Scientific Report 2005

Immunology

Control of V(D)J Recombination and Formation of the Antibody Repertoire in Normal and Autoimmune Mice

A.J. Feeney, C.R. Espinoza, J. Lamoureux, M. Cherrier, L. Watson, P. Lao, R.Z. MacDonald

A main focus of our laboratory is the molecular analysis of factors that influence the composition of the antibody repertoire and elucidation of the mechanisms that control the V(D)J rearrangement. In each precursor B lymphocyte, a different set of V, D, and J genes recombines to form exons for the light and heavy chains of the antibody molecule. Each locus has many V, D, and J genes, but the gene segments are not used equally. One of our goals is to understand the basis of this nonrandom use of gene segments.

We previously showed that much of this bias occurs because V genes undergo recombination with different intrinsic frequencies due to differences in the recombinase signal sequence, the binding site for the recombinase, flanking each gene segment. The recombinase signal sequence is composed of a relatively conserved heptamer and nonamer flanking a “spacer” of conserved length but only modestly conserved sequence. Few genes have consensus heptamers and nonamers, however, and changes in this natural variation in the recombinase signal sequence can greatly affect recombination frequency in vitro and in vivo. Surprisingly, even differences in the sequence of the spacer can greatly influence recombination frequency, and these differences also contribute to nonrandom use of genes in vivo.However, other factors clearly influence recombination frequencies, and currently, we are focusing on the role of transcription factors and chromatin modifications in controlling accessibility to V(D)J recombination and recombination frequency. Genes in loci that are undergoing recombination are often associated with histones that are acetylated. We hypothesized that the extent of histone modification affects the frequency of recombination of individual genes, and indeed we observed a correlation between the relative rearrangement frequency of several individual genes in vivo and the extent of acetylation of histones H3 and H4 associated with those genes as assessed by chromatin immunoprecipitation.

We also use a novel system in which certain immunoglobulin gene rearrangements can be induced in a nonlymphoid cell line after the transient transfection by vectors expressing a B cell–specific transcription factor, E2A or EBF, and the recombinases. We showed that E2A induces rearrangement of VκI genes. However, the VκII and VκIII genes, which are interspersed with the VκI genes within the IgVκ locus, seldom rearrange after E2A transfection. EBF induces VλJλ rearrangement, but mainly of only a single Vλ gene.

Thus, this induction of accessibility of genes is not uniform across the locus. Neighboring genes can be differentially induced to rearrange, suggesting localized control of accessibility for rearrangement. Current studies are aimed at elucidating the mechanism of this localized gene-specific control. Transfection with E2A does not induce much acetylation of the histones associated with VκI genes, but the rearranged genes are associated with acetylated histones, suggesting that once a V gene becomes accessible, it efficiently rearranges.

In other studies, we are examining the breakdown of B-cell tolerance in autoimmunity. When precursor B cells successfully recombine both heavy- and light-chain gene segments, they express a B-cell receptor for the first time. If the receptor is autoreactive, then the immature B cell normally continues to undergo light-chain V-J rearrangement until an innocuous receptor is made. This process is termed receptor editing and is an important checkpoint in B-cell tolerance. We have evidence that this process is not functioning the same in lupus-prone mice as in nonautoimmune mice, and we are investigating why this difference occurs. Such misregulation of this key checkpoint could lead to the release of autoreactive B cells into the periphery, where they can become activated to secrete autoantibodies and cause autoimmune disease.

Publications

Li, L., Salido, E., Zhou, Y., Battacharyya, S., Yannone, S.M., Dunn, E., Meneses, J., Feeney, A.J., Cowan, M.J. Targeted disruption of the Artemis murine counterpart results in SCID and defective V(D)J recombination that is partially corrected with bone marrow transplantation. J. Immunol. 174: 2420, 2005.