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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Mount Sinai School of Medicine
Schools of Medicine
New York
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Ren, S; Wong, B Y; Li, J et al. (1996) Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control of the alpha-globin locus control region. Blood 87:2518-24
Ren, S; Li, J; Atweh, G F (1996) CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells. Nucleic Acids Res 24:342-7
Cowley Jr, A W; Roman, R J (1996) The role of the kidney in hypertension. JAMA 275:1581-9
Jeha, S; Luo, X N; Beran, M et al. (1996) Antisense RNA inhibition of phosphoprotein p18 expression abrogates the transformed phenotype of leukemic cells. Cancer Res 56:1445-50
Luo, X N; Reddy, J C; Yeyati, P L et al. (1995) The tumor suppressor gene WT1 inhibits ras-mediated transformation. Oncogene 11:743-50
Luo, X N; Mookerjee, B; Ferrari, A et al. (1994) Regulation of phosphoprotein p18 in leukemic cells. Cell cycle regulated phosphorylation by p34cdc2 kinase. J Biol Chem 269:10312-8
Ren, S; Luo, X N; Atweh, G F (1993) The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity. Blood 81:1058-66
Mookerjee, B; Arcasoy, M O; Atweh, G F (1992) Spontaneous delta- to beta-globin switching in K562 human leukemia cells. Blood 79:820-5
Brickner, H E; Zhu, X X; Atweh, G F (1991) A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression. J Biol Chem 266:15363-8
Luo, X N; Arcasoy, M O; Brickner, H E et al. (1991) Regulated expression of p18, a major phosphoprotein of leukemic cells. J Biol Chem 266:21004-10

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