 protein complex, ChAHP. KSHV episomes detached from these sites when reactivation is triggered, and PAN RNA binds and inhibits CHD4 DNA binding in vitro. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible enhancer-promoter looping, and is therefore important for the ability of KSHV to establish and maintain latency in a “poised” state at specific host genomic loci. Overall design: The overall goal of these studies was to define the long-range chromatin interactions occurring within the KSHV genome as well as between the KSHV and human host cell genomes both during latency and viral reactivation. These were defined in an unbiased manner using Hi-C analysis, which is chromosome conformation capture (3C) combined with next-generation sequencing (NGS). A modified protocol, referred to as Capture Hi-C, was utilized to selectively enrich interactions involving KSHV by performing target enrichment with a custom-designed KSHV genomic capture probe library. Experiments were performed in three different KSHV naturally infected human primary effusion lymphoma (PEL) cell lines (TREx-F3H3-K-Rta BCBL-1, BC1 and BC3) and wild type or ORF57-Stop recombinant KSHV infected iSLK cells.
SAMPLE_TITLE: BCBL-1_TREx-K-Rta_latent_CHi-C_Rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "TREx-F3H3-K-Rta BCBL-1"}, {"TAG": "tissue", "VALUE": "human primary effusion lymphoma"}, {"TAG": "cell line", "VALUE": "TREx-F3H3-K-Rta BCBL-1"}, {"TAG": "cell type", "VALUE": "KSHV-positive human B cell lymphoma cells"}, {"TAG": "viral infection", "VALUE": "KSHV"}, {"TAG": "virus status", "VALUE": "latent"}]
Pool_Member_@attributes_sample_title: BCBL-1_TREx-K-Rta_latent_CHi-C_Rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: BCBL-1_TREx-K-Rta_latent_CHi-C_Rep1
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