The structure behind the switch

USC researchers uncover mechanism of class- switching in antibodies

A team of scientists from the Keck School of Medicine of USC has, for the first time, described a new, stable DNA structure in both mouse and human cells-one which differs from the standard Watson-and-Crick double helix and plays a critical role in the production of antibodies, or immunoglobulins.
The research will be published online in the journal Nature Immunology this week, and will appear in print in the journal’s May issue.

“The way in which the five different immunoglobulin classes are created is a nearly perfect system,” notes Michael Lieber, M.D., Ph.D., professor of pathology and biochemistry and the study’s principal investigator. “And yet, the DNA mechanism for how a cell switches from producing one class to producing another has remained a mystery for almost 20 years.”

The typical antibody molecule is shaped like the letter Y. The region at the end of each of the two short arms houses the receptors that recognize and bind with a specific foreign object, or antigen. These receptors are created via a well-described cutting-and-splicing mechanism that occurs within the nuclear DNA of B cells, which are key components of the immune system.

The long stem, or handle, of the Y determines to which immunoglobulin class an antibody belongs. It, too, is created via a B-cell nuclear cut-and-paste job, but the mechanics here are much more complicated-and until now, much less well understood.

An immunoglobulin’s class is important because it determines where in the body the antibody’s efforts will be concentrated. While immunoglobulin M (IgM) works mostly in the bloodstream, for instance, IgG can easily slip through a capillary’s walls and cross the placenta, and IgA can make itself at home in the lungs, the digestive tract and the body’s secretions (saliva, sweat, tears).

Although antibodies are needed in all areas of the body, they all begin life as IgM, explains Kefei Yu, Ph.D., the paper’s first author and a research associate at the USC/Norris Comprehensive Cancer Center. In order to go where they’re needed, the antibodies need to change their class-to go from being IgM to being IgG or IgA or IgE or IgD.

By undergoing this so-called class switch, Lieber explains, the body can send “the same antibody missile to different areas of the body.”

The switch is made by cutting the DNA so that the code for IgM and any of the other class types that might precede the desired immunoglobulin class are abolished.

What Lieber, Yu and their colleagues have found is that, in order for such a cut to be made, the DNA that codes for the desired class must first form a stable, relatively permanent bond with the RNA strand that is transcribing it. Only when this aptly named R-loop is present can the DNA be cut and spliced to create an antibody of a different immunoglobulin class.

This is not the normal process by which DNA is cut. Usually, an enzyme cuts DNA based on a particular nucleotide sequence; the sequence acts as a signal to the enzyme, pointing to the precise place the cut is to be made. But in immunoglobulin class switching, Yu explains, there is no specific signaling sequence-instead, as the Keck School scientists proved in their paper, it is the mere physical presence of the R-loop that tells the enzymes where the cut is to be made. “The protein enzyme is not recognizing a sequence, but rather an altered DNA structure,” Yu says.

This is also not the normal process by which DNA is transcribed. Generally, DNA being transcribed serves as a template for the creation of a protein or enzyme. The double-stranded DNA separates, and then an RNA strand begins to pair up with each individual DNA nucleotide on one of those strands, creating a sort of mirror image of the DNA; this is the transcript. During this process, only the leading edge of the RNA remains bonded to the DNA nucleotides it’s transcribing. The rest of the RNA strand hangs off like the tail of a kite; when the RNA reaches the end of the stretch of DNA to be transcribed, the entire RNA strand drops away from the DNA and leaves the nucleus.

Not so in immunoglobulin production, says Yu. For one thing the part of the DNA that’s transcribed during immunoglobulin class switching doesn’t actually produce anything-it’s called a silent transcript. And for another, the RNA strand remains firmly attached to each and every DNA nucleotide it touches-creating a sort of permanent RNA sandwich, with the RNA between two strands of DNA, though only attached to one of them. That’s the R-loop. And it is what makes immunoglobulin class switching remarkable and unique.

“The whole process is more sophisticated than we first thought,” Yu remarks.

And it may also be more illuminating than they thought. According to Yu and Lieber, the discovery of the R-loop may shed light on the development of B-cell cancers like myelomas. “We believe something may be going wrong during this class-switching recombination event that activates an oncogene,” says Yu. “That is not proven yet, but it is something we will be looking at in the laboratory.”

Kefei Yu, Frederic Chedin, Chih-Lin Hsieh, Thomas E. Wilson, Michael R. Lieber, “R-loops at immunoglobulin class switching regions in the chromosomes of stimulated B cells.” Nature Immunology, www.nature.com/natureimmunology.