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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

Targeted suppression of the reporter gene by epigenetic suppressors. a. Schematic diagram of suppressor and reporter gene vectors. GAL4: the GAL4 DNA binding domain; GBS: GAL4-binding site; KRAB: kruppel-associated box domain; NSL: nuclear localization signal; pCMV: cytomegalovirus (CMV) promoter; PA: SV40 polyadenylation signal; Sss1: methyltransferase gene from Spiroplasma sp. strain MQ1; vSET: the histone H3 lysine 27 methyltransferase SET domain. Synthetic factors use the GAL4 domain to bind to the GBS site in the target gene vector, where the suppressor domain suppresses the activity of the downstream CMV promoter through epigenetic mechanisms. b. Relative expression of the reporter gene. 293 T cells were transiently co-transfected with 250 ng suppressor vectors, 250 ng luciferase target vector, and 25 ng thymidine kinase promoter-Renilla luciferase reporter (pRL-TK) control vector. The empty pcDNA3.1 vector was used as the study control. Forty-eight hours post-transfection, cells were harvested for luciferase assay. For comparison, the pcDNA3.1 control vector was adjusted to 100%. Each error bar represents the 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 1: Targeted suppression of the reporter gene by epigenetic suppressors. a. Schematic diagram of suppressor and reporter gene vectors. GAL4: the GAL4 DNA binding domain; GBS: GAL4-binding site; KRAB: kruppel-associated box domain; NSL: nuclear localization signal; pCMV: cytomegalovirus (CMV) promoter; PA: SV40 polyadenylation signal; Sss1: methyltransferase gene from Spiroplasma sp. strain MQ1; vSET: the histone H3 lysine 27 methyltransferase SET domain. Synthetic factors use the GAL4 domain to bind to the GBS site in the target gene vector, where the suppressor domain suppresses the activity of the downstream CMV promoter through epigenetic mechanisms. b. Relative expression of the reporter gene. 293 T cells were transiently co-transfected with 250 ng suppressor vectors, 250 ng luciferase target vector, and 25 ng thymidine kinase promoter-Renilla luciferase reporter (pRL-TK) control vector. The empty pcDNA3.1 vector was used as the study control. Forty-eight hours post-transfection, cells were harvested for luciferase assay. For comparison, the pcDNA3.1 control vector was adjusted to 100%. Each error bar represents the 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: To optimize the potency of different epigenetic suppressors, we constructed a target vector by inserting a GAL4-binding site cluster sequence (GBS) [19] upstream of a cytomegalovirus (CMV) promoter that drives the expression of the reporter luciferase gene (Figure 1A). By measuring luciferase activity, we attempted to determine the best epigenetic suppressor for use in targeted gene silencing.


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)

Targeted suppression of the reporter gene by epigenetic suppressors. a. Schematic diagram of suppressor and reporter gene vectors. GAL4: the GAL4 DNA binding domain; GBS: GAL4-binding site; KRAB: kruppel-associated box domain; NSL: nuclear localization signal; pCMV: cytomegalovirus (CMV) promoter; PA: SV40 polyadenylation signal; Sss1: methyltransferase gene from Spiroplasma sp. strain MQ1; vSET: the histone H3 lysine 27 methyltransferase SET domain. Synthetic factors use the GAL4 domain to bind to the GBS site in the target gene vector, where the suppressor domain suppresses the activity of the downstream CMV promoter through epigenetic mechanisms. b. Relative expression of the reporter gene. 293 T cells were transiently co-transfected with 250 ng suppressor vectors, 250 ng luciferase target vector, and 25 ng thymidine kinase promoter-Renilla luciferase reporter (pRL-TK) control vector. The empty pcDNA3.1 vector was used as the study control. Forty-eight hours post-transfection, cells were harvested for luciferase assay. For comparison, the pcDNA3.1 control vector was adjusted to 100%. Each error bar represents the 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 1: Targeted suppression of the reporter gene by epigenetic suppressors. a. Schematic diagram of suppressor and reporter gene vectors. GAL4: the GAL4 DNA binding domain; GBS: GAL4-binding site; KRAB: kruppel-associated box domain; NSL: nuclear localization signal; pCMV: cytomegalovirus (CMV) promoter; PA: SV40 polyadenylation signal; Sss1: methyltransferase gene from Spiroplasma sp. strain MQ1; vSET: the histone H3 lysine 27 methyltransferase SET domain. Synthetic factors use the GAL4 domain to bind to the GBS site in the target gene vector, where the suppressor domain suppresses the activity of the downstream CMV promoter through epigenetic mechanisms. b. Relative expression of the reporter gene. 293 T cells were transiently co-transfected with 250 ng suppressor vectors, 250 ng luciferase target vector, and 25 ng thymidine kinase promoter-Renilla luciferase reporter (pRL-TK) control vector. The empty pcDNA3.1 vector was used as the study control. Forty-eight hours post-transfection, cells were harvested for luciferase assay. For comparison, the pcDNA3.1 control vector was adjusted to 100%. Each error bar represents the 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: To optimize the potency of different epigenetic suppressors, we constructed a target vector by inserting a GAL4-binding site cluster sequence (GBS) [19] upstream of a cytomegalovirus (CMV) promoter that drives the expression of the reporter luciferase gene (Figure 1A). By measuring luciferase activity, we attempted to determine the best epigenetic suppressor for use in targeted gene silencing.

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