Transcription of Bcl-x In erythroid progenitor cells

Bcl-x has been shown to be essential for survival and maturation of late erythroid progenitors and for survival of cells in the developing brain. During the late phases of erythroid differentiation there is a large increase in the Bcl-x protein and its mRNA in mouse and human erythroblasts. In mouse erythroblasts and other hematopoietic cells, the main transcription initiation sites form a cluster at positions −662, −653 and −643 relative to the ATG initiation codon. This cluster of sites accounts for about 70% of all transcripts in these cells and another 15 to 20% appear to be initiated at position −112. Human erythroblasts exhibit a doublet of transcription start sites at −660 and −654 relative to the initiation codon that is homologous to the main cluster in the mouse. In contrast to hematopoietic tissues, a large fraction of bcl-x transcripts isolated from mouse brain originates from start sites far upstream of those used in erythroblasts. To determine which sequences upstream of the coding region of the gene are important for regulation of transcription, plasmid constructs were made that contain various portions of this region of bcl-x (from −1 to −1211) placed just upstream of the firefly luciferase gene. These plasmids were used in transient transfection assays of bcl-x promoter function in the mouse erythroid cell line HCD57, a line that is dependent on erythropoietin, EPO, for viability. A DNA sequence between positions −196 and −96 was found to be essential for transcription, and this sequence was sufficient for transcription at nearly the maximum rate observed for any of the plasmids. Additional bcl-x sequence between −196 and −998 had little effect on the rate of transcription. However, plasmids containing the sequence between −998 and −1211 exhibited a significant reduction of transcription rate, indicating that there is a negative regulatory element in that region. Transient transfection experiments did not show evidence for localized regulatory sequences within the −1 to −1211 region that modulate transcription specifically in response to EPO.