The canonical BCL repressive mechanism of action in GC B

The canonical BCL6 repressive mechanism of action in GC streptozotocin receptor is mediated by recruitment of the SMRT, NCOR, and BCOR corepressors to the BCL6 BTB domain (Hatzi and Melnick, 2014). However, we find that Bcl6 BTB mutant mice are still mostly capable of forming early GC clusters. This phenotype is in contrast to the complete abrogation of GC formation observed in BCL6 RD2 mutant mice. Hence, whereby BCL6 mediates early steps in commitment to the GC fate and clustering into nascent GCs through the RD2 domain, once GCs are established, Bcl6 is then required to maintain the proliferation and survival by repressing genes through its BTB domain. These findings suggest a model of sequential and biochemically distinct biological functions of BCL6 at different GC B cell developmental stages (Figure S6). It is also possible that the RD2 domain might also contribute to later events in established GCs such as regulation of terminal differentiation (Fujita et al., 2004, Parekh et al., 2007), although such functions cannot be assessed in the RD2 animal model developed here. This pre-GC B cell differentiation deficiency caused by RD2 loss of function was explained at least in part by the finding that the BCL6 RD2 domain is required to repress expression of the key migration factors GRP183 and S1PR1. Downregulation of GRP183 is critically important for B cell migration into the follicle center (Gatto et al., 2009, Pereira et al., 2009). S1PR1 plays a key role in enabling B cell trafficking out of follicles (Cinamon et al., 2004). Our finding that BCL6 directly represses these genes in GC cells suggests a role for BCL6 at an early stage of the GC response, whereby BCL6 can enable “capture” of B cells within follicles to enable their clustering. At the same time, S1PR2 upregulation in GC B cells confines them to an S1P-low niche within follicles (Green et al., 2011). We observed that S1PR1 overexpression promoted GC-type B cell migration and antagonized S1PR2 (Figure S5G). Taken together, the data suggest that dynamic regulation and equilibrium of S1PR1 and S1PR2 is a critical event in the early pre-GC phase of the humoral immune response, controlled at least in part through BCL6 direct binding and repression of S1PR1. From a mechanistic standpoint, the BCL6 RD2 domain represses the GPR183 and S1PR1 loci by recruiting HDAC2, but not MTA3-NuRD, to suppress the enhancer activation mark H3K27ac at their distal regulatory elements. However, these data do not exclude the possibility that other as yet unknown corepressor proteins may bind with BCL6-HDAC2 repression complexes to these key target genes. Taken together with recent findings showing that BCL6 BTB domain corepressors distribute to different sets of genomic loci with unique functions in GC B cells (Hatzi et al., 2013), these data suggest that transcriptional programming by BCL6 is exquisitely compartmentalized through linkage of distinct biochemical functions to genes involved in specific immunological and biological functions.