To characterize and isolate the trans-acting protein factors specific human globin gene expression, we have prepared nuclear extracts from hemin-induced and uninduced K562 cells for use in a soluble cell-free in vitro system (a run-off assay). In order to examine the 5' promoter region which can affect transcriptional activity, the epsilon-globin gene promoter was truncated at 10 different sites by restriction enzymes. The variation in transcriptional activity of the epsilon-globin gene was observed depending on deleted region of promoter, suggesting the possible regions to which nuclear proteins involved in transcriptional process may bind. K562 nuclear extracts can not initiate transcription from beta- globin gene. However, when K562 nuclear extracts are supplemented with HeLa whole cell extracts, beta-globin gene transcription is observed in vitro. To identify the trans-acting proteins necessary for specific globin gene expression, the chromatographic fractionation of crude nuclear extracts of K562 cells are now in progress. The structural requirements for active gene transcription are being assessed by examining the binding characteristics of nuclear protein extracts from K562 cells to regions of the epsilon promoter and 2 kilobase 5'-flanking region, using gel-retardation electrophoresis, DNA- footprinting and ion exchange chromatography. To examine the functional requirements for transcription, a globin-hybrid gene system has been designed to facilitate the analysis, separation and recovery of viable cells in which the epsilon-globin gene is actively transcribed.
