Construction Of BCBL1 Cell Line Stably Inhibiting Histone Acetylase (HDAC1) And Preliminary Study Of KSHV Acetylation Modification Site

Background: Kaposi's sarcoma-associated herpesvirus(KSHV)is a causative agent for a variety of fatal lymphomas such as Primary Exudative Lymphoma(PEL)and Multicenter Kastman's Disease(MCD).After infection,the life cycle of KSHV is divided into latency and lytic replication.Like the thorough treatment of HIV,the key to eradicating KSHV is to shift the activation of KSHV in the latent status to the lytic replication status.Epigenetic modifications are crucial for activation of lytic replication.For example,inhibition of Histone deacetylase 1(HDAC1)enhances replication of KSHV genome.However,the mechanism of acetylation modification on the KSHV genome after HDAC1 inhibition is still not clear.In this study,a KSHV-infected B-cell lymphoma(BCBL1)stably inhibiting HDAC1 was constructed,and a preliminary identification of the KSHV acetylation sites was performed.Objective: Construction of a BCBL1 cell line stably inhibiting HDAC1 expression by using CRISPR/Cas9;Elucidating the effect of HDAC1 knockout on KSHV gene;Performing chromatin immunoprecipitation assay(ChIP)to determine the acetylation modification sites on KSHV genome after HDAC1 knockout.Methods: 1.The lentiviral expression vector LentiCRISPRv2-sgHDAC1 targeting human sgRNA of HDAC1 was constructed.The constructed LentiCRISPRv2-sgHDAC1 and lentiviral packaging plasmids were co-transfected into 293 T cells.2.BCBL1 cells were infection with sgHDAC1 lentivirus,the cells were then screened and maintained by using puromycin.Western Blotting was performed to detect the expression of HDAC1 protein and histone acetylation in the cells.Real-time quantitative PCR were performed to detect the mRNA levels of HDAC1 and the expression of KSHV lytic/latency genes in the cells;3.Optimize the condition for the ultrasonic disruption in the cell lines;4.Perform ChIP and verify the immunoprecipitation effect;perform preliminary analysis of the acetylation sites on the DNA by ChIP-qPCR technique.Results: 1.Three groups of LentiCRISPRv2-sgHDAC1 expression plasmids were constructed and packaged with lentivirus.2.Screening of infected BCBL1 cells showed that the expression level of HDAC1 was significantly decreased by 80%,and the expression level of KSHV genes were significantly increased.3.Obtained the optimized conditions for ChIP experiment and obtained preliminary data for the ChIP test.4.Preliminarily determined the sites of acetylation modification on KSHV genome by using ChIP-qPCR.Conclusions: 1.Two BCBL1 cell lines with significant decreased expression of HDAC1 were screened out.2.The expression of HDAC1 in BCBL1 was significantly decreased,while the expression levels of RTA and K8 in KSHV cleavage were significantly increased.3.The expression level of HDAC1 in BCBL1 was significantly decreased,while the level of acetylation of RTA and K7 in KSHV gene increased,which may be the epigenetic modification sites of KSHV.