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

Recent studies of erythropoietin (Epo) receptor signalling suggest that sig nals for mitogenesis, survival and differentiation are relayed efficiently by receptor forms lacking at least seven of eight cytoplasmic (phospho)tyro sine [(P)Y] sites for effector recruitment. While such receptor forms are k nown to activate Jak2 and a limited set of known immediate response genes ( IRGs), the complex activities they exert predict the existence of additiona l target genes. To identify such targets, a minimal Epo receptor chimera wa s expressed in Epo-responsive erythroid SKT6 cells, and genes whose transcr iption is induced via this active receptor form mere cloned by subtractive hybridization. Several known genes not previously linked to Epo signalling mere discovered to be Epo IRGs including two which may further propagate Ep o signals [Prl1 tyrosine phosphatase and receptor activator of of NF kappa B (Rank)], and three regulators of protein synthesis (EF1 alpha, eIF3-p66 a nd Nat1), Several Epo IRGs mere novel murine clones including FM2 and FM6 w hich proved to represent broadly expressed IRGs, and FM3 and FL10 which wer e induced primarily in haematopoietic cells. Interestingly, FL10 proved to correspond to a recently discovered regulator of yeast mating-type switchin g, and mas induced by Epo in vivo. Thus, several nem Epo signalling targets are described, which mag modulate haematopoietic cell development. (C) 200 0 Academic Press.