The hallmark of B lymphopoiesis is the sequential productive genomic rearrangement of immunoglobulin heavy (Ig) then light chain loci. Following in-frame Ig recombination, expression of the pre-BCR is associated with IL-7 dependent clonal expansion. However, pre-B cells must exit cell cycle before initiating Igk recombination. Failure to do so risks genomic instability and leukemic transformation. Downstream of the IL-7R, STAT5 drives proliferation by inducing cyclin D3 while simultaneously repressing Igk recombination. We have now elucidated three mechanisms by which STAT5 represses Igk. First, STAT5 binds the Igk intronic enhancer as a tetramer and recruits the histone methyltransferase Ezh2 that decorates the Jk and Ck regions with H3K27me3. STAT5 also represses the bromodomain containing epigenetic reader BRWD1. In the absence of BRWD1, Igk did not recombine efficiently and this was associated with diminished Jk accessibility and disordered nucleosome positioning at both Jk exons and the Igk enhancers. BRWD1 was required for clearing nucleosomes from extended GAGA motifs and for recruiting RAG1/2 to Jk. These findings suggest critical and comprehensive roles for BRWD1 in chromatin remodeling, gene transcription and in targeting recombination to Igk. Finally, STAT5 induces expression of cyclin D3, which both drives proliferation and represses Vk transcription. Confocal and super-resolution microscopy demonstrated that in pro-B cells, Vk was imbedded within a web of closely juxtaposed nuclear matrix-associated cyclin D3 and elongating-pol II (transcription centers). In these cells, Vk did not access the transcription centers. However, upon transition to the small pre-B cell stage, or when cyclin D3 was absent, one allele of Vk looped into e-pol II. In contrast, in small pre-B cells JkCk more commonly associated with e-pol II in a biallelic manner. These later findings suggest that Vk accessibility, and not JkCk accessibility, contributes to allelic exclusion. In toto, these findings suggest a comprehensive model of Igk regulation in which radically different mechanisms of repression, control of mobile Vk segments access to e-pol II and epigenetic regulation of a fixed JkCk platform, determine Igk accessibility to recombination, and ensure that proliferation and recombination remain mutually exclusive.
Aim 1. Define the functional importance of BRWD1-mediated chromatin remodeling in B lymphopoiesis.
Aim 2 : Determine how GAGA motif recognition by BRWD1 contributes to Igk recombination.
Aim 3. Determine the mechanisms regulating Vk accessibility.
The development of B cells (antibody secreting cells) is a highly ordered series of molecular processes. Failure in any one of these leads to immunodeficiency while disorder in these processes can cause leukemia. This project is focused on the transition between two B cell developmental mechanisms, cell division and immunoglobulin light chain recombination.
|Mandal, Malay; Hamel, Keith M; Maienschein-Cline, Mark et al. (2015) Histone reader BRWD1 targets and restricts recombination to the Igk locus. Nat Immunol 16:1094-103|
|Teng, Grace; Maman, Yaakov; Resch, Wolfgang et al. (2015) RAG Represents a Widespread Threat to the Lymphocyte Genome. Cell 162:751-65|