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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

Promoter histone marks and heterochromatin factor HP1a binding. Chromatin immunoprecipitation (ChIP) assay was performed with anti-trimethyl H3K4 (A), anti-trimethyl H3K9 (B), anti-dimethyl H3K27 (C) and anti-HP1a (D) antibodies. Input DNA of each group was amplified by real-time qPCR as a positive control. Stable copGFP clone cells were transiently transfected with 1 μg blank pcDNA3.1 or suppressor vectors. Forty-eight hours post-transfection, cells were harvested for ChIP assay. a: P <0.05 versus pcDNA3.1 blank vector; b P <0.05 versus KRAB group; and c P <0.05 versus Sss1 group.
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Figure 5: Promoter histone marks and heterochromatin factor HP1a binding. Chromatin immunoprecipitation (ChIP) assay was performed with anti-trimethyl H3K4 (A), anti-trimethyl H3K9 (B), anti-dimethyl H3K27 (C) and anti-HP1a (D) antibodies. Input DNA of each group was amplified by real-time qPCR as a positive control. Stable copGFP clone cells were transiently transfected with 1 μg blank pcDNA3.1 or suppressor vectors. Forty-eight hours post-transfection, cells were harvested for ChIP assay. a: P <0.05 versus pcDNA3.1 blank vector; b P <0.05 versus KRAB group; and c P <0.05 versus Sss1 group.

Mentions: The 3xKRAB suppressing domain, which uses a different mechanism to inhibit gene activity, also slightly increased CpG DNA methylation as compared with the reporter vector group. When fused with Sss1, CpG DNA methylation significantly increased, presumably reflecting the role of the DNA methylase activity. The induced de novo DNA methylation was also confirmed by bisulfite sequencing of a proximal CMV promoter fragment (Additional file 1: Figure S1).We then used a chromatin immunoprecipitation (ChIP) assay to examine promoter histone modifications in the three treatment groups (Sss1, KRAB, and Sss1/KRAB). We focused on histone H3 methylation at lysines 4, 9, and 27 (H3K4, H3K9, and H3K27). H3K4 is associated with an active promoter. We found that treatment with KRAB or Sss1/KRAB significantly reduced H3K4 methylation. Sss1 alone also decreased the level of H3K4 methylation (Figure 5A).Both H3K9 methylation and H3K27 methylation are suppressive markers on gene promoters. Transfection of the KRAB construct enhanced these two suppression signals in the CMV promoter (Figure 5B, C). Surprisingly, Sss1 was the strongest inducer of H3K27 methylation (Figure 5C), in addition to its DNA methylase activity. The vSET domain (2xvSET) increased H3K9 and H3K27 methylation marks in the gene promoter (Figure 5B, C).Heterochromatin Protein 1a (HP1a) functions as an epigenetic ‘gatekeeper’ to inhibit gene activity by binding to H3K9me marks. Using ChIP we found that Sss1, but not KRAB, induced the binding of HP1a to the gene promoter. The KRAB-Sss1 fusion protein recruited HP1a to the promoter at an intermediate level (Figure 5D).


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)

Promoter histone marks and heterochromatin factor HP1a binding. Chromatin immunoprecipitation (ChIP) assay was performed with anti-trimethyl H3K4 (A), anti-trimethyl H3K9 (B), anti-dimethyl H3K27 (C) and anti-HP1a (D) antibodies. Input DNA of each group was amplified by real-time qPCR as a positive control. Stable copGFP clone cells were transiently transfected with 1 μg blank pcDNA3.1 or suppressor vectors. Forty-eight hours post-transfection, cells were harvested for ChIP assay. a: P <0.05 versus pcDNA3.1 blank vector; b P <0.05 versus KRAB group; and c P <0.05 versus Sss1 group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Promoter histone marks and heterochromatin factor HP1a binding. Chromatin immunoprecipitation (ChIP) assay was performed with anti-trimethyl H3K4 (A), anti-trimethyl H3K9 (B), anti-dimethyl H3K27 (C) and anti-HP1a (D) antibodies. Input DNA of each group was amplified by real-time qPCR as a positive control. Stable copGFP clone cells were transiently transfected with 1 μg blank pcDNA3.1 or suppressor vectors. Forty-eight hours post-transfection, cells were harvested for ChIP assay. a: P <0.05 versus pcDNA3.1 blank vector; b P <0.05 versus KRAB group; and c P <0.05 versus Sss1 group.
Mentions: The 3xKRAB suppressing domain, which uses a different mechanism to inhibit gene activity, also slightly increased CpG DNA methylation as compared with the reporter vector group. When fused with Sss1, CpG DNA methylation significantly increased, presumably reflecting the role of the DNA methylase activity. The induced de novo DNA methylation was also confirmed by bisulfite sequencing of a proximal CMV promoter fragment (Additional file 1: Figure S1).We then used a chromatin immunoprecipitation (ChIP) assay to examine promoter histone modifications in the three treatment groups (Sss1, KRAB, and Sss1/KRAB). We focused on histone H3 methylation at lysines 4, 9, and 27 (H3K4, H3K9, and H3K27). H3K4 is associated with an active promoter. We found that treatment with KRAB or Sss1/KRAB significantly reduced H3K4 methylation. Sss1 alone also decreased the level of H3K4 methylation (Figure 5A).Both H3K9 methylation and H3K27 methylation are suppressive markers on gene promoters. Transfection of the KRAB construct enhanced these two suppression signals in the CMV promoter (Figure 5B, C). Surprisingly, Sss1 was the strongest inducer of H3K27 methylation (Figure 5C), in addition to its DNA methylase activity. The vSET domain (2xvSET) increased H3K9 and H3K27 methylation marks in the gene promoter (Figure 5B, C).Heterochromatin Protein 1a (HP1a) functions as an epigenetic ‘gatekeeper’ to inhibit gene activity by binding to H3K9me marks. Using ChIP we found that Sss1, but not KRAB, induced the binding of HP1a to the gene promoter. The KRAB-Sss1 fusion protein recruited HP1a to the promoter at an intermediate level (Figure 5D).

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