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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

Suppression of the reporter gene by epigenetic ‘two-hit’ suppressors. a. Schematic diagram of the two-hit suppressor vectors. Two epigenetic suppressor domains are fused with the GAL4 domain. After binding to the target vector, the synthetic factors suppress the target gene using two distinct epigenetic pathways. b. Relative expression of the reporter gene. Forty-eight hours post-transfection, cells were harvested for luciferase assay as described in the Figure 1 legend. Each error bar represents the standard error of mean (SEM) of three independent experiments. a: P <0.05 as compared with the pcDNA3.1 control vector; b: P <0.05 as compared with the Sss1 group.
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Figure 2: Suppression of the reporter gene by epigenetic ‘two-hit’ suppressors. a. Schematic diagram of the two-hit suppressor vectors. Two epigenetic suppressor domains are fused with the GAL4 domain. After binding to the target vector, the synthetic factors suppress the target gene using two distinct epigenetic pathways. b. Relative expression of the reporter gene. Forty-eight hours post-transfection, cells were harvested for luciferase assay as described in the Figure 1 legend. Each error bar represents the standard error of mean (SEM) of three independent experiments. a: P <0.05 as compared with the pcDNA3.1 control vector; b: P <0.05 as compared with the Sss1 group.

Mentions: Gene suppressors tested in our system inhibit their target genes using distinct epigenetic mechanisms. We were curious if these epigenetic suppressors can be engineered as a super suppressor that would inhibit target genes at the maximum activity. We then tested the suppressive activity of a vector containing both DNA methylation and H3K27 methylation activities. We constructed three fusion suppressors and tested their potency in 293 T cells (Figure 2A).We first fused CpG methylase Sss1 with H3K27 methyltransferase domain vSET. After co-transfection with the reporter vector, we did not observe an additive or synergistic effect of these two epigenetic suppressor domains (Figure 2B), probably because of the weak activity of vSET in our system (Figure 1B).We also examined the suppressive effect of combining the DNA methylase Sss1 with KRAB. We constructed two fused targeting vectors as Sss1-KRAB and KRAB-Sss1 expression cassettes. Both fusion cassettes showed a significantly higher inhibition rate of target gene expression than did the Sss1 cassette alone (Figure 2B). There were no significant differences in gene silencing when the Sss1 enzyme was inserted in front of KRAB or at the C-terminus of KRAB. We did not observe enhanced inhibition when the vSET suppression domain was linked to CpG methylase Sss1.


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)

Suppression of the reporter gene by epigenetic ‘two-hit’ suppressors. a. Schematic diagram of the two-hit suppressor vectors. Two epigenetic suppressor domains are fused with the GAL4 domain. After binding to the target vector, the synthetic factors suppress the target gene using two distinct epigenetic pathways. b. Relative expression of the reporter gene. Forty-eight hours post-transfection, cells were harvested for luciferase assay as described in the Figure 1 legend. Each error bar represents the standard error of mean (SEM) of three independent experiments. a: P <0.05 as compared with the pcDNA3.1 control 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 2: Suppression of the reporter gene by epigenetic ‘two-hit’ suppressors. a. Schematic diagram of the two-hit suppressor vectors. Two epigenetic suppressor domains are fused with the GAL4 domain. After binding to the target vector, the synthetic factors suppress the target gene using two distinct epigenetic pathways. b. Relative expression of the reporter gene. Forty-eight hours post-transfection, cells were harvested for luciferase assay as described in the Figure 1 legend. Each error bar represents the standard error of mean (SEM) of three independent experiments. a: P <0.05 as compared with the pcDNA3.1 control vector; b: P <0.05 as compared with the Sss1 group.
Mentions: Gene suppressors tested in our system inhibit their target genes using distinct epigenetic mechanisms. We were curious if these epigenetic suppressors can be engineered as a super suppressor that would inhibit target genes at the maximum activity. We then tested the suppressive activity of a vector containing both DNA methylation and H3K27 methylation activities. We constructed three fusion suppressors and tested their potency in 293 T cells (Figure 2A).We first fused CpG methylase Sss1 with H3K27 methyltransferase domain vSET. After co-transfection with the reporter vector, we did not observe an additive or synergistic effect of these two epigenetic suppressor domains (Figure 2B), probably because of the weak activity of vSET in our system (Figure 1B).We also examined the suppressive effect of combining the DNA methylase Sss1 with KRAB. We constructed two fused targeting vectors as Sss1-KRAB and KRAB-Sss1 expression cassettes. Both fusion cassettes showed a significantly higher inhibition rate of target gene expression than did the Sss1 cassette alone (Figure 2B). There were no significant differences in gene silencing when the Sss1 enzyme was inserted in front of KRAB or at the C-terminus of KRAB. We did not observe enhanced inhibition when the vSET suppression domain was linked to CpG methylase Sss1.

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