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Silencing of the Genes
By Marina Chicurel

Featured Article.

You can't always judge a gene by its sequence. Without suffering a single mutation, the VHL gene, for example, can predispose its host to cancer of the kidney, pancreas, eye, brain, spinal cord, and adrenal glands when parts of the gene become decorated with molecules known as methyl groups. Von Hippel-Lindau disease, or VHL, is an inherited disease in which tumors develop in one or more parts of the body. Once methylated, the gene becomes silent and stops performing its normal function of cell growth.

‘Its potential is absolutely tremendous.’

According to a recent study published in Nature Genetics, aberrant methylations seem to be scattered all over the genomes of cancer cells. Joseph Costello, of the Ludwig Institute for Cancer Research in Melbourne, Australia, suggests that a typical human tumor cell carries about 600, and as many as 4,500, abnormal methylations in DNA sequences that are frequently associated with controlling gene activity. Although a clear causal link between methylation and cancer exists for only a handful of tumors, some researchers speculate that abnormal methylation or gene-silencing could prove as important as mutations in causing cancer.

It is not surprising that gene silencing gone awry may have such dire consequences. When it works normally, gene silencing acts as a master coordinator of normal development. Silencing is passed from generation to generation of dividing cells, conferring a molecular memory of the genes that cells turn off as they acquire more specialized functions. Gene silencing also seems to help protect genomes from foreign invaders. A spate of new studies is revealing gene silencing's far-ranging effects, adding a new layer of complexity to our understanding of the way genes work. The research is also offering new ways of manipulating genes to advance basic science, improve agricultural products, and treat disease. "Its potential is absolutely tremendous," says Moshe Szyf, a pharmacologist and cancer researcher at McGill University in Montreal, Canada.

Researchers have implicated genes that control silencing in several genetic diseases such as Rett's syndrome, one of the major causes of mental retardation in women. In addition, new studies are bolstering the link between cancer and aberrant gene silencing. Several studies, for example, have shown that DNA methylation can act as an early trigger for cancer by contributing to the inactivation of tumor suppressor genes such as VHL.

The mottling exhibited by some of these kernels indicates partial silencing of the color gene. Over three generations, the gene becomes more and more silenced, leading to lighter and lighter mottling. No changes take place in the DNA sequence.

Mis-targeted methylation can also contribute to cancer by paving the way for genetic mutations. In some cancers, researchers have observed abnormal methylation in genes that repair DNA, suggesting that cells could become cancerous when they accumulate mutations due to the lack of repair enzymes.

Stephen Baylin, of the Johns Hopkins University School of Medicine, in Baltimore, Maryland, thinks that as much as half of the alterations in cancer could be related to gene silencing. "Over the last 5 years there's an emerging awareness that the epigenetic changes in cancer may be as frequent as the genetic changes," he says.

But others feel it is too early to speculate. Robert Weinberg, of the Whitehead Institute at the Massachusetts Institute of Technology, remains skeptical of the role of gene silencing in cancer. He acknowledges that a few very clear examples have been discovered, but questions whether these are rare or the tip of an iceberg. "It's just impossible to know at this stage," he says.

But Baylin and colleagues are so convinced of the importance of silencing in cancer, that they have begun developing therapies to block it. In collaboration with Thomas Wolffe, of the National Institute of Child Health and Human Development, and researchers at the National Cancer Institute, Baylin is setting up clinical trials to test the effects of drugs that inhibit gene silencing on cancer patients. Based on Baylin's findings in isolated tumor cells, the researchers plan to use 5-azacytidine, a blocker of methylation, together with phenyl butyrate, a blocker of histone deacetylation, to re-activate the patients' silenced tumor suppressor genes. Baylin has begun a clinical trial in patients with leukemia and myelodysplastic syndrome who have failed previous treatment. A future trial will target patients with all solid tumor types. Wolffe is recruiting patients with non-small-cell lung carcinoma and small-cell lung cancer for his trial.

So much of the information of life is packed into the genome's sequence that, in the past three decades, biologists have conducted numerous studies of DNA from this perspective. At the same time, a growing number of observations have piled up that cannot be explained through sequences alone. For example, the same pigment gene that colors one corn plant's kernels with solid purple will sometimes barely tinge the kernels of a genetically identical plant.

This has spurred interest in understanding the chemical modifications, chromosome packaging and the function of RNAs—the molecules that carry out the instructions encoded in DNA—that affect a gene's behavior.

One common theme in gene silencing is that it relies on the modification of DNA, RNA, or both. Gene silencing at the DNA level is called transcriptional gene silencing, or TGS. TGS often begins when transcription factors—proteins that regulate gene activity by recognizing specific sequences—bind and temporarily block RNA production. These factors then attract a group of proteins that convert the inhibition into long-term silencing. Through a chemical modification called deacetylation, DNA-packaging proteins, or histones, re-package the gene to conceal it from activating proteins. Then, acting as a photographic fixative, methylation seals the gene's fate.

But, as explained by Adrian Bird, of the University of Edinburgh, Scotland, there are variations to this basic theme. In some organisms, for example, methylation is not part of the silencing process. In other cases, methylation seems to act as the developer rather than the fixative, guiding histone deacetylation and chromosome remodeling. One possibility is that histone deacetylation and DNA methylation are part of a self-propagating cycle that helps insure that once a gene is switched off, it stays off.

Although researchers argue about the details, most agree this general scheme is at the core of how some cells become skin cells and others become nerve cells during development. As cells become specialized for their particular jobs in the adult organism, they must turn many genes off. A cell destined to become a skin cell must turn off nerve-specific genes, for example. And it must also ensure that its daughter cells follow its lead. In other words, cell development requires cells to have a memory—to remember the specialization path they have chosen and pass this information on to their offspring. Not surprisingly, in addition to gene silencing mechanisms, cell development makes use of gene activating mechanisms to keep certain genes continuously active.

Preventing a gene from producing RNA is one way of silencing a gene, but preventing its RNA from performing its functions can be just as potent. This general form of silencing is called PTGS, post-transcriptional gene silencing. As with TGS, the mechanisms of PTGS are still not well understood. In general, however, PTGS occurs when RNA molecules trigger the degradation of other RNAs that contain similar sequences. Sometimes RNA molecules may even affect DNA directly.

Two features of PTGS—signal amplification and action at a distance—have sparked much excitement among researchers, especially genetic engineers. In the roundworm C. elegans, for example, scientists have silenced genes that produce many RNAs by injecting only a handful of RNA silencers. Even more remarkable, the RNA injected into the tail has silenced genes as far away as the head. In an article in Nature Cell Biology, Julia Bosher and Michel Labouesse, of the INSERM in France, call this form of PTGS a genetic magic wand because "it acts at a distance, is powerful, and is still mysterious."

Several researchers are now trying to harness the power of PTGS to elucidate the functions of genes. For several years, biologists have relied on genetic knockouts, particularly in mice, to tease out the roles of individual genes within an organism. But creating these knockouts is time-consuming and expensive, and knockouts can be made only in a few organisms. With PTGS, biologists may soon be able to more readily knock out gene function in organisms ranging from single-celled parasites to mice.

As the molecular details of gene silencing begin to emerge, it appears that a shared set of principles governs silencing in such diverse processes as cell development and viral defense, in organisms ranging from yeast to mice. "People are very excited by the unification of such disparate information," says Wolffe.

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Bosher, J.M. & Labouesse, M. RNA interference: Genetic wand and genetic watchdog. Nat Cell Biol 2, E31-E36 (February 2000).
Costello, J.F. et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet 24, 132-138 (February 2000).
Jones, P.A. & Laird, P.W. Cancer epigenetics comes of age. Nat Genet 21, 163-167 (February 1999).
Jorgensen, R.A. et al. An RNA-based information superhighway in plants. Science 279, 1486-1487 (March 6, 1998).
Lyko, F. & Paro, R. Chromosomal elements conferring epigenetic inheritance. BioEssays 21, 824-832 (October 1999).
Ng, H.-H. & Bird, A. DNA methylation and chromatin modification. Curr Opin Genet Dev 9, 158-163 (April 1999).
Wolffe, A.P. & Matzke, M.A. Epigenetics: Regulation through repression. Science 286, 481-486 (October 15, 1999).

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