In the Front Seat of VDJ Recombination

New studies from the Alt Lab, published in an article in the October 8 issue of Nature (Ba et al., “CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning”), have provided a major advance with respect to our knowledge of how diverse antibodies are generated during the development of progenitor B lymphocytes.  It has been known for decades that this process involves the assembly of exons encoding antigen-binding antibody heavy chain variable regions from component VH, D, and JH segments by the process of V(D)J recombination.   Chromosomal V(D)J recombination is a cut-and-paste reaction initiated by the RAG endonuclease (RAG).  The VH, D, and JH segments are encoded in consecutive clusters along the chromosome.   RAG initiates V(D)J recombination by binding JH-containing chromatin to form a chromatin-based recombination center (RC). 

Recent work published in Nature from the Alt Lab revealed that upstream D-containing chromatin is moved past the RAG-bound RC  via a process termed chromatin loop extrusion to allow RAG to find and bind a D segment and then cleave the D and JH for subsequent joining to form a RAG-bound DJH RC.   While this work showed a fundamental role for chromatin loop extrusion in the in the D to JH joining process, two major questions remained unanswered until this most recent publication.

The first question was what drives the mechanism that propels upstream chromatin past the RC.   A strong candidate was the ring protein complex known as cohesin, which has a major role in organizing chromatin throughout the genome.  Cohesin was proposed by others to be the driving force for the process of loop extrusion that segregates the genome into many defined chromatin loops.  The Alt Lab had provided suggestive evidence for such a role for cohesin in chromatin loop extrusion during V(D)J recombination.  However, while cohesin was shown to extrude DNA loops in vitro, its role in chromatin loop extrusion in vivo was still not firmly established. 

The new studies from Ba et al. firmly established the role of cohesin in chromatin loop extrusion during V(D)J recombination by specifically degrading Rad21, a cohesin component, in early B cell lines that were G1 arrested and undergoing V(D)J recombination.  While cohesin degradation is lethal to most cycling cells, it had little effect on the G1-arrested pro-B line viability.  This allowed the authors to show that all V(D)J recombination was abrogated in these cohesin-deficient lines, except the joining of one D segment to the JH, which is known to access the JH  by a mechanism not involving loop extrusion, and thus served as a positive control.

The second major question addressed by this new study was the mechanism by which the hundreds of VH segments, spread over many megabases (Mbs), access the DJH RC.   The Ds and JHs lie in the same chromosomal domain, which is formed by the interaction of sets of elements on either side that are bound to the CTCF chromatin-looping factor, while the VHs lie further upstream separated into a different domain.  These elements, which segregate the entire genome into well-defined loops, are referred to as CTCF-binding elements (CBEs). Prior work from the Alt Lab showed that RAG chromatin scanning is terminated at the borders of CBE domains and greatly impeded by CTCF-bound CBEs.  Moreover, the VH domain is peppered with hundreds of CBEs which themselves would impede scanning.   If scanning were responsible for VH utilization across the locus, how could it make it through these many, many impediments?

The Ba et al. study hypothesized that regulation of the ability of CBEs to block scanning could represent a new mechanism of long-range chromatin gene regulation via changes in chromatin architecture.   The progenitor B cell lines used to study D to JH recombination are blocked at a developmental stage in which VH segments do not rearrange to DJH intermediates.   Thus, to address this potential mechanism by which normal B cells can move on to the VH to DJH rearrangement step, the authors tested the effect of depleting CTCF to remove potential CTCF-bound CBE impediments to V(D)J recombination.  

CTCF degradation in other cycling cell types also results in rapid loss of cell viability; however, in G1-arrested progenitor B cells lines CTCF depletion again resulted in only modest effects on viability and little effect on V(D)J recombination. This allowed the researchers to obtain clear-cut experimental results that were quite remarkable.  CTCF degradation allowed RAG to scan through the entire several-Mb long VH locus to generate diverse repertoires of VH(D)JH rearrangements across the entire locus, yielding overall repertoires similar to those generated in normal pro-B cells in the bone marrow.

Thus, the study implicates cohesin as the driver of loop extrusion-mediated RAG chromatin scanning and further demonstrates that the regulation of cohesin complex gene activity can modulate chromatin loop extrusion as a new mechanism for long-range regulation of V(D)J recombination.  Such regulation could occur via a process that inactivates CBE impediments to scanning or potential mechanisms that could allow cohesin to circumvent such impediments during scanning, or both.  These findings of new mechanisms of long-range gene regulation in the context of chromatin architecture could have broader implications for mechanisms that regulate gene expression in heath and disease genome-wide, including mechanisms of chromosomal translocations in B cell and other malignancies.