title: DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.
abstract: Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.
authors.affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; School of Biosciences, Faculty of Science, The University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor, Malaysia.
authors.forename: Siew Ching
authors.initials: SC
authors.lastname: Ngai
authors.affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; Genetics & Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia.
authors.forename: Rozita
authors.initials: R
authors.lastname: Rosli
authors.affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; Genetics & Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia.
authors.forename: Akram
authors.initials: A
authors.lastname: Al Abbar
authors.affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; Genetics & Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia.
authors.forename: Syahril
authors.identifier: 0000-0003-2718-6266
authors.initials: S
authors.lastname: Abdullah
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D022041:Euchromatin / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells; D042421:Histone Code / Q000187:drug effects / Q000235:genetics*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D019076:Transgenes / Q000187:drug effects
chemical_list: D022041:Euchromatin; D056572:Histone Deacetylase Inhibitors; D049452:Green Fluorescent Proteins; D006655:Histone Deacetylases