Researchers in the laboratory of Frederick Alt of the Howard Hughes Medical Institute and Program in Cellular and Molecular Medicine (PCMM) at Children's Hospital Boston have made important strides towards resolving a long-standing question about how different classes of antibodies are made. Led by Junchao Dong, Rohit A. Panchakshari, and Tingting Zhang in collaboration with teams at several other major research centers, the Alt lab adapted a highly specific and sensitive genetic assay they originated in 2011 to propose the existence of a guiding principle behind the production of viable antibodies by lymphocytes called B cells.

These findings, published online in Nature on August 26, 2015, suggest that a novel mechanism guides the directionality of antibody class switch recombination (CSR), a crucial process in immunity.

The Alt lab has a long track record of research on the two main ways antigen-activated B cells make diverse antibodies to respond to the widest possible range of antigens: Somatic hypermutation mutates the antigen binding variable region encoding exon to greatly increase the affinity and specificity of B cell antibodies; CSR changes the antibody constant regions exon that specifies how the antibody attacks and eliminates a pathogen.

Specifically, CSR takes place through a DNA breakage-and-joining mechanism: the antibody gene's DNA is broken by an enzyme in (at least) two places: 1) in a switch region just downstream of the DNA encoding the antigen-binding antibody variable region and 2) farther down the chromosome, within switch regions that flank alternating gene segments that encode different constant regions. When DNA is excised from between the two breaks and the newly exposed ends are rejoined, the resulting gene produces a functional antibody that is not only specific to an antigen, but further customized to act in certain locations in the body and to recruit specific immune cells and molecules. If the DNA between the breaks is instead inverted (reversed) and rejoined, the resulting "antisense" gene produces no antibody.

However, given what we know about the joining of DNA breaks, deletional and inversional joining are theoretically equally likely; there should be a 50/50 split between them. One suggestion had been that productive (deletional) joining really does take place only 50% of the time, and we simply have enough B cells to protect ourselves from immune threats without the other half of the antibodies they might produce.

Dr. Alt explains that this question of efficiency caught his lab's attention: "Maybe we lose the contribution of half our B cells -- we have lots of B cells.  Or is there really some elegant unknown mechanism that drives switching so that it works mainly in a productive deletional orientation?"

Asking -- much less answering -- this question was no small matter, because switch regions are extremely long and repetitive, and previously no one had been able to look at junctions inside them.  As Dr. Alt explains, "Everything people had known about class switching, which has been in textbooks for years, comes from looking at events at the edges of the switch regions, or maybe a few junctions from tumors, where you could find something in the middle."

So the lab capitalized on its own previous technological innovation to shed some light on the question.  In 2011 the Alt lab published a paper on its development of a technique they called high-throughput genome-wide translocation sequencing (HTGTS). After breaks are made using DNA-cutting enzymes, this specific and sensitive assay makes it possible to identify sequences across the genome to which the ends of those breaks are joined. 

For the current research, the team adapted HTGTS to increase by several orders of magnitude the amount of data on where switch regions are being joined during CSR. The workers in Alt's lab selected used normal CSR breaks in the upstream switch region as bait to find out where the join to the repetitive sequences in the long downstream switch regions. To provide some context, the assays previously used to explore switch region junctions were quite limited in scope.  For example, earlier papers might have presented data on 25 or 50, or at most 100 junctions.  Here the Alt lab applied HTGTS to obtain data on 30-40,000 junctions.

The main finding was dramatic: instead of the predicted 50/50 split between deletional (productive) and inversional (non-productive) joins, a full 90-95% of the joins occurred in the deletional orientation, i.e., the orientation that produces viable antibodies. 

Dr. Alt's team also explored several related questions designed to reveal when and where this directionality and bias towards efficiency does not operate.

While the assays above created breaks only at switch regions, the lab also used other "designer" enzymes engineered to break the DNA in a specific location bordering switch regions. Those breaks were rejoined as generally predicted: about half in the productive orientation and half inversional/non-productive.

In addition, the HTGTS assays described above were performed within the same chromosome.  When breaks were made in analogous points on different chromosomes -- even at switch regions -- no directional bias was found.

The fact that joins across the entire switch region are so clearly biased in a productive direction argues strongly for the existence of an unprecedented mechanism that pushes them in that direction. 

The team in the Alt lab hypothesizes that some as-yet-unrecognized organizational features of the antibody locus orient switch regions on the same chromasome to optimize them for deletional joining to make antibodies; they further show that "normal" cellular DNA joining pathways then take over to fuse them and enforce the orientation preference. 

Dr. Alt puts the findings of the new Nature paper in context: "No one had conceived of a mechanism that supported directionality, nor did anybody have the technology to test directionality before. We developed an assay to follow DNA breaks in an antibody gene switch region and find where they join to another switch region.  With other assays you couldn't conceive of looking at this because they were insufficiently sensitive and could not work on the long and highly repetitive switch regions where CSR actually happens."

The Alt lab has already begun work to define and characterize the mechanisms responsible for CSR directionality, which will likely have ramifications not just in the field of immunology, but also in genome organization and DNA repair at large.