B and T lymphocyte development is driven by V(D)J recombination, a process by which gene segments at the antigen receptor loci are repeatedly rearranged to create a vast repertoire of antigen receptor loci that recognize and protect against foreign pathogen. Given the deleterious consequences of aberrantly repaired double strand breaks (DSBs) and their contribution to leukemia and lymphoma, B and T cells must tightly regulate the recombination process. Control is exerted at three main levels: lineage specificity (e.g., completed rearrangements of B cell loci occur only in B cells and not T cells), coordinated, ordered rearrangement within a given lineage (e.g., the heavy chain locus in B cells rearranges before the light chain loci), and allelic exclusion (cell surface expression of only one functionally rearranged allele). The Skok lab has been interested in trying to understand the mechanisms underlying coordinated recombination between individual alleles and loci during lymphocyte development. We previously published that inter-locus pairing between Igh and Igk is involved in coordinating rearrangement at the pre-B cell stage so that Igh rearrangement finishes when Igk rearrangement starts. At the Igh locus, loss of intra-chromosomal connections and altered locus accessibility occur as a result of an inter-chromosomal interaction with pericentromerically located Igk alleles. Igk (located on chromosome 6) directs the unrearranged Igh allele (located on chromosome 12) to pericentromeric heterochromatin where the two loci associate, mediated by the 3'enhancer (3'E?) of the Igk locus. Repositioning of the unrearranged Igh locus to pericentromeric heterochromatin limits access to RAG proteins while decontraction provides a physical barrier to further V gene rearrangement2. However, the mechanism by which Igk is repositioned to pericentromeric regions (which is the initial step in this process) are not known. Recent data generated by Hans-Martin Jack and colleagues indicate that Ig? mRNA has a role in enforcing allelic exclusion at the Igh locus. Intriguingly, this group showed that the presence of a stable noncoding Ig? mRNA can perform the same function as a functional Ig? mRNA. These findings prompted us to analyze Igh and Igk in individual developing B cells from two mouse lines (Ter5hi and Ter5lo) that express differential levels of stable noncoding Ig? mRNA. Our preliminary experiments identify a role for Ig? mRNA in repositioning Igk to pericentric regions and in regulating Igk cleavage. We now aim to explore this in greater depth to test our hypothesis that Ig? mRNA acts in trans to regulate nuclear accessibility and allelic exclusion of Igk, which in turn is important for control of Igh nuclear accessibility and allelic exclusion.
My lab demonstrated that one Igk allele directs one Igh allele to a common pericentromeric cluster, where the two transiently associate, and that pairing of the two loci in this repressive nuclear compartment at the pro- to pre-B cell transition induces decontraction of the Igh locus, which is important for establishing allelic exclusion and preventing ongoing cleavage. Intriguingly, we have now discovered that Ig? mRNA is involved in this regulation. In this application we aim to identify the salient features by which Ig? mRNA alters accessibility to control two important aspects of recombination: allelic exclusion and coordinate rearrangement of the Igh and Igk loci.