Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation (SHM) and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages (TBMs) and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentization of non-compatable functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet, how the pre-BCR mediates these functions remains unclear. Herein, we demonstrate that the pre-BCR initiated a feed-forward amplification loop mediated by the transcription factor IRF4 and the chemokine receptor CXCR4. CXCR4 ligation by CXCL12 activated the mitogen-activated protein kinase (MAPK) ERK which then directed the development of small pre-and immature B cells including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin 7 (IL-7) had only modest effects on B cell developmental transcriptional and epigenetic programs. These data demonstrate a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Thymocyte development requires a complex orchestration of multiple factors. Ablating either Tcf-1 or HEB in CD4+CD8+-thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and reduced late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that Tcf-1 and HEB share ~7000 DNA binding-sites genome- wide and promote chromatin accessibility. The binding of both Tcf-1 and HEB is required at these shared sites for epigenetic and transcriptional gene regulation. Binding of Tcf-1 and HEB to their conserved motifs in enhancer regions of T-cell differentiation and survival genes promotes their expression. Binding to sites that lack conserved motifs in promoter regions of cell-cycle genes limits proliferation. Tcf-1 alone displaces nucleosomes to allow for chromatin accessibility. Importantly, Tcf-1 inhibits Notch-signaling to protect HEB from Notch mediated proteasomal degradation. Thus, Tcf-1 shifts nucleosomes and safeguards HEB to enable their cooperation in establishing the epigenetic and transcription profile of CD4+CD8+-thymocytes.

Transcription factor (TF) networks determine cell fate in hematopoiesis. However, how TFs cooperate with other regulatory mechanisms to instruct transcription remains poorly understood. Here we show that in small pre-B cells, the lineage restricted epigenetic reader BRWD1 closes early development enhancers and opens the enhancers of late B lymphopoiesis to TF binding. BRWD1 regulates over 7000 genes to repress proliferative and induce differentiation programs. However, BRWD1 does not regulate the expression of TFs required for B lymphopoiesis. Hypogammaglobulinemia patients with BRWD1 mutations have B-cell transcriptional profiles and enhancer landscapes similar to those observed in Brwd1-/- mice. These data indicate that, in both mice and humans, BRWD1 is a master orchestrator of enhancer accessibility that cooperates with TF networks to drive late B-cell development.

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