Scripps Research Logo

The Feeney Lab


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. One major long term focus of our lab has been 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. 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. We have published the first deep sequencing study of the initially created Igh repertoire, and current work is addressing changes in the repertoire as pro-B cells differentiate, as well as understanding in more depth the factors which regulate V gene usage such as the epigenetic and transcriptional local environment of each V gene. We are currently extending our analysis to examine the Igκ repertoire, and we are utilizing analysis of genomic DNA rather than cDNA to get a more accurate analysis of the repertoire.

Epigenetic control of accessibility for V(D)J recombination and regulation of early B cell differentiation

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. This has permitted the identification of histone modifications that change as B cells transition through each stage of differentiation. Of great interest is the changing extent of H3K4me1 and H3K27ac, the marks of active enhancers as cells progress from pro-B cells to pre-B cells. ChIP-seq is also being performed for key transcription factors, as it is likely that they may direct the epigenetic and transcriptional changes that take place as B cells differentiate.

Modification of novel regulatory elements within the Igκ locus and the Igh locus with CRISPR/Cas9 genome editing

Our epigenetic studies of pro-B cells and pre-B cells have revealed many locations with the epigenetic marks of enhancers (H3K4me1) in the V region portions of the Igh and Igκ locus, respectively. A few locations within the Igκ locus have these enhancer marks in pro-B cells, and these regions also have several important transcription factors bound to them. We hypothesize that these are key elements in the stage-specific process of locus contraction which brings all the Vκ genes into closer proximity to the Jκ genes, to which one Vκ gene will rearrange. We are focusing on the regions with the highest level of the epigenetic marks of enhancers at the pro-B cell stage, hypothesizing that they may initially orchestrate the long-range interactions and proper folding of the Igκ locus. We are now using CRISPR/Cas9 technology to delete these putative regulatory elements in a cell line that we can induce to undergo Ig rearrangement. This is allowing us to test the role of these novel enhancer elements on the composition of the Igκ repertoire, and on the 3D structure of the Igκ locus. We have also deleted one of these novel enhancer elements, E88, in mice. Since we are also interested in determining how the various transcription factors and epigenetic marks regulate the structure of the receptor loci and the repertoire made from each locus, we are mutating individual transcription factor binding sites within these novel enhancer elements.

In the Igh locus, we have also identified 4 regions with epigenetic marks of enhancers within the VH portion of the Igh locus. We have deleted one of these (NE1) in mice and are analyzing the repertoire in pro-B cells from these mice. In collaboration with the lab of Dr. Amy Kenter, University of Illinois at Chicago, we are analyzing the changes in the long range interactions resulting from the deletion of this NE1 element in mice, and also from deletion of both NE1 and NE2 enhancer elements in a cultured pro-B cell line.

3-dimensional looping mediated by CTCF and ncRNA at the Ig 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 multi-looped rosette-like structure 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 have analyzed CTCF binding at the other large receptor loci, and have shown that some CTCF is bound in a lineage and stage-specific manner in the antigen receptor loci, while other CTCF sites display more general binding patterns. We are deleting several CTCF sites which we hypothesize are influencing the structure of the Ig locus, and we are determining how their absence affects the resulting repertoire in a cell line in which we can induce Ig rearrangement.

We have also performed the first RNA-seq on pro-B cells to determine the complete transcriptional profile of the germline transcription (non-coding RNA) throughout the Igh locus. By chromosome conformation capture (3C), we have demonstrated that regions of high transcription interact directly with the Eμ enhancer, the promoter of the strong Iμ non-coding RNA. We hypothesize the act of transcription brings V gene promoters throughout the Ig locus to the same transcription factory as Iμ which is adjacent to the DJ rearrangement to which one V gene will ultimately rearrange. We propose that a different subset of V gene promoters and intergenic regions will produce ncRNA in each pro-B cell at any given moment, and that this dynamic and stochastic juxtapositioning of ncRNA transcribed regions and the DJ rearrangement will result in a diverse set of V genes used in Igh rearrangement. To definitively assess the role of ncRNA of individual V genes in accessibility for rearrangement, we are deleting promoters of V genes and determining the ability of the promoter-less V genes to undergo rearrangement.