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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

DNA methylation of the CMV promoter. a. The schematic diagram of the CMV promoter and the location of cytosine-phosphate-guanine dinucleotide (CpG) islands. After treatment with sodium bisulfite, genomic DNA was amplified with PCR primer JH1351 and JH1370. Red bar: CpG islands that were sequenced. b. DNA methylation of the CMV promoter using methylation-specific PCR (MSP). Stable clone cells that have the genomically integrated GBS-pCMV-copGFP were transiently transfected with synthetic suppressor vectors. Genomic DNA was extracted and amplified with primers that specifically recognize the methylated CpGs (top panel). Total genomic DNAs were amplified with primers that recognize both unmethylated and methylated CpGs. c. Efficiency of de novo DNA methylation by synthetic suppressors. Stable GBS-pCMV-copGFP clone cells were transiently transfected with 1 μg suppressor vectors. Forty-eight hours post-transfection, cells were harvested for bisulfate sequencing. DNA methylation was calculated as the average percentage of methylated CpGs/(methylated CpGs + unmethylated CpGs) from five CpG islands (110, 122, 141, 165, and 174). *P <0.05 as compared with cells transiently transfected with pcDNA3.1 empty vector.
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Figure 4: DNA methylation of the CMV promoter. a. The schematic diagram of the CMV promoter and the location of cytosine-phosphate-guanine dinucleotide (CpG) islands. After treatment with sodium bisulfite, genomic DNA was amplified with PCR primer JH1351 and JH1370. Red bar: CpG islands that were sequenced. b. DNA methylation of the CMV promoter using methylation-specific PCR (MSP). Stable clone cells that have the genomically integrated GBS-pCMV-copGFP were transiently transfected with synthetic suppressor vectors. Genomic DNA was extracted and amplified with primers that specifically recognize the methylated CpGs (top panel). Total genomic DNAs were amplified with primers that recognize both unmethylated and methylated CpGs. c. Efficiency of de novo DNA methylation by synthetic suppressors. Stable GBS-pCMV-copGFP clone cells were transiently transfected with 1 μg suppressor vectors. Forty-eight hours post-transfection, cells were harvested for bisulfate sequencing. DNA methylation was calculated as the average percentage of methylated CpGs/(methylated CpGs + unmethylated CpGs) from five CpG islands (110, 122, 141, 165, and 174). *P <0.05 as compared with cells transiently transfected with pcDNA3.1 empty vector.

Mentions: We used several epigenetic approaches to examine how these suppressors inhibit expression of pCMV reporters. We first compared the status of DNA methylation in treated cells using sodium bisulfite sequencing (Figure 4A).In the control cells that received only the reporter vector, there was minimal DNA methylation of CpG dinucleotides near the transcription initiation site (17.6 to 27.5%). In three groups of cells that contain the CpG methylase domain Sss1 insert, there was an increase in CpG methylation of 40 to 60% (Figure 4B, C).


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)

DNA methylation of the CMV promoter. a. The schematic diagram of the CMV promoter and the location of cytosine-phosphate-guanine dinucleotide (CpG) islands. After treatment with sodium bisulfite, genomic DNA was amplified with PCR primer JH1351 and JH1370. Red bar: CpG islands that were sequenced. b. DNA methylation of the CMV promoter using methylation-specific PCR (MSP). Stable clone cells that have the genomically integrated GBS-pCMV-copGFP were transiently transfected with synthetic suppressor vectors. Genomic DNA was extracted and amplified with primers that specifically recognize the methylated CpGs (top panel). Total genomic DNAs were amplified with primers that recognize both unmethylated and methylated CpGs. c. Efficiency of de novo DNA methylation by synthetic suppressors. Stable GBS-pCMV-copGFP clone cells were transiently transfected with 1 μg suppressor vectors. Forty-eight hours post-transfection, cells were harvested for bisulfate sequencing. DNA methylation was calculated as the average percentage of methylated CpGs/(methylated CpGs + unmethylated CpGs) from five CpG islands (110, 122, 141, 165, and 174). *P <0.05 as compared with cells transiently transfected with pcDNA3.1 empty vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: DNA methylation of the CMV promoter. a. The schematic diagram of the CMV promoter and the location of cytosine-phosphate-guanine dinucleotide (CpG) islands. After treatment with sodium bisulfite, genomic DNA was amplified with PCR primer JH1351 and JH1370. Red bar: CpG islands that were sequenced. b. DNA methylation of the CMV promoter using methylation-specific PCR (MSP). Stable clone cells that have the genomically integrated GBS-pCMV-copGFP were transiently transfected with synthetic suppressor vectors. Genomic DNA was extracted and amplified with primers that specifically recognize the methylated CpGs (top panel). Total genomic DNAs were amplified with primers that recognize both unmethylated and methylated CpGs. c. Efficiency of de novo DNA methylation by synthetic suppressors. Stable GBS-pCMV-copGFP clone cells were transiently transfected with 1 μg suppressor vectors. Forty-eight hours post-transfection, cells were harvested for bisulfate sequencing. DNA methylation was calculated as the average percentage of methylated CpGs/(methylated CpGs + unmethylated CpGs) from five CpG islands (110, 122, 141, 165, and 174). *P <0.05 as compared with cells transiently transfected with pcDNA3.1 empty vector.
Mentions: We used several epigenetic approaches to examine how these suppressors inhibit expression of pCMV reporters. We first compared the status of DNA methylation in treated cells using sodium bisulfite sequencing (Figure 4A).In the control cells that received only the reporter vector, there was minimal DNA methylation of CpG dinucleotides near the transcription initiation site (17.6 to 27.5%). In three groups of cells that contain the CpG methylase domain Sss1 insert, there was an increase in CpG methylation of 40 to 60% (Figure 4B, C).

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