A Role for Twist in Thymocyte Development.

Abstract Twist-1 and Twist-2 are basic helix-loop-helix (bHLH)- containing transcription factors important for mesoderm and muscle formation, and for osteoblast differentiation. The Twist proteins were previously shown to interact both physically and genetically with Runx2, a member of the core binding factor family of transcription factors (Bialek et al. Dev. Cell6: 423, 2004). Twist-1 and Twist-2 transiently inhibit Runx2 function during bone formation by inhibiting DNA binding by the Runx2 Runt domain. Since the Runt domains of the core binding factors are highly conserved, this raised the possibility that the Twist proteins may also regulate Runx1 function in hematopoiesis. To address this hypothesis we used Charlie Chaplin (CC) mice, which contain a hypomorphic Twist-1 allele generated by N-ethyl-N-nitrosourea (ENU) mutagenesis (Justice, Nat. Rev. Genet.1: 109, 2000). The CC allele encodes a single amino acid substitution (Ser192Pro) in the Twist box of Twist-1, which normally interacts with the Runt domain. CC/CC mice die from an unknown cause shortly after birth, and have small thymuses. No other hematopoietic organs show significant differences in cellularity. The number of bone marrow hematopoietic progenitors is almost identical among CC/CC, CC/+, and +/+ littermates as assessed by flowcytometry and colony forming assays. The ratios of CD4/CD8 single positive cells, and DN1-4 thymocytes are unperturbed, suggesting that T cell maturation is normal in CC/CC fetuses. However, CC/CC thymuses are reconstituted by wild type bone marrow at a lower efficiency than are wild type thymuses in fetal thymus organ culture assays, suggesting that Twist-1 may have an important non-cell autonomous role in thymocyte development.