Double-stranded siRNA targeted to the huntingtin gene does not induce DNA methylation.
RNA interference is an evolutionarily conserved mechanism of post-transcriptional gene silencing. Small interfering RNAs (siRNA) of 21-23 nucleotides generated from processing double-stranded RNA (dsRNA) by ribonuclease III, Dicer, are widely used for selective sequence-specific gene silencing in a broad range of organisms. In plants, siRNA is associated with de novo RNA-directed DNA methylation (RdDM) at the homologous target genomic region. To examine RdDM in somatic cells, human glioblastoma cell lines were treated with siRNAs homologous to the human huntingtin gene responsible for Huntington's disease. Methylation of CpG dinucleotides in the plasmid vectors expressing the dsRNAs and homologous genomic region was investigated by bisulfite-mediated genomic sequencing. Target regions of the siRNA in the huntingtin gene showed no significant change in the pattern of DNA methylation, and no CpG methylation was observed on the plasmid vectors. These results indicate that siRNA is not directly linked to DNA methylation at the target huntingtin genomic locus in human cells.
Park
Chang Won
CW
Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Chen
Zongyu
Z
Kren
Betsy T
BT
Steer
Clifford J
CJ
D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D020868:Gene Silencing; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D000071058:Huntingtin Protein; D008957:Models, Genetic; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012313:RNA