Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells

To date there has not been a study directly comparing relative Ig kappa rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Ig kappa repertoire from the same pre-B cell pool. We find that similar to 20% of V kappa genes have rearrangement frequencies >= 2-fold up or down in RNA vs. DNA libraries, including many members of the V kappa 3, V kappa 4, and V kappa 6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual V kappa genes rearranged at very different frequencies, and also displayed very different J kappa usage. Regression analysis revealed that the greatly unequal V kappa gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many V kappa genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Ig kappa rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of J kappa-distal V kappa genes, particularly V kappa 10-96 and V kappa 1-135. Regression analysis indicates that PU.1 binding is the highest predictor of V kappa gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iE kappa(-/-) pre-B cells reveal that iE kappa actively influences V kappa gene usage, particularly V kappa 3 family genes, overlapping with a zone of iE kappa-regulated germline transcription. These represent new roles for iE kappa in addition to its critical function in promoting overall Ig kappa rearrangement. Together, this study provides insight into many aspects of Ig kappa repertoire formation.