This grant proposal represents continuation of studies initiated in our laboratory to study the regulation of the human alpha-globin and beta- globin genes in normal erythroid cells and in erythroleukemic cells. The first part of this project is an investigation of the regulatory elements of the alpha-globin genes that are responsible for its well known enhancer- independent expression upon transfer into heterologous cells. We have identified a regulatory element within the 5' end of the structural gene that is responsible for this enhancer-independent expression. We believe that this element may play an important role in the control of expression of this gene in vivo. We propose to dissect this regulatory element and study its interactions with trans-acting regulatory protein in erythroid and non-erythroid cells. We also propose to study the interaction of this element with the newly described regulatory domain located far upstream of the alpha-globin gene cluster (i.e. alphaLCR). The second part of this project deals with the study of the molecular basis of delta to beta-globin gene switch that occurred in K562 erythroleukemia cells grown in culture in our laboratory. Whereas K562 erythroleukemic cells are known to express the zeta, alpha, epsilon, gamma and delta-globin genes in an inducible manner, the expression of the human beta-globin genes has not been previously described in these cells. We have observed a spontaneous and stable delta to beta-globin switch in a variant of this cell line we designated K562-BM. Other isolates of K562 cell do not undergo the same switch when grown under the same culture conditions. We show that this switch is a result of changes in the trans-acting environment of these cells. A construct containing a beta-globin promoter linked to a reporter gene is expressed efficiently upon transfection into K562-BM cells. The same construct is inactive when transfected into classical K562 cells. We propose to study the molecular mechanism of this switch of the trans-acting environment of the cells using DNase I hypersensitivity studies, gel retardation, DNase I foot-printing, site- directed mutagenesis and transient expression studies. The study of this interesting variant of K562 erythroleukemia cells may improve our understanding of factors involved in normal beta-globin gene regulation.
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