14 DAY TRIAL // Biologists at Washington University in St. Louis have made an important breakthrough in understanding a pathway plant cells take to silence unwanted or extra genes using short bits of RNA. Basically, they have made it possible to see where, and how, the events in the pathway unfold within the cell, and seeing is believing, as the old saying goes.
Craig Pikaard, Ph.D., Washington University professor of biology in Arts & Sciences and his collaborators have described the roles that eight proteins in Arabidopsis plants play in a pathway that brings about DNA methylation, an epigenetic function that involves a chemical modification of cytosine, one of the four chemical subunits of DNA. Without proper DNA methylation, higher organisms from plants to humans have a host of developmental problems, from dwarfing in plants to certain tumors in humans, and death in mice.
One role of DNA methylation is to turn off repetitive genes, such as transposable elements that can move or spread throughout a genome and disrupt other gene functions if left unchecked. There is also interest in DNA methylation because understanding how some genes are selectively silenced and how silenced alleles can be turned on again may someday have practical benefits. For instance, tumor suppressor genes that normally help keep cells from dividing uncontrollably are often silenced by DNA methylation and histone (proteins that wrap DNA) modifications in cancer cells, contributing to tumor growth. And certain blood disorders resulting from defective genes expressed in adults might be alleviated if versions of those same genes that are only expressed very early in development, but are then silenced in adults, could only be turned on again.
"The pathway we are studying is part of an interesting phenomenon that occurs in plants, and reportedly in humans, too, called RNA-directed DNA methylation," Pikaard explained. "This pathway takes place in the nucleus, and it involves short RNAs, called small interfering RNAs -- siRNAs."
Those little tykes, just 24 nucleotides long, are somehow responsible for methylation of DNA sequences that match the sequence of the siRNAs, but not without a lot of help from their friends. The friends in this case are the team of eight known proteins of the RNA-directed DNA methylation pathway.
Using an impressive toolkit of sophisticated techniques, Pikaard and his collaborators not only have described the locations of the eight proteins in the pathway but also have provided the sequence of events that leads to methylation. It is a twisted and ultimately circular path, but Pikaard and his colleagues are the first researchers to literally see the pathway and thereby provide a clearer understanding of the steps leading to methylation and gene silencing.
A paradoxical aspect of the pathway is that siRNAs direct DNA methylation but DNA methylation is also required for the production siRNAs. "It's a circular pathway. You have to produce the siRNA in order to have them come back and methylate the loci, which somehow induce more siRNA production involving Pol IVa [RNA Polymerase IVa]". Pikaard said.
A combination of genetic mutants, transgenes, antibodies, RNA-FISH and DNA-FISH were the key to the study. "This toolkit is really powerful," Pikaard said. "It enabled us to look at a complex pathway and figure out not only the order of events but also the spatial organization of the pathway in the nucleus. Our hope for the future is to develop tools that will enable us to watch the pathway function in live cells using fluorescent proteins and time-lapsed microscopy, to learn even more."