Common lymphoid progenitors (CLPs) are the first bone marrow precursors in which V(D)J recombinase activity is up-regulated. Here, we show that loss of the transcription factor E47 produces a reduced CLP population that lacks V(D)J recombinase activity and D-JH rearrangements in vivo. Apart from a profound arrest before the pro–B cell stage, other downstream lymphoid progeny of CLPs are still intact in these mice albeit at reduced numbers. In contrast to the inhibition of recombinase activity in early B lineage precursors in E47-deficient animals, loss of either E47 or its cis-acting target Erag (enhancer of rag transcription) has little effect on recombinase activity in thymic T lineage precursors. Taken together, this work defines a role for E47 in regulating lineage progression at the CLP stage in vivo and describes the first transcription factor required for lineage-specific recombinase activity.

V(D)J recombinase activity is expressed in bone marrow (BM) multipotent progenitors (MPPs) that retain the potential to differentiate into B cells in BM and T cells in thymus. However, it remains unclear whether MPPs in each organ exhibit distinct transcriptional requirements for recombinase activity, or whether thymic MPPs imprint the requirements of upstream BM MPPs from which they originate. Here we use V(D)J recombination reporter mice to examine transcriptional requirements in BM versus thymic multipotent subsets. Within BM, the frequency of recombinase+ cells increases from 5% in MPPs to 15% in common lymphoid progenitors (CLPs) and 20% in pre-pro B cells. Within thymus, 10% of early thymic progenitors (ETPs) are recombinase+. While the E47 transcription factor is expressed in both BM and thymus, E47 deficiency selectively ablates V(D)J recombination in the former. BM MPPs, CLPs and pre-pro B cells from E47 knockouts lack detectable recombinase activity. In the thymus, E47 deficiency reduces ETP numbers but V(D)J recombinase activity is maintained. Loss of the related E protein HEB does not diminish recombinase activity in ETPs while functional inhibition of E47:HEB complexes leads to a 40% reduction in TCRβ rearrangements. Thus, V(D)J recombinase activity is regulated differently in MPPs in BM and thymus. E protein transcription factors are essential for recombinase activity in multipotent BM MPPs and CLPs but are dispensable in multipotent thymic ETPs.

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Uncommitted bone marrow precursors replenish the immune system throughout life, and it is of great interest to understand the mechanisms that control their functional integrity. We show that E47 knockout (KO) mice have major defects in multipotent LSKs, a subset that contains self‐renewing hematopoietic progenitors. We have found that LSKs from E47 KO mice have skewed levels of the flk2/flt3 cytokine receptor as well as the cell cycle regulator p21. 70% of wild type LSKs are flk2+ versus only 30% of E47 KO LSKs. Moreover, p21 expression is reduced by 50% in the latter mice. Two populations thought to be downstream of LSKs, common lymphoid progenitors (CLPs) and early thymic progenitors (ETPs) are both reduced ~4–10‐fold in E47 KO animals. While V(D)J recombinase activity, an indicator of lymphoid lineage progression, is normally expressed in ~50% of CLPs and ETPs, interruption of E47 impairs this process. We have shown that loss of E47 ablates recombinase activity in CLPs and now show that loss of E47:HEB complexes ablates recombinase activity in ETPs. Specifically, inhibition of E47:HEB leads to a 40% reduction in recombination activity as assessed at the single cell level. Together, these data indicate a key role for E47 in the functional integrity of LSKs, and the ability of these cells to effectively repopulate downstream lymphoid compartments. Support from: NIH, Elsa U Pardee Cancer Research Foundation, and USIDNET.