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Targeted gene suppression by inducing de novo DNA methylation in the gene promoter.

Ma AN, Wang H, Guo R, Wang YX, Li W, Cui J, Wang G, Hoffman AR, Hu JF - Epigenetics Chromatin (2014)

Bottom Line: However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach.In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter.Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

View Article: PubMed Central - HTML - PubMed

Affiliation: King's Lab, Shanghai Jiao Tong University School of Pharmacy, 800 Dongchuan Road, Shanghai 200240, China ; Stanford University Medical School, VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA.

ABSTRACT

Background: Targeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a 'super suppressor' by combining the activities of two suppressors that function through distinct epigenetic mechanisms.

Results: Gene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter.

Conclusions: Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

No MeSH data available.


Related in: MedlinePlus

Epigenetic suppression of the copGFP reporter gene. a. Schematic diagram of the copGFP reporter gene system. b. Inhibition of the transiently-transfected copGFP gene. 293 T cells were transiently co-transfected with copGFP reporter and suppressor vectors. Forty-eight hours post-transfection, copGFP expression was analyzed by luminometer. Each error bar represents the standard error of mean (SEM) of three independent experiments. *Indicates P <0.05 versus pcDNA3.1 control vector. c. Inhibition of the copGFP gene that has been stably integrated in the genome of the target cell. Expression of the copGFP reporter gene was quantitated by real-time quantitative PCR. Each sample was analyzed in quadruplicate. a: P <0.05 as compared with 293 T cells transiently transfected with pcDNA3.1 empty vector; b: P <0.05 as compared with the Sss1 group.
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Figure 3: Epigenetic suppression of the copGFP reporter gene. a. Schematic diagram of the copGFP reporter gene system. b. Inhibition of the transiently-transfected copGFP gene. 293 T cells were transiently co-transfected with copGFP reporter and suppressor vectors. Forty-eight hours post-transfection, copGFP expression was analyzed by luminometer. Each error bar represents the standard error of mean (SEM) of three independent experiments. *Indicates P <0.05 versus pcDNA3.1 control vector. c. Inhibition of the copGFP gene that has been stably integrated in the genome of the target cell. Expression of the copGFP reporter gene was quantitated by real-time quantitative PCR. Each sample was analyzed in quadruplicate. a: P <0.05 as compared with 293 T cells transiently transfected with pcDNA3.1 empty vector; b: P <0.05 as compared with the Sss1 group.

Mentions: In addition to the luciferase reporter, we also examined the inhibition of a second reporter protein copGFP that has a relatively long half-life in host cells (Figure 3A). Using fluorescence microscopy, the suppressors tested showed varying inhibition of the expression of the pCMV-copGFP cassette. Quantitation of copGFP fluorescence revealed an inhibition pattern similar to that seen in the luciferase reporter system (Figure 3B). In general, the KRAB suppressor, whether constructed as a single unit or as three unit modules, showed the best inhibition among the tested domains. The combination Sss1 and KRAB suppressor constructs showed immediate inhibition of the copGFP reporter expression, but the vSET domain cassette did not show significant inhibition in this reporter system.We then used a lentiviral delivery system to insert the pCMV-copGFP cassette into the genome of 293 T cells. The suppressors were transiently transfected into cell clones that stably expressed the copGFP gene. Using this system, we found that the KRAB construct induced the greatest inhibition among the suppressors tested (Figure 3C). The Sss1 insert modestly inhibited the expression of the copGFP inserted genes, while the H3K27 methyltransferase vSET domain did not inhibit the stably-expressed copGFP.


Targeted gene suppression by inducing de novo DNA methylation in the gene promoter.

Ma AN, Wang H, Guo R, Wang YX, Li W, Cui J, Wang G, Hoffman AR, Hu JF - Epigenetics Chromatin (2014)

Epigenetic suppression of the copGFP reporter gene. a. Schematic diagram of the copGFP reporter gene system. b. Inhibition of the transiently-transfected copGFP gene. 293 T cells were transiently co-transfected with copGFP reporter and suppressor vectors. Forty-eight hours post-transfection, copGFP expression was analyzed by luminometer. Each error bar represents the standard error of mean (SEM) of three independent experiments. *Indicates P <0.05 versus pcDNA3.1 control vector. c. Inhibition of the copGFP gene that has been stably integrated in the genome of the target cell. Expression of the copGFP reporter gene was quantitated by real-time quantitative PCR. Each sample was analyzed in quadruplicate. a: P <0.05 as compared with 293 T cells transiently transfected with pcDNA3.1 empty vector; b: P <0.05 as compared with the Sss1 group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150861&req=5

Figure 3: Epigenetic suppression of the copGFP reporter gene. a. Schematic diagram of the copGFP reporter gene system. b. Inhibition of the transiently-transfected copGFP gene. 293 T cells were transiently co-transfected with copGFP reporter and suppressor vectors. Forty-eight hours post-transfection, copGFP expression was analyzed by luminometer. Each error bar represents the standard error of mean (SEM) of three independent experiments. *Indicates P <0.05 versus pcDNA3.1 control vector. c. Inhibition of the copGFP gene that has been stably integrated in the genome of the target cell. Expression of the copGFP reporter gene was quantitated by real-time quantitative PCR. Each sample was analyzed in quadruplicate. a: P <0.05 as compared with 293 T cells transiently transfected with pcDNA3.1 empty vector; b: P <0.05 as compared with the Sss1 group.
Mentions: In addition to the luciferase reporter, we also examined the inhibition of a second reporter protein copGFP that has a relatively long half-life in host cells (Figure 3A). Using fluorescence microscopy, the suppressors tested showed varying inhibition of the expression of the pCMV-copGFP cassette. Quantitation of copGFP fluorescence revealed an inhibition pattern similar to that seen in the luciferase reporter system (Figure 3B). In general, the KRAB suppressor, whether constructed as a single unit or as three unit modules, showed the best inhibition among the tested domains. The combination Sss1 and KRAB suppressor constructs showed immediate inhibition of the copGFP reporter expression, but the vSET domain cassette did not show significant inhibition in this reporter system.We then used a lentiviral delivery system to insert the pCMV-copGFP cassette into the genome of 293 T cells. The suppressors were transiently transfected into cell clones that stably expressed the copGFP gene. Using this system, we found that the KRAB construct induced the greatest inhibition among the suppressors tested (Figure 3C). The Sss1 insert modestly inhibited the expression of the copGFP inserted genes, while the H3K27 methyltransferase vSET domain did not inhibit the stably-expressed copGFP.

Bottom Line: However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach.In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter.Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

View Article: PubMed Central - HTML - PubMed

Affiliation: King's Lab, Shanghai Jiao Tong University School of Pharmacy, 800 Dongchuan Road, Shanghai 200240, China ; Stanford University Medical School, VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA.

ABSTRACT

Background: Targeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a 'super suppressor' by combining the activities of two suppressors that function through distinct epigenetic mechanisms.

Results: Gene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter.

Conclusions: Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

No MeSH data available.


Related in: MedlinePlus