Identification of critical residues for the function of the bHLH transcription factor SCL in hematopoietic stem cells
Porcher, C.; Schlaeger, T.M.; Flitter, S.; Orkin, S.H.; Vyas, P.
Blood 102(11): 131a
The basic helix-loop-helix (bHLH) transcription factor SCL/Tal-1 is essential for specification of hematopoietic stem cells during development. Studies of the prototype of this family, the muscle-specific protein MyoD, suggest that bHLH proteins bind DNA as dimers and regulate transcription of lineage-specific target genes by recruitment of co-activators or co-repressors. We have challenged this concept by showing that SCL does not need its DNA-binding activity for specification of hematopoietic stem cells (Porcher et al, Development 1999). In addition, the only domain crucial for this function is the heterodimerisation (or HLH) region. Therefore, the mechanism of action of SCL in stem cells relies, in large part, on protein-protein interactions mediated by its HLH domain. Characterization of residues within the SCL HLH domain necessary for specification of hematopoietic stem cells is of importance to understand protein complex formation and to identify protein partners of SCL involved in this central biological process. To address these questions, we have conducted an exhaustive mutational analysis of the SCL HLH region using both loss-of-function and gain-of-function approaches. We have generated chimeric proteins in which each individual domain of the SCL HLH region was substituted by the corresponding domain of other bHLH proteins. We have also engineered point mutations into the SCL HLH domain. These chimeric proteins were introduced into SCL-null ES cells by retroviral-mediated gene transfer and hematopoietic potential was tested by in vitro differentiation into embryoid bodies. The function of the mutants was quantitatively assessed by measuring rescue of primitive hematopoiesis. These loss-of-function studies demonstrated that one key residue in helix2 and, unexpectedly, most of the amino acids in the loop are essential for SCL function in early hematopoietic development. To validate these results, we have performed a gain-of-function study. We have introduced these key SCL residues into the MyoD HLH domain with the aim of converting this muscle-specific HLH domain into one capable of rescuing hematopoiesis when introduced into SCL-/- ES cells. With these modifications, MyoD HLH region was able to functionally substitute for SCL HLH region. Furthermore, we have shown by co-immunoprecipitation and mammalian two-hybrid experiments that the critical residue in SCL helix2 mediates binding of SCL to its known partner, the LIM-only protein LMO2. Like SCL, LMO2 is required for specification of hematopoietic stem cells. Our data provide evidence, for the first time, of a functional interaction between these two proteins in stem cells. In conclusion, we have identified functionally critical residues in SCL which we hypothesize serve as docking sites for interactions with crucial regulators of hematopoietic stem cell development. Identification of LMO2 as a protein binding to one of these residues supports our hypothesis. Surprisingly, the majority of the residues in the loop are important for SCL activity. Therefore, this region plays a crucial functional role rather than just being a bridge between two helices. These studies provide a biochemical and genetic platform to unveil the nature of a hematopoietic stem cell-specific protein complex.