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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 at MOI of 3.2 at different time points. The GFP expression was measured, at days 0.25 (6 hours), 1, 2, 3, 5, and 7 after transduction as the percentage of GFP-expressing cells within the total events acquired. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the untreated group is reported as ∗∗∗P < 0.001.
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fig2: GFP expression of HPCs transduced with LV/CMV/GFP at MOI of 3.2 at different time points. The GFP expression was measured, at days 0.25 (6 hours), 1, 2, 3, 5, and 7 after transduction as the percentage of GFP-expressing cells within the total events acquired. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the untreated group is reported as ∗∗∗P < 0.001.

Mentions: The transduction efficiency and duration of transgene expression of HPCs transduced with LV were examined in vitro. The GFP expression was determined at 6 hours and at days 1, 2, 3, 5, and 7 after transduction. Figure 2 shows rapid transgene silencing in HPCs transduced with the LV. The GFP expression increased from 1.57% at 6 hours to 7.34% at day 1 after transduction. The highest level of transgene expression was read at day 2 after transduction (17.08%) (P < 0.001). The GFP expression decreased starting from day 2 after transduction, reaching values of 4.07% at day 7 after transduction (P < 0.001). LV transgene silencing was also observed in many cell types although the rate of silencing varies depending on the cell types [4, 13, 14]. Since the GFP expression started to decrease at day 2 after transduction, therefore, day 3 was chosen as the day of transgene silencing for the following experiments.


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 at MOI of 3.2 at different time points. The GFP expression was measured, at days 0.25 (6 hours), 1, 2, 3, 5, and 7 after transduction as the percentage of GFP-expressing cells within the total events acquired. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the untreated group is reported as ∗∗∗P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: GFP expression of HPCs transduced with LV/CMV/GFP at MOI of 3.2 at different time points. The GFP expression was measured, at days 0.25 (6 hours), 1, 2, 3, 5, and 7 after transduction as the percentage of GFP-expressing cells within the total events acquired. Data are presented as mean ± standard deviation in triplicate. Statistical difference between groups compared to the untreated group is reported as ∗∗∗P < 0.001.
Mentions: The transduction efficiency and duration of transgene expression of HPCs transduced with LV were examined in vitro. The GFP expression was determined at 6 hours and at days 1, 2, 3, 5, and 7 after transduction. Figure 2 shows rapid transgene silencing in HPCs transduced with the LV. The GFP expression increased from 1.57% at 6 hours to 7.34% at day 1 after transduction. The highest level of transgene expression was read at day 2 after transduction (17.08%) (P < 0.001). The GFP expression decreased starting from day 2 after transduction, reaching values of 4.07% at day 7 after transduction (P < 0.001). LV transgene silencing was also observed in many cell types although the rate of silencing varies depending on the cell types [4, 13, 14]. Since the GFP expression started to decrease at day 2 after transduction, therefore, day 3 was chosen as the day of transgene silencing for the following experiments.

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