Research

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

Epigenetic control of V(D)J recombination

A main focus of our laboratory is the molecular analysis of factors which influence the composition of the antibody repertoire, and the elucidation the mechanisms which control the V(D)J rearrangement process (1).  In each precursor B lymphocyte, a different set of V, D, and J genes recombine to form exons for the light and heavy chains of the antibody molecule. Although there are many V, D, and J genes at each locus, these gene segments are not used equally. One of our goals is to understand the basis of this non-random gene utilization. 

We have previously shown that much of this bias is due to the fact that V genes undergo recombination with different intrinsic frequencies due to differences in sequence of the binding site for the recombinase flanking each gene segment. However, other factors clearly influence recombination frequencies, and some of our current studies are focusing on the role of transcription factors and chromatin modifications in controlling accessibility to V(D)J recombination and recombination frequency.

We have uncovered a novel role for a transcription factor in promoting V(D)J rearrangement. Pax5 is a transcription factor that is essential for B cell development. In the absence of Pax5, V_H to DJ_H rearrangement is severely impaired. We have shown that there are Pax5 binding sites in the coding regions of many V_H genes (2). In collaboration with X. Zhang and M. Cooper, we have also shown that Pax5 binds to the recombinase proteins, RAG1 and RAG2. Hence we propose that Pax5 may recruit RAG1/2 to the RSS, or may stabilize the interaction of the RAG complex with its binding site. Other roles of Pax5 in forming the antibody repertoire formation are being investigated.

The V(D)J rearrangement process is precisely controlled such that heavy chain genes rearrange before light chain genes, and kappa light chain genes rearrange before lambda genes. It is likely that the structure of chromatin, the histone proteins that are intimately associated with DNA, plays an important role in the control of accessibility of V, D and J genes of the various loci.  Genes in loci which are undergoing V(D)J recombination are often associated with histones which are acetylated, and we have shown that V_H genes that rearrange more often are more highly enriched in acetylated histones.  However, there are many other post-translational modifications of histones in addition to acetylation which may influence the accessibility of genes to undergo V(D)J rearrangement .  Therefore, we have recently analyzed the dynamic patter of methylation of lysines 4, 27, 36 and 79 on histone 3 (H3) on immunoglobulin genes during the course of B cell differentiation. 

In general, methylation of K4, K36 and K79 in histone 3 is associated with active genes, whereas methylation of K27 is associated with repressed genes.  We found that trimethylation of lysine 4 (H3K4me3) is associated primarily with J genes which are actively undergoing germline transcription and rearrangement.  In contrast, methylation of lysine 27 (H3K27me3) and lysine 36 (H3K36me2) are the main histone modifications seen at high level on V genes.  The distal and proximal halves of the large 2.5 Mb immunoglobulin V_H locus are under distinct control. Distal V_H genes do not undergo rearrangement in the absence of the transcription factors Pax5 or YY1, or the Polycomb protein Ezh2, and show reduced rearrangement in the absence of IL7.  Ezh2 was of particular interest since it is the histone methyltransferase that methylates H3K27.  We therefore performed ChIP (Chromatin immunoprecipitation) as well as ChIP-on-chip on pro-B cells, and showed that H3K27me3 was exclusively present on proximal V_H genes, whereas H3K36me2 had the reciprocal pattern.   We showed that the H3K27me2 was absent in Pax5-deficient mice, thus linking those two phenotypes both of which are unable to rearrange distal V_H genes.  IL7 signaling did not affect H3K27me3, but it did increase the active modification H3K36me2.  Pro-B cells in fetal mice preferentially rearrange proximal V_H genes, and these pro-B cells lacked H3K27me3.  Based on the phenotype of the Ezh2-deficient pro-B cells, and on our ChIP data, we propose that the repressive modification H3K27me3 which is present exclusively on proximal V_H genes is necessary to facilitate rearrangement of the distal V_H genes, thus providing a diverse repertoire of antibodies making full use of all of the V_H genes (3).

The light chain loci undergo rearrangement at the pre-B cell stage of differentiation, after heavy chain rearrangement has been successfully completed, and rearrangement at the kappa light chain locus occurs before rearrangement at the lambda light chain locus.  We have shown that this ordered rearrangement is epigenetically controlled.  Histone modifications occur first on the kappa genes, are not present at high levels on lambda genes until the immature B cell stage, the stage at which receptor editing is initiated.  We further demonstrated that signaling through the pre-BCR is necessary to initiate epigenetic modifications at the kappa locus, since mice impaired in the ability to transduce a pre-BCR signal show no histone methylation or acetylation on kappa gene segments.  In addition, the intronic kappa enhancer is also necessary for these modifications, as determined using mice in which the intronic kappa enhancer was deleted.

 

Influence of B cell receptor repertoire on B cell fate decisions

B cells in the spleen are divided into functionally distinct subsets. We are investigating differences in the antibody repertoires between the marginal zone B cells, which respond to blood-borne pathogens, and follicular B cells, the largest population of splenic B cells. We previously showed that B cells made in fetal and neonatal life lack an enzyme, TdT, which greatly diversifies the adult antibody repertoire. We now have evidence that B cells generated early in ontogeny are preferentially selected into the marginal zone compartment, suggesting that the fetal/neonatal repertoire of antibodies, which is very different from that generated in adults, may be particularly useful against blood-borne pathogens.  Furthermore, our recent data using bone marrow chimeric mice show that preferential selection of B cells with the fetal/neonatal repertoire occurs not only in the marginal zone B cell compartment, but also in an earlier transitional compartment.  Conversely, selection of the adult-type TdT-positive repertoire is observed preferentially in the follicular compartment, and especially in the mature recirulating compartment of B cells.  Our data suggest that B cell fate decisions are influenced by the repertoire of the B cells at several branch points during B cell differentiation. Furthermore, our data suggest novel alternative pathways of differentiation for B cells.

 

Misregulation of receptor editing in lupus-prone mice

When precursor B cells successfully recombine both heavy chain 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 demonstrated that this process is not functioning as efficiently in lupus prone mice as in nonautoimmune mice using B cell receptor transgenic mice (4), and we are investigating why this is occurring. 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.

We are currently extending these studies to lupus-prone mice that we have bred which have the B cell receptor transgenes “knocked-in”, i.e, the immunoglobulin light and heavy chain transgenes have been targeted to the endogenous immunoglobulin light and heavy chain loci, respectively.  This permits the full extent of receptor editing to occur, and these studies are revealing more aspects of misregulation of receptor editing and B cell development in the lupus-prone mice.

 

Feeney, A.J., Goebel, P. and Espinoza, C.R. Many levels of control of V gene rearrangement frequency. Immunol Rev. 200:44-56, 2004.

 

Zhang, Z., Espinoza, C.R., Yu, Z., Stephan, R., He, T., Williams, G.S., Burrows, P.D., Hagman, J., Feeney, A.J., and Cooper, M.D. Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V(H)-to-DJ(H) rearrangement of immunoglobulin genes. Nat Immunol. 7:616-624, 2006.

 

Lamoureux, J.L., Watson, L.C., Cherrier, M., Skog, P., Nemazee, D. and Feeney, A.J. Reduced receptor editing in lupus-prone MRL/lpr mice. J Exp Med. 204(12):2853-64, 2007.

 

Xu, C.-R., Schaffer. L., Head, S.R. and Feeney, A.J. Reciprocal methylation of H3K27 and H3K36 on distal and proximal IgV_H genes is modulated by IL7 and Pax5. PNAS. In press,  2008.

 

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