The long term goal of this project is to define the molecular mechanisms that control the switch from fetal (gamma) to adult (beta) globin synthesis; this switch is controlled at the level of globin gene transcription. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) continue to express gamma-globin genes in adult RBCs, and frequently have mutations just upstream from the gamma-globin genes that probably cause the HPFH phenotype. We intend to use these mutations to define the cis-acting DNA sequences and trans-regulatory factors that control gamma- globin transcription in erythroid cells, as follows: 1) We will define the cis-acting DNA sequences in the human beta-globin gene cluster that silence the gamma-globin gene in mouse erythroleukemia (MEL) cells. gamma-globin genes are minimally expressed in """"""""adult"""""""" erythroid MEL cells when introduced on whole chromosomes or cosmids. We will define the sequences that normally silence gamma-globin transcription in MEL cells using stable transfection techniques, and will determine whether these elements interact with HPFH mutations. 2) We will further define an S1 nuclease hypersensitive site (S1-HSS) in the -210 region of the gamma-globin promoter, and define how this site is altered by HPFH mutations. The region from -216 to -208 contains an S1-HSS in supercoiled plasmids; this site is destabilized by some HPFH mutations. We will define the sequences that contribute to the formation of the S1-HSS with site-directed mutations, and will examine the relevance of the site for the binding of potential regulatory factors. 3) We will characterize trans-acting factors that discriminate between wild-type and HPFH-associated gamma-globin alleles. We have identified a MEL cell protein(s) that binds with increased affinity to a gamma-globin allele with the -202 c -> G HPFH substitution. This protein will be characterized, purified, and cloned. A newly region will be further characterized. Functional analysis of this cloned protein will examine its relevance for globin gene switching. 4) We will further characterize the structure and function of the murine beta-globin locus control region (LCR). The region upstream from murine beta-globin LCR 5' HS-2 will be sequenced and examined with a variety of functional assays. Structure/function relationships with the human beta- globin LCR will be established.
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