The Ig? locus is responsible for the production of a large variety of Ig light chain rearrangements, but how these processes are regulated is unknown. The generation of a diverse Ig? repertoire is facilitated by contraction of the locus at the time of Ig? rearrangements. These changes in the 3-dimensional structure of the Ig? locus brings V? genes throughout the locus in closer proximity with the J? genes to which one V? gene will rearrange in each pre-B cell. However, the factors involved in orchestrating locus compaction and long-range looping interactions of the Ig? locus in pre-B cells are unknown. Also, it is not known how the 3D structure affects V? gene utilization. The Feeney lab has performed ChIP-seq in RAG-/-?+ pre-B cells and RAG-/- pro-B cells for a wide variety of histone modifications, including H3K4me1, the epigenetic marker of enhancer elements. Surprisingly, in addition to the three known enhancers at the 3' end of the Ig? locus, many H3K4me1 peaks were observed within the large V? part of the locus in pre-B cells, and most were not present in pro-B cells. This developmental stage-specificity of the H3K4me1 peaks suggests that these regions may play a role in regulating V? rearrangement. In addition, two published studies revealed regions within the V? locus that are preferential sites of long-range interactions with iE?, the enhancer located just 2 kb downstream of the J? genes. Furthermore, the Feeney lab showed that the most predominant long-range interaction 'hubs' from those two studies are characterized by regions of high H3K4me1, and ChIP-seq data revealed that these 'hubs' bound multiple key transcription factors (TFs) such as Pax5, EBF, E2A, Ikaros, PU.1 or YY1. We therefore hypothesize that some of these novel enhancer-like elements are important in regulating the composition of the Ig? repertoire, possibly through orchestrating the 3D configuration of the compacted Ig? locus. To test this hypothesis, we will delete specific enhancer-like regions, and mutate individual TF binding sites within these regions, in an Abelson (Abl) pre-B cell line using CRISPR/Cas9 homologous recombination genome editing technology. Culture of the Abl pre-B cell lines with the Abl-kinase inhibitor STI571 for 48 hours robustly induces a diverse repertoire of Ig? rearrangements. We will assay the effects of the deletions or mutations on long-range interactions by 3C (chromosome conformation capture) and 4C. Changes in the Ig? repertoire will be assayed by TaqMan real time PCR and by deep sequencing on an Ion Torrent PGM. If our hypothesis is correct, then deletion of key TF binding sites and/or larger regulatory regions with binding sites for several key TFs will affect the long-range looping patterns within the Ig? locus, and will affect the resulting Ig? repertoire. We wil determine whether the effect of the deletion on the resulting Ig? repertoire will be predominantl on genes in the vicinity of each putative regulatory region or more globally. If we should observe any of these effects, this would demonstrate a highly novel function for these elements bearing the epigenetic characteristics of traditional enhancers.
The establishment of a diverse repertoire of antibodies is essential for the proper functioning of the immune system to combat a wide variety of pathogens. Here we will study new regulatory elements that may orchestrate the V(D)J recombination process by which the light chain genes of antibodies are assembled. The large scale movement of the Ig loci during rearrangement places it in close proximity to other genes including oncogenes, and inappropriate regulation of the double-strand DNA breaks that occur during V(D)J recombination can then result in translocations of those oncogenes with Ig genes resulting in lymphomas and leukemias.
