Faculty

Ann Feeney 
Professor
Immunology and Microbial Science
TSRI - 1992

Education 
Ph.D., Cornell University, 1976

Research Focus 
Epigenetic and Genetic Control of V(D)J Rearrangement, and B Cell Repertoire Formation in Normal and Autoimmune Mice

The antibody repertoire is highly diverse, and much of this diversity is due to the existence of many V, D and J genes. In each precursor lymphocyte, a unique combination of V, D and J gene segments recombine to form antibody genes. In addition, marked diversity is created at the junctions of these gene segments during recombination. This V(D)J recombination process is under tight lineage-specific and developmental stage-specific control. A main focus of our lab is the molecular analysis of the epigenetic and genetic mechanisms which regulate accessibility of the V, D, and J immunoglobulin (Ig) gene segments for V(D)J recombination, and elucidation of the factors which influence the composition of the initial antibody repertoire. We also are assessing differences in repertoire formation and B cell tolerance in autoimmune mice as compared to non-autoimmune mice.

Although there are many V, D, and J genes at each locus, we have previously shown that different gene segments rearrange with quite different relative frequencies in pro-B cells in vivo. One of our goals is to understand the basis of this non-random gene utilization. We have shown that much of this bias in rearrangement frequency is due to differences in sequence of the binding site for the recombinase (RSS) flanking each gene segment. However, other factors clearly influence recombination frequencies. Current studies are focusing on the role of transcription factors and chromatin modifications in controlling accessibility to V(D)J recombination and also their role in non-random gene rearrangement.

Epigenetic control of accessibility for V(D)J recombination

We are analyzing the chromatin modifications that accompany B cell differentiation in vivo in an effort to understand the mechanism of lineage-specific and stage-specific control of accessibility of Ig genes, as well as to understand the control of rearrangement on the level of individual genes. By isolating pro-B cells and pre-B cells from adult or newborn mice, we can directly assess the histone posttranslational modifications (e.g acetylation, methylation) at individual genes at the various loci by chromatin immunoprecipitation (ChIP) and also on a genome wide level by ChIP-on-chip. We have shown that the extent of acetylation of histones surrounding individual genes correlates with their rearrangement frequency in vivo. We analyzed many different histone modifications, especially methylations, at each step of B cell differentiation. This has permitted the identification of histone modifications that change as B cells transition through each stage of differentiation. We are currently assessing the role of these histone modifications in V(D)J recombination and in B cell development. For example, we have shown by ChIP-chip that H3K27me3, added by the Polycomb protein Ezh2, is present on proximal VH genes exclusively, and absence of Ezh2 prevents rearrangement of distal V_H genes, suggesting a functional consequence of this modification during IgH chain rearrangement. We have also demonstrated that several histone modifications associated with active genes are predominantly expressed on J genes, with much lower levels on V or D genes. This is of particular relevance for H3K4me3, which can be bound by the RAG2 recombinase, and thus this interaction could help recruit RAG2 to J segments, and thus provide ordered rearrangement beginning with J genes.

3-dimensional looping mediated by CTCF at the Ig loci

The very large Ig and TCR loci have to undergo contraction via multiple loop formation to bring the enormous megabase V loci near the small (D)J loci to facilitate effective recombination to V genes throughout the locus. Since the insulator CTCF has been demonstrated to mediate long-distance chromosomal looping, we asked whether CTCF was bound throughout the V loci, and thus was a candidate for being involved in the locus contraction. We observed that indeed there were many CTCF sites throughout the Ig loci, primarily in the V loci, and that these sites were also bound by cohesin primarily in a lineage-specific and stage specific manner. Ongoing experiments are testing whether CTCF and its related proteins do mediate looping throught the Ig loci and thus facilitate the formation of a diverse repertoire of antibodies utilizing a full range of V genes.

Role of transcription factors in controlling accessibility

A related issue is to determine what controls the change in these histone modifications. A likely set of candidates is the transcription factors such as E2A, EBF and Pax5 which are critically involved in B cell differentiation. We have previously shown that ectopic expression of E2A in a non-lymphoid cell line induced a specific subset of Vκ genes, the Vκ I family, to undergo rearrangement, while leaving the surrounding Vκ II and Vκ III genes unrearranged. We also showed that EBF induces one particular Vλ gene to undergo rearrangement. This demonstrates that the induction of accessibility of genes is not uniform across a locus, but that neighboring genes can be differentially induced to rearrange, suggesting localized control of accessibility for rearrangement. Similarly, we are investigating mice heterozygous for transcription factors, and find that individual genes are preferentially rearranged or inhibited in progenitor B cells from these mice as compared to wild type B cell progenitors. Current studies are aimed at elucidating the mechanism of this localized gene-specific control.

Pax5 is another transcription factor that is essential for B cell differentiation, specifically at the stage of V_H to D_HJ_H rearrangement. We have found Pax5 binding sites in many V_H genes by EMSA, and ChIP analysis shows that Pax5 is bound to these sites in vivo. Furthermore, Pax5 also binds to the V(D)J recombinase, RAG 1 and RAG2. We are addressing the hypothesis that Pax5 may aid in V_H rearrangement by recruiting or stabilizing RAG binding to the RSS.

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, potentially including a direct transitional 1 to marginal zone B cell pathway.

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.

Selected References 
Xu, C.R., Schaffer. L., Head, S.R. and Feeney, A.J. Reciprocal patterns of methylation of H3K36 and H3K27 on proximal vs. distal IgVH genes are modulated by IL7 and Pax5. Proc Natl Acad Sci USA 105:8685-8690, 2008.

Carey, J.B., Moffatt-Blue, C.S., Watson, L.C., Gavin, A.L. and Feeney, A.J. Repertoire-based selection into the marginal zone compartment during B cell development. J. Exp. Med. 205:2043-2045, 2008. Evaluated by the Faculty of 1000 Biology.

Degner, S.C., Wong, T.P., Jankevicius, G., and Feeney, A.J. Cutting Edge: Developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development. J. Immunol. In press.

Xu, C.-R. and Feeney, A.J. The epigenetic profile of immunoglobulin genes is dynamically regulated during B cell differentiation and is modulated by pre-BCR signaling. J. Immunol. In press.