The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.

Background:The Sox2-protein interactome in ESC has not been identified. Results: ESC that exogenously express Oct4, Sox2, Klf4, and c-Myc self-renew. This permitted the identification of the Sox2-interactome in ESC. Conclusion: Sox2 associates with Ͼ70 proteins, and the knockdown of the Sox2-associated protein Smarcd1 induces the differentiation of ESC. Significance: This is the first description of the Sox2-interactome in undifferentiated ESC.

SUMMARYDefects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1 -/-mice have a nearly complete perinatal lethality. Histologic examination of Ets1-/-embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1 -/-mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.

The transcriptional co-factor Friend of GATA1 (FOG-1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N-terminus. To test the importance of FOG-1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG-1/NuRD interaction (FOG-1 R3K5A). Homozygous FOG-1 R3K5A mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG-1 R3K5A/R3K5A megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG-1/NuRD interaction is required for the earlier described 'GATA Switch'. In addition, ablation of FOG-1/ NuRD interaction led to inappropriate expression of mast cell and eosinophil-specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG-1 R3K5A/R3K5A megakaryocytes, suggesting that FOG-1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG-1/NuRD interaction for the re-enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.

Summary The bHLH transcription factor ATOH7 (Math5) is essential for establishing retinal ganglion cell (RGC) fate. However, Atoh7-expressing retinal progenitor cells (RPCs) can give rise to all retinal cell types, suggesting that other factors are involved in specifying RGCs. The basis by which a subpopulation of Atoh7-expressing RPCs commits to an RGC fate remains uncertain but is of critical importance to retinal development since RGCs are the earliest cell type to differentiate. To better understand the regulatory mechanisms leading to cell-fate specification, a binary genetic system was generated to specifically label Atoh7-expressing cells with GFP. FACS-purified GFP+ and GFP- cells were profiled by RNA-seq. Here, we identify 1,497 transcripts that were differentially expressed between the two RPC populations. Pathway analysis revealed diminished growth factor signaling in Atoh7-expressing RPCs, indicating that these cells had exited the cell cycle. In contrast, axon guidance signals were enriched, suggesting that axons of Atoh7-expressing RPCs were already making synaptic connections. Notably, many genes enriched in Atoh7-expressing RPCs encoded transcriptional regulators, and several were direct targets of ATOH7, including, and unexpectedly, Ebf3 and Eya2. We present evidence for a Pax6-Atoh7-Eya2 pathway that acts downstream of Atoh7 but upstream of differentiation factor Pou4f2. EYA2 is a protein phosphatase involved in protein-protein interactions and posttranslational regulation. These properties, along with Eya2 as an early target gene of ATOH7, suggest that EYA2 functions in RGC specification. Our results expand current knowledge of the regulatory networks operating in Atoh7-expressing RPCs and offer new directions for exploring the earliest aspects of retinogenesis.