While transcriptional gene silencing has been observed in plants, its existence in mammals has heretofore remained undemonstrated. However, a report in this week’s issue of Science has changed that.
Spurred on by the research of others, David Looney, Kevin Morris (who is now working at the Beckman Research Institute), and colleagues from the University of California, San Diego have conducted and published experiments indicating that promoter-directed siRNAs can inhibit transcription of an integrated proviral EF1A promoter-GFP reporter gene, as well as transcription of endogenous EF1A.
“There’s a group in Australia [at St. Vincent’s Hospital] under K. Suzuki … and they haven’t published this data but they presented a poster [at a conference in 2002] showing what they thought was transcriptional gene silencing of the 5’ LTR of HIV,” Morris told RNAi News, commenting on how his research came about.
Having spent much time working on HIV/AIDS, and with an interest in inducing TGS in mammalian cells, Morris said he met with Suzuki to discuss the Australian group’s work. “They were targeting the promoter of HIV, and the promoter of HIV is also found within the transcript of HIV,” he said. “They had these siRNAs targeting the LTR, and they were seeing a thousand-fold knockdown of HIV.
“It became pretty clear that something was going on that was different than what we’ve seen with siRNAs targeting the mRNA … but it was difficult to tease apart whether they were seeing TGS or PTGS,” Morris added. As a result, he began his own research to determine whether TGS occurs in human cells.
According to the Science paper, Morris et al. “chose to target an EF1A promoter-GFP reporter gene integrated into the genome of human 293FT cells with a lentiviral feline immunodeficiency virus vector. SiRNA EF52 is homologous to an EF1A promoter sequence essential for transcription,” they noted in the paper.
“A second siRNA homologous to exon 2 of the GFP coding region was designed to target post-transcriptional mRNA destruction,” the UCSD researchers wrote. “We transduced 293FT cells with the EF1A-GFP vector, allowed 24 hours for integration, then transfected with either EF52, GFP, or a control siRNA matching human CCR5.”
Forty-eight hours later, the researchers found that the siRNAs targeting the GFP mRNA transcript “reduced expression relative to the control as measured by quantitative ... RT-PCR. Potent inhibition of GFP expression was also seen with siRNA EF52 targeting the EF1A promoter,” Morris and colleagues wrote in the Science paper.
“The mRNA was extremely potent — we saw huge knockdown,” Morris said. “We saw this in 293 cells. We also did it in human [peripheral blood mononuclear cells] — that data is not presented in the paper — and saw a knockdown, as well.”
Further analysis confirmed that the inhibition was occurring at the promoter level. Additionally, the silencing was associated with DNA methylation of the targeted sequence, and the researchers determined that the silencing required either active transport of the siRNAs into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction.
While the data suggest that siRNA-directed TGS is conserved in mammals, Morris stressed that it remains unclear “how conserved it is across all promoters. Does it work against … any other promoter for any other gene? We don’t know, but it does open up an avenue to suggest that there’s a pathway conserved within the nucleus that allows this to occur,” he said.
Morris noted that questions have been raised as to why people working with shRNAs targeting promoters haven’t seen significant DNA methylation or TGS. “It turns out there’s this enzyme called Drosha which apparently works with Exportin-5 to bind and pull hairpin siRNAs out of the nucleus for processing by Dicer,” he said. “Our siRNAs were just sense and antisense constructed through the Ambion methodology and transfected directly into the cells. This suggests that if you are going to use this in the future, you have to have your siRNAs expressed from separate promoters.”
One of those researchers working with shRNAs is John Rossi, also of the Beckman Research Institute, who has a paper on the subject under review at a peer-reviewed journal. “We looked at the idea that siRNAs could actually target site-specific methylation of DNA based on what was known in plants,” Rossi told RNAi News.
He said he used the RASSF1A tumor-suppressor gene, which is shut off by methylation in almost every primary tumor but is un-methylated and active in HeLa cells, and “just expressed short-hairpin RNAs covering various parts of the promoter region, and were able to detect region-specific methylation directed by the siRNAs.”
Rossi said that the processing mechanism of the RNAs remains unknown, but noted that the observations of TGS using promoter-directed RNAs made by Morris and colleagues are nonetheless important.
“Nobody understands the mechanism by which de novo methylation takes place, and a very selective methylation takes place during aging, development, genomic imprinting, and even X-chromosome inactivation,” he said. “It’s thought that, perhaps, RNA that’s transcribed through promoter regions, for instance, can initiate [these events].”
Rossi, who is now working with Morris, noted that he is planning on looking into proteins in the nucleus that bind siRNAs to see if “we can identify Argonaute family members that might be involved in this process.”