The broad objective of this proposal is to analyze the mechanism(s) by which the transcription of the c-myc proto-oncogene is maintained in undifferentiated leukemia cells and irreversibly shut off during terminal differentiation. Both the promonomyelocytic leukemia cell line HL-60 and the monoblastic cell line U-937 can be induced to differentiate by a variety of agents, such as dimethyl sulfoxide, and are well suited to study this question. Since the presence of c-myc is critical for the maintenance of a proliferating, nonterminally differentiated state in these cells, an elucidation of mechanisms which lead to a permanent reduction in the expression of this gene has significant implications in the development of biological modifiers that might be used therapeutically for certain tumors. During my post-doctoral fellowship I discovered a novel nuclear protein that is present in undifferentiated leukemia cells and which binds in vitro 1,500 bp upstream of the c-myc P1 promoter. The binding of this factor in vivo to its cognate sequence potentiates expression from a chimeric c-myc CAT gene by approximately 80%. Importantly, binding activity of the factor disappears with commitment to differentiation (in extracts from cells treated with dimethyl sulfoxide), in contrast to the unaltered constitutive binding activity of a number of other c-myc regulatory factors during this process. Recently we have purified this transactivating factor from HL-60 cell extracts and have performed sequence analysis of a number of peptides generated by enzymatic and chemical cleavage. With this information we now propose to obtain a full-length cDNA clone using pools of degenerate oligonucleotide probes to hybridize with an HL-60 cell cDNA library. Alternatively, the double-stranded cognate sequence will be used as a probe on phage plaques induced to produce fusion proteins. With this clone we will be able to analyze molecular interactions of this protein that are associated with DNA binding and transactivation of c-myc. With the use of cDNA probes and specific antibodies, experiments will be performed to determine whether loss of the factor's binding activity during differentiation occurs because of a change in its synthesis or a modulatory modification. In addition, the linkage of its binding activity to c-myc expression and to cellular differentiation will be studied by its selective removal from undifferentiated cells (via transfected anti-sense oligonucleotides). Conversely, the transactivating factor will be over- expressed in cells induced to differentiate with dimethyl sulfoxide to determine whether its presence will have an impact on the reduction of c- myc transcriptional initiation and commitment to differentiation. These studies will elucidate a new control mechanism that regulates expression of a gene that plays a critical role in the growth of many neoplastic cells.
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| Duncan, R; Bazar, L; Michelotti, G et al. (1994) A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev 8:465-80 |
