Gene expression pattern and downregulation of DNA methyltransferase 1 using siRNA in porcine somatic cells

Abstract

DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through their influence on gene expression. Manipulation of the DNA methylation marks of differentiated cells will allow a better understanding of the different molecular processes associated with chromatin structure and gene expression. The objective of this study was to identify small interfering RNAs (siRNAs) with the ability to reduce DNA methyltransferase 1 (Dnmt1) mRNA and consequently decrease Dnmt1 protein as well as DNA methylation in porcine cells. Fibroblasts from four porcine fetuses were established and cultured in 5% CO2 in air at 38 degrees C. Optimal transfection conditions were evaluated using a FITC-labeled control siRNA. Four Dnmt1-specific siRNAs were evaluated upon transfection of each cell line. A nonsilencing siRNA was used as a negative control. The expression patterns of Dnmt1 were analyzed by Q-PCR. The combination of 1 microg of siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Downregulation of Dnmt1 varied between siRNAs. Transfection of porcine cells with highly effective siRNAs resulted in a drastic reduction of Dnmt1 mRNA and a slight decrease in protein production. However, this small reduction in the protein concentration induced significant genomic hypomethylation. These data suggest that although Dnmt1 mRNA abundance plays an important role during protein regulation, Dnmt1 enzyme is mainly posttranscriptionally regulated. Subsequent use of these cells for cloning, differentiation, and cancer studies will provide insight as to how methylation of the DNA affects genomic reprogramming.

Figure 1

Specificity of the anti-Dnmt1 antibody in porcine fibroblast cells. Analysis of porcine fibroblasts (PF) by Western blot demonstrated that the antibody binds to a protein of the expected molecular weight for Dnmt1 (A). PF cells incubated with the anti-Dnmt1 and labeled with an Alexa Fluor 488 antibody showed, as predicted, the specificity of the antibody for nuclear epitopes (B).

Figure 2

Porcine fibroblasts stained with propidium iodide (A). Cells incubated with anti-5-methylcytidine antibody and labeled with Alexa Fluor 488 (B). The staining pattern shows the specificity of the antibodies for nuclear structures and their affinity for areas of highly compacted chromatin.

Figure 3

Relative level of Dnmt1 mRNA (A) and methylated DNA in porcine fibroblast cells (B). Different letters indicate significant difference in the level of gene expression of Dnmt1 or DNA methylation after seeding (p < 0.05). Solid and dotted arrow indicates that cells reached 80% and 100% confluence, respectively. Bars indicate SEM.

Figure 4

Gene expression of Dnmt1 in PF1, PF2, PF3, and PF4 transfected with siRNA1, siRNA2, siRNA3, or siRNA4. Gray and black columns represent the level of gene expression of Dnmt1 in control and siRNA-treated cells, respectively. Different letters indicate significant difference in the expression level of Dnmt1 over time. An asterisk indicates significant difference in the level of Dnmt1 between cells transfected with specific or nonsilencing siRNA at every given time point (p < 0.05). Bars indicate SEM.

Figure 5

Relative levels of Dnmt3a and Dnmt3b RNA of PF1, PF2, and PF4 transfected with siRNA4, siRNA3, and siRNA2, respectively. Gray and black columns represent the relative level of the transcript in control and siRNA-treated cells, respectively. Different letters indicate significant difference in the level of the transcript between cells transfected with specific or nonsilencing siRNA at every given time point (p < 0.05). Bars indicate SEM.

Figure 6

Relative levels of DNA methylation of PF1, PF2, and PF4 transfected with siRNA4, siRNA3, and siRNA2, respectively. Gray and black columns represent the DNA methylation level of control and siRNA-treated cells, respectively. Different letters indicate significant difference in the level of methylated DNA over time. An asterisk indicates significant difference in level of DNA methylation between cells transfected with specific or nonsilencing siRNA at every given time point (p < 0.05). Bars indicate SEM. AUF, arbitrary units of fluorescence.