Runx1/Cbfß2 complexes are required for lymphoid tissue inducer cell differentiation at two developmental stages.

Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer's patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfß transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfß2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4ß7 integrin (α4ß7)(+) IL-7R α-chain (IL-7Rα)(+) population, can be subdivided into Rorγt-expressing IL-7Rα(high) cells and nonexpressing IL-7Rα(mid) cells. Whereas Id2 and Rorγt are required to direct α4ß7(+)IL-7Rα(mid) cells to become α4ß7(+)IL-7Rα(high) cells, Runx1/Cbfß2 complexes are necessary for the emergence of α4ß7(+)IL-7Rα(mid) cells. In addition, the loss of Cbfß2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4ß7(+)IL-7Rα(+) LTi cells at anlagen. Our results thus revealed that Runx1/Cbfß2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.

Phosphorylation of Runx1 at Ser249, Ser266, and Ser276 is dispensable for bone marrow hematopoiesis and thymocyte differentiation.

Runx1, one of three mammalian runt-domain transcription factor family proteins, is essential for definitive hematopoiesis. Based on transfection assays, phosphorylation of Runx1 at the three serine residues, Ser249, Ser266, and Ser276, was thought to be important for trans-activation activity of Runx1. By using "knock-in" gene targeting, we generated mouse strains expressing mutant Runx1 protein that harbored a combined serine-to-alanine substitution at either of two residues, Ser249/Ser266 or Ser249/Ser276. Either mutation resulted in a lack of major phosphorylated form of Runx1. However, while loss of definitive hematopoiesis and impaired thymocyte differentiation was observed following the loss of Runx1, these phenotypes were rescued in those mice lacking the major phosphorylated form of Runx1. These results not only challenge the predicted regulation of Runx1 activity by phosphorylation at these serine residues, but also reaffirm the effectiveness of "knock-in" mutagenesis as a powerful tool for addressing the physiological relevance of post-translation modifications.

Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.

Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I-restricted thymocytes into CD4+CD8- helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.