Program in Cellular and Molecular Medicine (PCMM) | Chromatic Loops

By Paul Guttry

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 continue their groundbreaking work at the nexus of genetics and immunology, specifically the response of antigen-activated B cells to the enormous variety of possible threats, including bacteria, viruses, and parasites.

Two reports from the Alt Lab in Nature (the first in September 2019 and a second online on October 30, 2019 with a Nature “News and Views” covering both) present major advances in chromatin regulation, showing that two distinct types of antibody gene recombination, occurring at different developmental stages, both depend upon reeling long loops of chromatin past recombination centers to align substrate gene segments in the processes known as V(D)J recombination and class switch recombination (CSR).

Chromatin is the combination of DNA and proteins that compresses and streamlines the long DNA molecule, strengthening and protecting it while regulating genetic processes like replication and expression.

The Nature report published by the Alt Lab in September 2019 presented new insights into the role of chromatin loop extrusion in V(D)J recombination, the process responsible for creating a vast repertoire of specific antibodies.

The new Nature report, entitled Fundamental roles of chromatin loop extrusion in antibody class switching, was published online in Nature on October 30, 2019. Alt lab member and first author Xuefei Zhang and his team focused on the role of chromatin loop extrusion in CSR, which uses the specific antibodies produced by V(D)J recombination to create the right type of antibody in a specific anatomical location. For example, if you have a bacterial infection in your mouth, V(D)J recombination makes the right antibody, but how do you actually get rid of that pathogen? The activated B cells that started making (for example) an IgM antibody instead make IgA antibodies, which can be secreted across the epithelium into the oral cavity to fight your infection.

What scientists formerly understood about CSR was difficult to explain using any known biological recombination mechanism. They knew CSR to be initiated by activation-induced cytidine deaminase (AID), which begins double-strand breaks (DSBs) in one (or more) of hundreds of targets in the “donor” upstream IgM switch region and in a downstream “acceptor” S region. The AID enzyme is attracted by transcription, which in the donor region is likely routine; but transcription in the acceptor region depends on immune activation of a nearby upstream promoter. In turn, the promoter must contact and become activated by enhancers at the far downstream end of the Igh locus.

The Alt Lab had previously shown that CSR takes place in a productive deletional orientation, i.e., not only is a stretch of DNA removed, but the portions that remain in the genome produce a functional protein. Because at the time, no known mechanism was capable of aligning CSR DSB ends for deletional joining (such as the enzyme that performs this task in V(D)J recombination), and given other unique mechanistic constraints, the Alt lab in a 2015 Nature paper concluded that CSR involves an “unprecedented mechanism" that depends in part on chromosomal organization.

After several years of intensive work on the subject, Xuefei Zhang and his team now propose cohesin-mediated loop extrusion as that “unprecedented" deletional joining mechanism; in fact, loop extrusion is likely involved in CSR at two levels.

First, a basel loop of DNA/chromatin is formed by loop extrusion, bringing widely separated enhancers together with the donor IgM switch region to form what they term a "class switch recombination center"; all potential acceptor regions are retained in the extruded loop. Then cytokines produced by other types of immune cells prime a promoter upstream of the "acceptor" switch region and constant region within the extruded loop, and extrusion moves it into the class switch recombination center, where its promoter becomes activated and the acceptor switch region is highly transcribed. The transcriptionally activated promoter region then also loads cohesin, the ring-shaped protein complex that drives extrusion and helps organize the genome during basic processes like replication, repair, and condensation. Cohesin tightly aligns the acceptor and donor regions, preparing them to be broken by AID and rejoined as described above.

Second, cohesin-mediated loop extrusion helps address the long-standing question of how most AID-initiated DSBs in CSR are joined in deletional — rather than inversional — orientation. This was another mystery, as DSBs were previously thought to be joined simply by diffusion, depending on their location on the chromosome; in fact, across the genome, most DSBs are rejoined with little regard to direction. The cohesin loop extrusion-mediated joining mechanism proposed by the Alt lab relies on two cohesin rings putting tension on the regions between them. Thus, two breaks can occur at different times and places along the two switch regions, since one or both ends will be reeled into an opposing cohesin ring, where extrusion stalls. Then another break in the other stalled switch region strand under tension, goes through the same process and the correct ends becomes aligned in a cohesion ring with the first break end for productive deletional joining.

Together , the studies show that impeding extrusion as it passes a V(D)J or CSR recombination center promotes the access of the impeded regions to the enzymes that initiate these processes. These findings indicate that impeded extrusion over a recombination center promote substrate chromatin accessibility, as first proposed by the Alt lab decades ago. A further ramification of this finding, beyond the normal V(D)J and CSR mechanisms, is that, by impeding loop extrusion at a recombination center, either for V(D)J recombination or CSR, you can force those mechanisms to target unlikely sequences, or as Dr. Alt puts it, “we can turn something that’s really nothing into a real substrate just by slowing things down.”

The latter finding raises implications of the general loop extrusion CSR mechanism for diseases involving expansions and deletions of other genomic sequences. Repeated targeting by such a recombination mechanism could greatly enlarge a small DNA sequence. One familiar example might be Huntington’s disease, which is associated with a repeat in a CAG sequence that tends to expand. For genetic diseases like Huntington’s and spinal muscular atrophy, the mechanisms remain unknown. However, as many enzymes and other activities can cause genome breaks; factoring in “accidental” slowing of chromatin loop extrusion in particular regions of the genome may eventually help explain some genetic diseases.

This dynamic new loop extrusion-mediated CSR model constitutes a dramatic leap forward from the current static representation of CSR not only in the academic literature, but in textbooks as well.

In fact, without exaggeration, the new findings and model from the Alt Lab have the potential to reshape the CSR field and have a formative influence on the emerging field of chromatin loop extrusion-mediated gene regulation.

The CSR paper also contains a link to an animation of the chromatin loop extrusion mechanism that regulates CSR via changes in chromatin architecture. In the words of the Nature editor, the animation of the Alt lab’s new CSR model “is worth more than a thousand words.” The paper comes out in print on November 14, 2019.