Subtraction cloning and initial characterization of novel epo-immediate response genes

Recent studies of erythropoietin (Epo) receptor signalling suggest that signals for mitogenesis, survival and differentiation are relayed efficiently by receptor forms lacking at least seven of eight cytoplasmic (phospho)tyrosine [(P)Y] sites for effector recruitment. While such receptor forms are known to activate Jak2 and a limited set of known immediate response genes (IRGs), the complex activities they exert predict the existence of additional target genes. To identify such targets, a minimal Epo receptor chimera was expressed in Epo-responsive erythroid SKT6 cells, and genes whose transcription is induced via this active receptor form were cloned by subtractive hybridization. Several known genes not previously linked to Epo signalling were discovered to be Epo IRGs including two which may further propagate Epo signals [Prl1 tyrosine phosphatase and receptor activator of of NFkappaB (Rank)], and three regulators of protein synthesis (EF1alpha, eIF3-p66 and Nat1). Several Epo IRGs were novel murine clones including FM2 and FM6 which proved to represent broadly expressed IRGs, and FM3 and FL10 which were induced primarily in haematopoietic cells. Interestingly, FL10 proved to correspond to a recently discovered regulator of yeast mating-type switching, and was induced by Epo in vivo. Thus, several new Epo signalling targets are described, which may modulate haematopoietic cell development.