Transcriptional Gene Silencing in Nucleus Shown
A new gene-silencing technique that takes place in the nucleus of human cells, has been demonstrated by researchers at the University of California, San Diego (UCSD) School of Medicine and the VA San Diego Healthcare System. The technique, called transcriptional gene silencing (TGS), provides a new research tool to study gene function and, if continuing studies prove the concept, it could potentially become a method for therapeutic modification or the expression of disease-producing genes.
Selected for speedy publication in the August 5, 2004 edition of Science Express, the study describes, for the first time, the ability to shut down a gene literally before it is born in the nucleus of a cell. The benefit over previous gene-silencing techniques is that the nuclear version may have the potential to last considerably longer than current methods that act in the cytoplasm, the cellular area outside the nucleus.
The new technique, and older gene-silencing methods that have given rise in recent years to a multi-million dollar pharmaceutical industry, utilizes ribonucleic acid (RNA), the cousin of DNA. Specifically, researchers use synthetic, short pieces of RNA called short interfering RNA (siRNA), to shut down genes. The synthetic versions are patterned after naturally occurring siRNA in the body that may act as a defense against gene sequences that come from viruses or other genetic parasites.
The study’s senior author, David J. Looney, M.D., associate professor of medicine at UCSD and the VA San Diego Healthcare System, said the new technique provides a new tool for research investigation aimed at elucidating the effects of different genes, and has the potential to modify gene expression in disease, such as knocking out expression of genes required for tumor growth. He cautioned, however, that further studies are needed to prove the general applicability of this concept.
An understanding of siRNA begins with a look at theway by which genes work. First, a “promoter” region within the gene must be active in order to allow the genetic information encoded in the DNA to be copied (transcribed) into a single strand of RNA called messenger RNA (mRNA). During normal transcription, the mRNA leaves the nucleus and travels to the cytoplasm of the cell, where it works with another cellular component called the ribosome to make proteins.
Technology developed about four years ago introduced synthetic siRNA into the cytoplasm of cells to silence specific genes. This technique was called post-transcriptional gene silencing (PTGS). However, PTGS is transient, with siRNA lasting only a few days in the cytoplasm. Although this is enough time for short-term research projects, the use of siRNA for therapeutic applications, such as treatment for viral infections like HIV, probably require multiple siRNA treatments or the use of a gene therapy approach.
UCSD researchers used either lentiviral vectors (molecular ferries) to open up the nuclear membrane, or special transfection reagents which direct the transfected synthetic siRNA to the nucleus. This allowed siRNA access to the promoter, where it stopped the first part of the gene-making process called transcription, before it began. Previous research with siRNA used in the nucleus of plants has indicated that this effect can be long lasting, giving rise to the hope that it will be similarly long lasting in humans. Until now, however, scientists have been unable to detect activity of siRNA directed against gene promoters in the nucleus of human cells.
Kevin V. Morris, Ph.D., the study’s first author and a post-doctoral fellow in Looney’s lab, noted that “theoretically, one could envision targeting virtually any gene at the level of the promoter and silencing that gene. This has implications in most biological processes in which one would want to down regulate the expression of a gene, such as those genes involved in virus infections such as HIV, as well as human cancers and certain genetic disorders.”
In continuing studies, the Looney lab and others in the country will investigate this new method’s persistence within the human-cell nucleus, its successful targeting of human promoters, and whether it is feasible to use this technique to inhibit HIV or other viruses.
In addition to Looney and Morris, the authors were Simon W.-L. Chan, Ph.D., UCLA Department of Molecular, Cell and Developmental Biology; and Steven E. Jacobsen, Ph.D., UCLA Department of Molecular, Cell and Developmental Biology, and the UCLA Molecular Biology Institute.
The study was supported by the National Institutes of Health.