Epigenetic, genetic and transcriptional control of B cell differentiation and B cell repertoire formation
A main focus of our lab is the molecular analysis of the epigenetic, genetic and transcriptional mechanisms which regulate accessibility of the V, D, and J immunoglobulin (Ig) gene segments for V(D)J recombination, and that regulate the differentiation of B cell progenitors. In our repertoire formation studies, we are elucidating 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. Cutting edge technologies such as ChIP-seq, RNA-seq, deep sequencing of the antibody repertoire and CRISPR/Cas9 genome editing are utilized in our research. 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. 3D-FISH and chromosome conformation capture (3C) and 4C are being used to study the changes in the 3-dimensional structure of the Igh and Igκ loci. A related study is to determine which transcription factors control the changes in the epigenetic profile and in the 3-dimensional structure of the receptor loci at the developmental stage at which they undergo rearrangement. Current studies are aimed at elucidating the mechanism by which transcription factors control V(D)J rearrangement and B cell differentiation. We have identified regions with the epigenetic marks of enhancers within the Vκ portion of the Igκ locus, and we are using CRISPR/Cas9 technology to delete these novel enhancer-like regulatory elements in a cell line that we can induce to undergo Igκ rearrangement. We are also studying the epigenetic regulation of later steps in B cell differentiation.