Regulation of the Signal-Dependent E Protein HEBAlt Through a YYY Motif Is Required for Progression Through T Cell Development.

The E protein transcription factors E2A and HEB are critical for many developmental processes, including T cell development. We have shown that the Tcf12 locus gives rise to two distinct HEB proteins, with alternative (HEBAlt) and canonical (HEBCan) N-terminal domains, which are co-expressed during early T cell development. While the functional domains of HEBCan have been well studied, the nature of the HEBAlt-specific (Alt) domain has been obscure. Here we provide compelling evidence that the Alt domain provides a site for the molecular integration of cytokine signaling and E protein activity. Our results indicate that phosphorylation of a unique YYY motif in the Alt domain increases HEBAlt activity by 10-fold, and that this increase is dependent on Janus kinase activity. To enable in vivo studies of HEBAlt in the T cell context, we generated ALT-Tg mice, which can be induced to express a HA-tagged HEBAlt coding cassette in the presence of Cre recombinases. Analysis of ALT-Tg mice on the Vav-iCre background revealed a minor change in the ratio of ISP cells to CD8+ SP cells, and a mild shift in the ratio of T cells to B cells in the spleen, but otherwise the thymus, spleen, and bone marrow lymphocyte subsets were comparable at steady state. However, kinetic analysis of T cell development in OP9-DL4 co-cultures revealed a delay in early T cell development and a partial block at the DN to DP transition when HEBAlt levels or activity were increased. We also observed that HEBCan and HEBAlt displayed significant differences in protein stability that were resolved in the thymocyte context. Finally, a proteomic screen identified STAT1 and Xpo1 as potential members of HEBAlt-containing complexes in thymocytes, consistent with JAK-induced activation of HEBAlt accompanied by translocation to the nucleus. Thus, our results show that the Alt domain confers access to multiple layers of post-translational control to HEBAlt that are not available to HEBCan, and thus may serve as a rheostat to tune E protein activity levels as cells move through different thymic signaling environments during T cell development.

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development, and is limited to immature CD4-CD8- thymocytes. The major proliferative event is initiated at the 'ß-selection' stage following successful rearrangement of Tcrß, and is triggered by and dependent on concurrent signaling by Notch and the pre-T cell receptor (TCR); however, it is unclear how these signals cooperate to promote cell proliferation. Here, we found that ß-selection-associated proliferation required the combined activity of two Skp-cullin-F-box (SCF) ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for polyubiquitination and proteasomal degradation. We found that Notch signals induced the transcription of Fbxl1, whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of ß-selected thymocytes.

A key role for IL-7R in the generation of microenvironments required for thymic dendritic cells.

Interleukin-7 receptor (IL-7R) signaling is critical for multiple stages of T-cell development, but a role in the establishment of the mature thymic architecture needed for T-cell development and thymocyte selection has not been established. Crosstalk signals between developing thymocytes and thymic epithelial cell (TEC) precursors are critical for their differentiation into cortical TECs (cTECs) and medullary TECs (mTECs). In addition, mTEC-derived factors have been implicated in the recruitment of thymic dendritic cells (DCs) and intrathymic DC development. We therefore examined corticomedullary structure and DC populations in the thymus of Il7r-/- mice. Analysis of TEC phenotype and spatial organization revealed a striking shift in the mTEC to cTEC ratio, accompanied by disorganized corticomedullary structure. Several of the thymic subsets known to have DC potential were nearly absent, accompanied by reductions in DC cell numbers. We also examined chemokine expression in the Il7r-/- thymus, and found a significant decrease in mTEC-derived CCR7 ligand expression, and high levels of cTEC-derived chemokines, including CCL25 and CXCL12. Although splenic DCs were similarly affected, bone marrow (BM) precursors capable of giving rise to DCs were unperturbed. Finally, BM chimeras showed that there was no intrinsic need for IL-7R signaling in the development or recruitment of thymic DCs, but that the provision of wild-type progenitors enhanced reconstitution of thymic DCs from Il7r-/- progenitors. Our results are therefore supportive of a model in which Il7r-dependent cells are required to set up the microenvironments that allow accumulation of thymic DCs.

Targeted Disruption of TCF12 Reveals HEB as Essential in Human Mesodermal Specification and Hematopoiesis.

Hematopoietic stem cells arise from mesoderm-derived hemogenic endothelium (HE) during embryogenesis in a process termed endothelial-hematopoietic transition (EHT). To better understand the gene networks that control this process, we investigated the role of the transcription factor HEB (TCF12) by disrupting the TCF12 gene locus in human embryonic stem cells (hESCs) and inducing them to differentiate toward hematopoietic outcomes. HEB-deficient hESCs retained key features of pluripotency, including expression of SOX2 and SSEA-4 and teratoma formation, while NANOG expression was reduced. Differentiation of HEB-/- hESCs toward hematopoietic fates revealed a severe defect in mesodermal development accompanied by decreased expression of regulators of mesoendodermal fate choices. We also identified independent defects in HE formation at the molecular and cellular levels, as well as a failure of T cell development. All defects were largely rescued by re-expression of HEB. Taken together, our results identify HEB as a critical regulator of human mesodermal and hematopoietic specification.

The robustness of a signaling complex to domain rearrangements facilitates network evolution.

The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.