Title: Regulation of E Protein Activity During Dendritic Cell Development
Description: Dendritic cells are a key population in the immune system. There are two main subsets: conventional DC (sometimes called classical DC, cDCs), which survey tissues for pathogens and activate naive T cells, and plasmacytoid dendritic cells (pDCs), which produce high amounts of IFNα in response to viral products. Both subsets begin development in the bone marrow from common dendritic progenitor (CDP), and driven by Flt3 signaling induced by the growth factor Flt3L. The CDP gives rise to a cDC committed preDC which exits the bone marrow and seeds peripheral organs to give rise to cDCs, while pDCs finish development in the bone marrow and migrate into the periphery as mature cells. pDC development is directed by E2-2, a member of the E protein family of basic helix-loop-helix transcription factors that are obligate dimers and bind an E-box sequence. E proteins are antagonized by Id proteins, and Id family member Id2 is required for cDC development. The apparently cell intrinsic choice of pDC vs. cDC fate is determined by the net activity of E proteins.

Loss of E2-2 affects only pDC development, but its expression is not as specific, as E2-2 is also expressed in cDCs, macrophages and B cells. E2-2 expression in cDCs was particularly puzzling, considering the dependence of cDCs on Id2, the E2-2 antagonist. We strived to address the discrepancy between the very specific activity of E2-2 in pDC development and its broader expression. This work shows that the E2-2 locus encodes two independently regulated isoforms. E2-2Long, the more active isoform is expressed only in pDCs, and E2-2Short, the less transcriptionally active isoform, is expressed more broadly. Moreover, E2-2Long is required for optimal pDC development. Homozygous loss of just E2-2Long leads to lower pDC frequency in the spleen and phenotypically affected pDCs in the periphery. Although E2-2 is essential for pDC development, no signal has yet been identified that induces E2-2 expression to influence pDC over cDC fate. Given the essential nature of E2-2 in pDC development, we aimed to understand better how E2-2 is regulated during pDC fate specification.

We examined an E2-2Long reporter and found that E2-2Long expression precedes pDC commitment in early progenitors. E2-2 is only upregulated in committed pDCs, and actively shut down in cDCs through the expression of Id2. Analysis of E2-2 in an in vitro time controlled model of dendritic cell development showed that all dendritic cells (both cDCs and pDC) go through an E2-2 expressing stage only to resolve into E2-2- cDCs and E2-2+ pDCs. Early E2-2 expression and E2-2 upregulation upon pDC commitment hinted at the presence of an E2-2 controlled cis-regulatory module. ChIP-Seq data showed only one peak of E2-2 binding located 150 kb downstream of the TCF4 gene. Heterozygous deletion of this region in the in vitro DC development model led to impaired pDC development and a fail to undergo the E2-2+ stage described above. The regulatory element is essential for E2-2 upregulation during DC development. This work describes two new mechanisms of E protein regulation during dendritic cell development: cell specific differential isoform usage and a distal cis regulatory element responsible for enforcing and upregulating E2-2 expression.

These new mechanisms can lead to better understanding of how this family of broadly expressed and pleiotropic transcription factors is regulated, with implications in overall development, not only dendritic cell fate decision.