![]() Precipitated DNA was then assayed by real-time PCR, using primers for the KSHV ORF50 promoter region (5′-CCC GCC CAG AAA CCA GTA G-3′ and 5′-TGC GGA GTA AGG TTG ACT TTT TAA-3′) and for normalization to the cellular actin DNA signal (5′-GCC ATG GTT GTG CCA TTA CA-3′ and 5′-GGC CAG GTT CTC TTT TTA TTT CTG-3′). The cell lysate was then subjected to phenol-chloroform extraction and ethanol precipitation. Briefly, 10 6 BCBL1 cells were resuspended in SDS lysis buffer (1% SDS, 20 mM NaCl, 4 mM EDTA, 20 mM Tris, pH 8.0) with proteinase K for at least 6 h at 50☌. ![]() The intracellular KSHV DNA copy number was determined by quantitative PCR (qPCR) analysis of purified total genomic DNA. Isolation of KSHV genomic DNA and quantification of its copy number. 293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. KSHV-positive PEL cells (BCBL1, BC-1, and BC-3) and both Epstein-Barr virus (EBV)- and KSHV-positive cells (JSC-1) were grown in RPMI medium (Gibco BRL) containing 10% heat-inactivated fetal bovine serum and the antibiotics penicillin and streptomycin (50 U/ml). We also show that the histone deacetylase inhibitor sodium butyrate (NaB) leads to a rapid loss of Rad21 and other cohesin subunits from chromatin, suggesting that cohesins play a regulatory role in the transcriptional repression of the KSHV immediate early lytic gene cluster. ![]() ![]() We also show that this region contains a bivalent histone modification pattern that is disrupted upon Rad21 depletion. We show that CTCF and cohesins bind to the region upstream of the divergent promoter for ORF50 and ORF45. We found that depletion of core cohesin subunits, especially Rad21, causes reactivation of latent KSHV immediate early gene expression and viral DNA replication. ![]()
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