By David N. Leff
Before the immune system's antibody-generating B cells can send their myriad sleuths out after alien antigens, they have to matriculate at Foreign Service School.
Crowning its curriculum is a course called Class Switching. Before qualifying to enroll in this crucial subject, the freshman B cell has to come to grips with its first antigen. After that rite of passage, the hazed B cell signs up for one of four classes, which will determine where in the body it will be assigned.
In class switching, the now-toughened antibodies (also known as immunoglobulins, or Ig) are posted to one of four membrane-protected sites in the body to pursue their pathogen-hunting missions. Antibodies are shaped like the letter "Y." To apprehend their prey, two forked tops of the Y terminate in variable-region pockets. These active sites vary to match the one target among many the mature antibody will finger. Then it switches class accordingly, as managed by the Y's single stem or handle - its constant region. It sends immunoglobulin receptors out to different parts of the body.
Molecular biologist and immunologist Michael Lieber, at the University of Southern California (USC) in Los Angeles, explained: "IgM targets the blood cells. IgG is good for slipping across capillary membranes, or crossing a placenta, and IgA for crossing mucosal membranes - like the gastrointestinal tract, the genitourinary tract, salivary, sweat and tear glands - as well as going into the lungs. And IgE deals with parasites and allergens. That class switching," he continued, "is what this is all about."
Introducing RNA/DNA Hybrid Structures
Lieber, a professor of pathology and biochemistry at USC, is senior author of a paper in today's issue of Science, dated May 12, 2000. Its title: "Stable RNA/DNA hybrids in the mammalian genome: Inducible intermediates in immunoglobulin switch recombination." Accompanying this article is a commentary titled "A touch of antibody class," by molecular geneticist and microbiologist Janet Stavnezer at the University of Massachusetts Medical School in Worcester.
"Class switching is important for people, and for mice of course," Stavnezer told BioWorld Today, "to make antibodies that can clear pathogens. If they can't do that, they can make only IgM, and that isn't as effective as IgA or IgG or IgE. So that's the first thing.
"The second thing," she went on, "is that we don't understand the mechanism of this process. We understand somewhat how it's regulated - how cells decide whether they're going to make IgE or IgG, for example. But we don't understand the mechanism of that regulation.
"We've known for a while," Stavnezer continued, "that RNA is transcribed from the constant-region genes prior to switching - and that these transcripts are needed for switching - but we don't know why. Now Lieber's paper provides a mechanistic explanation for why the transcripts are needed. It doesn't completely solve the question, but it moves us a lot further toward understanding their possible role.
"These RNA/DNA hybrids that the paper's about," Stavnezer explained, "are formed at switch regions on B-cell chromosomes. Now Lieber shows that the RNaseH enzyme transgene will digest away those RNA/DNA hybrids, and thereby reduce class switching. So that's the first evidence that these hybrids are important for class switching. And - another first - that they are present in cell nuclei."
Stavnezer described how this unique, class-switching RNA/DNA hybrid is formed: "The RNA starts upstream of the switch region, and is transcribed through that region. But then that RNA sticks with the DNA. Usually," she pointed out, "when transcripts are made, they fall off the DNA. This one doesn't because of the switch region. For some reason the RNA does not dissociate after transcription from the DNA. So that then prevents the other DNA strand from snapping back, and going back to the DNA duplexes that are normally present after an RNA transcript is synthesized. So that leaves what we call an R-loop.
"At some point," she pointed out, "that switch region - both strands of the DNA - the one that would be single stranded, and the other being the RNA/DNA hybrid, would be cut by an endonuclease. Lieber proposes," she observed, "that the RNA/DNA hybrid serves as a target for the endonuclease, which initiates the switch recombination."
Lieber and his co-authors made a transgenic mouse that expresses RNaseH, an enzyme that destroys this R-loop structure. They showed that the animal had difficulty undergoing class switching.
Attention Virologists, Immunologists, Companies
"This RNA mouse that we made," he suggested, "might be of interest to people in the retroviral community, because those retroviruses also have RNA/DNA hybrid structures like this R-loop. And our transgenic mouse that expresses the RNaseH gene, might attack the retroviral intermediates and alter their ability to propagate.
"It will certainly be of interest to people in the immunological field," Lieber added, "because they might be able to alter the immune response, using a strategy we report. Individuals who want to change the spectrum of IgG, A or E relative to IgM might wish to take advantage of the knowledge in this Science paper about how class switching occurs. It's a mechanism that's been pretty mysterious for almost 20 years really, and this provides some insights on that.
"There might be certain infectious agents or allergens where IgM would be more useful that IgA, E or G as a way of neutralizing the antigens. There are other ways a company could do that kind of thing," Lieber observed, "but I think this is worth drawing it to their attention as one way to achieve that."
USC, he said, "has applied for patents on our transgenic mouse that expresses RNaseH, as well as on the RNA/DNA hybrid - just the fact that it exists in the chromosome. It turns out," he concluded, "that Burkitt's lymphoma - one type of B-cell cancer - represents a chromosomal translocation to these fragile, unusual R-loop sites, and our current work is directed toward that."