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DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Ngai SC, Rosli R, Al Abbar A, Abdullah S - Biomed Res Int (2015)

Bottom Line: Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

GFP expression of HPCs transduced with LV/CMV/GFP and treated either with 2 μM 5-azaC + 50 nM TSA (Cocktail 1) or with 2 μM 5-azaC + 150 nM TSA (Cocktail 2) to prevent and to reverse GFP silencing. The expression was measured at day 5 after transduction. Untreated cells were served as the control. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the transduced untreated group is reported as ∗P < 0.050.
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fig6: GFP expression of HPCs transduced with LV/CMV/GFP and treated either with 2 μM 5-azaC + 50 nM TSA (Cocktail 1) or with 2 μM 5-azaC + 150 nM TSA (Cocktail 2) to prevent and to reverse GFP silencing. The expression was measured at day 5 after transduction. Untreated cells were served as the control. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the transduced untreated group is reported as ∗P < 0.050.

Mentions: Therefore, the effect of the combination of both drugs in preventing and reversing the transgene silencing was investigated after analyzing their individual effects. 5-azaC and TSA combinations (Cocktail 1: 2 μM 5-azaC + 50 nM TSA; Cocktail 2: 2 μM 5-azaC + 150 nM TSA) were added a day after transduction to prevent GFP silencing or on the day of GFP silencing to reverse the silenced GFP. The GFP expression in the reversion study was 3.56% and 10.67% for Cocktail 1 and Cocktail 2, respectively (Figure 6). Unfortunately, the readings were not statistically different from the transduced untreated cells (9.27%). The GFP expression was low for the cells treated with Cocktail 1 in the prevention study. However, significantly high expression (70.00%) was achieved for the transduced cells treated with Cocktail 2 (P = 0.006).


DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Ngai SC, Rosli R, Al Abbar A, Abdullah S - Biomed Res Int (2015)

GFP expression of HPCs transduced with LV/CMV/GFP and treated either with 2 μM 5-azaC + 50 nM TSA (Cocktail 1) or with 2 μM 5-azaC + 150 nM TSA (Cocktail 2) to prevent and to reverse GFP silencing. The expression was measured at day 5 after transduction. Untreated cells were served as the control. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the transduced untreated group is reported as ∗P < 0.050.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4417590&req=5

fig6: GFP expression of HPCs transduced with LV/CMV/GFP and treated either with 2 μM 5-azaC + 50 nM TSA (Cocktail 1) or with 2 μM 5-azaC + 150 nM TSA (Cocktail 2) to prevent and to reverse GFP silencing. The expression was measured at day 5 after transduction. Untreated cells were served as the control. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the transduced untreated group is reported as ∗P < 0.050.
Mentions: Therefore, the effect of the combination of both drugs in preventing and reversing the transgene silencing was investigated after analyzing their individual effects. 5-azaC and TSA combinations (Cocktail 1: 2 μM 5-azaC + 50 nM TSA; Cocktail 2: 2 μM 5-azaC + 150 nM TSA) were added a day after transduction to prevent GFP silencing or on the day of GFP silencing to reverse the silenced GFP. The GFP expression in the reversion study was 3.56% and 10.67% for Cocktail 1 and Cocktail 2, respectively (Figure 6). Unfortunately, the readings were not statistically different from the transduced untreated cells (9.27%). The GFP expression was low for the cells treated with Cocktail 1 in the prevention study. However, significantly high expression (70.00%) was achieved for the transduced cells treated with Cocktail 2 (P = 0.006).

Bottom Line: Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus