Related Experiment Videos
Inhibition of poly(ADP-ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA
Abstract:
The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.
Insights
Poly(ADP-ribosyl)ation protects DNA from methylation. Inhibiting this process with 3-aminobenzamide causes hypermethylation of foreign DNA and reduces gene expression, suggesting new experimental strategies.
Area of Science:
- Molecular Biology
- Epigenetics
- Biochemistry
Background:
- Poly(ADP-ribosyl)ation is a crucial post-translational modification involved in DNA repair and genomic stability.
- DNA methylation, particularly at CpG islands, plays a significant role in gene regulation and epigenetic control.
- Understanding the interplay between poly(ADP-ribosyl)ation and DNA methylation is essential for comprehending cellular processes and disease mechanisms.
Purpose of the Study:
- To investigate the influence of poly(ADP-ribosyl)ation on the methylation status of foreign DNA (plasmid) transfected into mammalian cells.
- To determine if inhibiting poly(ADP-ribosyl)ation affects the methylation pattern of the transfected plasmid DNA.
- To assess the functional consequences of altered DNA methylation on reporter gene expression.
Main Methods:
- Transfection of L929 mouse fibroblast cells with a pVHCk plasmid containing the chloramphenicol acyltransferase (CAT) gene.
- Inhibition of poly(ADP-ribosyl)ation using 3-aminobenzamide treatment.
- Bisulfite sequencing to analyze the methylation status of cytosine residues on the plasmid DNA.
- Quantification of CAT reporter gene expression.
Main Results:
- Transfected plasmid DNA remained unmethylated in control cells but exhibited significant methylation in cells treated with 3-aminobenzamide.
- Inhibition of poly(ADP-ribosyl)ation led to the acquisition of new methyl groups on the plasmid DNA.
- Increased DNA methylation on the plasmid correlated with a decrease in CAT reporter gene expression.
Conclusions:
- Poly(ADP-ribosyl)ation plays a protective role against the hypermethylation of both genomic and transfected DNA.
- Inhibition of poly(ADP-ribosyl)ation can induce DNA hypermethylation, potentially mimicking pathological conditions.
- 3-aminobenzamide serves as a valuable tool for experimentally inducing DNA hypermethylation and studying its effects.