The generation of the B cell receptor gene repertoire is accomplished in a lineage and developmentally regulated manner through the ordered assembly of variable (V), diversity (D) and joining (J) gene segments which are dispersed over the 2.8Mb IgH locus leading to mono-allelic expression of antigen-receptor. The lymphocyte-specific recombination activating gene (RAG) 1 and 2 proteins catalyze the initiation of the VDJ recombination by recognizing and cutting short, conserved recombination signal sequences that flank germline V, D, and J segments followed by the processing and re-ligation of the DMA ends by repair factors of the non-homologous end-joining machinery. Multiple lines of evidence support an accessibility framework whereby the chromosomal structure and chromosomal positioning of immunoglobulin loci undergo lineage and developmental alterations, to allow recombination and enforce allelic exclusion. Yet, the sheer number of VH genes and their chromosomal span has hampered genetic evaluation of certain aspects of accessibility. This proposal aims to evaluate a the role of cis-acting elements in controlling VDJ recombination via the characterization of mutant IgH loci and the creation of simplified endogenous loci towards a long-term effort to dissect the minimal set of cis-acting elements that regulate recombination. Specifically, is there a role for the intergenic region between V and D gene segments in recombination? This will be addressed by analysis of the effect of germ-line deletion of this sequence. Second, can simplified endogenous loci recapitulate critical aspects recombination? This question is being addressed by creation and characterization of mice with only limited numbers of V, D and J segments and monitoring efficiency of recombination, ordered rearrangement, and allelic exclusion. Third, what is the function of germline-sense and anti-sense transcription in controlling accessibility? This will be addressed by using minimal loci to delete sense promoters and further characterize the nature of antisense including mapping of transcriptional start sites. Understanding the cis-regulatory elements in VDJ recombination will enhance our understanding of lymphoid development and the control of gene expression in general.
|Guo, Chunguang; Yoon, Hye Suk; Franklin, Andrew et al. (2011) CTCF-binding elements mediate control of V(D)J recombination. Nature 477:424-30|
|Giallourakis, Cosmas C; Franklin, Andrew; Guo, Chunguang et al. (2010) Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination. Proc Natl Acad Sci U S A 107:22207-12|