Cloning and characterization of a potential transcriptional activator of human gamma-globin genes

Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse eryth roleukemia (MEL) cells initially produce predominantly or exclusively human gamma -globin and switch to human beta globin expression as time in cultur e advances. One explanation for the initially predominant expression of gam ma -globin gene in these hybrids is the presence of trans-acting factors th at activate gamma -globin gene transcription. We used differential display of hybrids before and after the gamma to beta switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for pot ential gamma gene activators. Identically sized amplicons which were presen t in fetal liver erythroblasts and in the hybrids expressing only gamma -gl obin but were absent in the adult erythroblasts and in the same hybrids aft er they had switched to beta globin expression were cloned and sequenced. F ifty pairs of cDNAs fitting these criteria were chosen for further analysis . The sequences of the two members of 48 pairs differed from each other, re vealing the low efficiency of this experimental approach. One clone pair co ded for human proteosome subunit X. The second pair coded for a protein con taining an acidic domain in the N-terminus and three consecutive CDC10/SW16 /ankyrin repeats in the C-terminus. Transactivation assays in the yeast hyb rid system and transient transfection assays in COS cells showed that a pot ent trans-activating domain resides in the N-terminus of this protein. Nort hern blot and RT-PCR assays showed that this gene is expressed in several f etal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of gamma mRNA in HFE X MEL cell hybrids that undergo the gamma to beta switch, suggestin g that this new gene encodes a protein that may function as gamma gene acti vator. (C) 2001 Academic Press.