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Sox6 belongs to the Sry (sex-determining region Y)–related high-mobility-group–box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.

Analysis of individual cells within phenotypically-uniform stem and progenitor populations reveals heterogeneity of cell states and affords exploration of their differential contribution to fate transitions. Herein, we investigate the transcriptional composition of lineage commitment transitions in the mouse hematopoietic system, obtained either spontaneously or driven by individual transcription factors (TF), to define alternative molecular states at the emergence of erythroid and myeloid lineage identity. To this end, we transcriptionally profile hundreds of individual cells across a spectrum of lineage states by quantitative RT-PCR on a Fluidigm platform. We explore the dual binary (on-off) vs. continuous (expression level) nature of PCR data and respectively calculate odds ratio, and Spearman correlation on co-expressing cells, for any pair of genes, in order to infer the circuitry underlying alternative routes to lineage commitment. This approach identifies Ddit3 as a specific target of GATA-driven networks that antagonizes Cebpa transcriptionally at the emergence of myeloid fate. Knockdown and knockout approaches indicate a requirement for Ddit3 in early erythroid decisions from stem cells. Enforced expression of Ddit3 in granulocytic-monocytic progenitors (GMP) results in down-regulation of individual Cebpfamily members and their downstream transcriptional programmes, including myelo-monocytic cytokine receptors. Notably, the resulting cells have robust self-renewal potential and express an early erythroid signature that is augmented in the presence of interleukin-3 and erythropoietin. Taken together, the data suggest that Ddit3 can nucleate incipient erythroid-affiliated programmes in early erythro-myelopoiesis, and may contribute to fate choice through antagonism with Cebp networks. Interestingly, ex-vivo culture and expression profiling of individual wild-type and DDIT3-enforced GMP suggest that the erythroid-primed state captured through Ddit3 expression may be found in a small proportion of native GMP, suggesting some fluidity of lineage identity in early progenitors. Disclosures: No relevant conflicts of interest to declare.