The K562 leukemic cell line can be used as a model system to increase our understanding of events associated with developmental expression and regulation in normal marrow progenitor cells. We are working to establish an in vitro transcription system in which the transcription pattern of the globin genes mimick that observed in vivo, i.e. expression of embryonic and fetal, but not adult globins. As an initial approach, in vitro transcriptional analysis using a whole cell and nuclear extract made from K562 cells was carried out. We have demonstrated that all cloned globin genes (beta, epsilon, and gamma) are transcribed in vitro indicating a lack of differential transcription of these genes. Entertaining the possibility that differential transcription of genes may require some type of DNA conformation, K562 chromatin was used as a DNA template. While the results are still preliminary, we find that proper transcriptional control similar to that observed in vivo can now be demonstrated. These results were obtained by S1 nuclease mapping wherein we observed in vitro transcription of the fetal but not adult globin genes. We are currently exploring several approaches to increase the sensitivity of our transcription assay to insure that this important observation is not a result of our inability to detect adult globin transcription. These include utilization of a K562 nuclear extract for transcription, altering transcription conditions so chromatin is more accessible to transcription factors, and using high specific activity SP6 globin RNA probes to detect accurate transcription. The establishment of an in vitro transcription system which mimicks the K562 transcription pattern observed in vivo will enable us to examine molecules which surround a gene and its control region when that gene is in an active state and when it and other genes are in a repressed state.
