The Feeney Lab

Research

Epigenetic, genetic and transcriptional control of B cell differentiation and B cell repertoire formation

Control of V(D)J Rearrangement and B Cell Repertoire Formation

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. 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.

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 some of this bias in rearrangement frequency is due to differences in the sequence of the binding site for the recombinase (RSS) flanking each gene segment. However, other factors clearly have a major influence on recombination frequencies. Current studies are focusing on the role of transcription factors, chromatin modifications and the 3-dimensional structure of the locus in controlling accessibility of the receptor loci to V(D)J recombination and in regulating non-random V gene rearrangement. Deep sequencing technology is greatly aiding our studies of antibody repertoire composition.

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, we can directly assess the histone post-translational modifications (e.g., acetylation, methylation) on a genome wide level by ChIP-seq. We have analyzed many different histone modifications at each step of early B and T 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 in the newborn and in the adult.

3-dimensional looping mediated by CTCF at the Ig and TCR loci

The very large Ig and TCR loci have to undergo contraction via the formation of multiple loops to bring the enormous megabase V loci near the small (D)J loci to facilitate effective recombination to V genes throughout the locus. This locus contraction occurs at the specific stage of B or T cell differentiation at which that locus is undergoing V(D)J rearrangement. Since the insulator-binding protein CTCF has been demonstrated to mediate long-distance chromosomal looping, we hypothesized that CTCF would be a good candidate for being involved in the contraction of Ig and TCR loci. We demonstrated by ChIP-seq that there were many CTCF sites bound throughout all of the large Ig and TCR loci, primarily in the V region part of the loci. Furthermore, we showed that cohesin was bound to CTCF at these sites primarily in a lineage-specific and stage-specific manner. To assess whether these sites are involved in the 3D structure of the Igh locus, we performed 3D-FISH after knockdown of CTCF in pro-B cells, and we showed that CTCF did result in a decrease in locus contraction, although not as completely as deficiency in the transcription factor YY1. In addition, by 3C (chromosome conformation capture) we determined that a CTCF-mediated loop is present containing the DH and JH genes, as well as the intronic enhancer. This CTCF/cohesin loop thus creates a domain in which DJ rearrangement can occur in a physically separate compartment from the large VH locus. We are currently analyzing the other large receptor loci, and we are also interested in determining how the various transcription factors and epigenetic marks might interact with the CTCF/cohesin complex in regulating the structure of the receptor loci. Furthermore, we demonstrated that germline transcription at the Igh locus is increased when we knockdown CTCF, and current studies are aimed at determining the mechanism by which CTCF regulates transcription.

Role of transcription factors in controlling accessibility

A related issue is to determine what controls the change in the epigenetic profile and in the 3-dimensional structure of the receptor loci at the developmental stage at which they undergo rearrangement, with particular emphasis on the Igh locus. A likely group of candidates are transcription factors such as E2A, EBF and Pax5, all of which are critically involved in B cell differentiation. Other candidates are YY1 and Ezh2, which are involved in locus compaction at the Igh locus. We have previously shown that E2A and EBF can exert localized control of rearrangement of individual V genes. Current studies are aimed at identifying where the transcription factors bind, and elucidating the mechanism by which transcription factors control V(D)J rearrangement and B cell differentiation.

Epigenetic misregulation in lymphoma

A specific mutation in the catalytic site of Ezh2 is found in Germinal Center B cell Diffuse Large B Cell Lymphoma. Ezh2 is the histone methyltransferase that methylates lysine 27 of histone H3, an epigenetic mark of repressed genes. Current studies are aimed at determining the role of Ezh2 in germinal center B cell development, and how this is misregulated in patients with the mutated Ezh2.

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 by adding N region diversity to the antigen-binding CDR3 portion of the antibody molecule. We now have evidence that B cells generated early in ontogeny, and B cells generated in the adult with a fetal-type repertoire lacking N region diversity, 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. 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.

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, 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.