title: Cloning and characterization of a potential transcriptional activator of human gamma-globin genes.
abstract: Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human gamma-globin and switch to human beta globin expression as time in culture advances. One explanation for the initially predominant expression of gamma-globin gene in these hybrids is the presence of trans-acting factors that 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 potential gamma gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only gamma-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to beta globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal 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, suggesting that this new gene encodes a protein that may function as gamma gene activator.
authors.affiliation: Division of Hematology, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: Z
authors.initials: Z
authors.lastname: Duan
authors.forename: E
authors.initials: E
authors.lastname: Skarpidi
authors.forename: Q
authors.initials: Q
authors.lastname: Li
authors.forename: T
authors.initials: T
authors.lastname: Papayannopoulou
authors.forename: G
authors.initials: G
authors.lastname: Stamatoyannopoulos
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018929:Cell Culture Techniques; D002678:Chimera / Q000235:genetics; D003001:Cloning, Molecular; D004900:Erythroblasts / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000235:genetics; D005333:Fetus / Q000166:cytology; D015870:Gene Expression; D020869:Gene Expression Profiling; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014018:Tissue Distribution; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
chemical_list: C435903:ANKRD49 protein, human; D012333:RNA, Messenger; D015534:Trans-Activators; D005914:Globins; D005319:Fetal Hemoglobin