Expression of the c-myc proto-oncogene is linked to proliferation ad differentiation in normal cells, and aberrant expression of c-myc is characteristic of many neoplastic states, including lymphomas, leukemias, and small cell lung carcinomas. Work in Dr. Groudine's laboratory uncovered a novel mode of eukaryotic transcriptional control, a conditional block to transcription elongation. This mechanism controls the amount of initiated transcripts that elongate past sites of arrest in exon 1 to produce full length c-myc transcripts. During the past granting period, Dr. Groudine and his colleagues have shown that, in mammalian cells, the primary site of polymerase arrest in the c-myc gene and other model templates occurs in the promoter-proximal region (+30 from the start site), and that the simple combination of proximal upstream activators and core promoter elements are sufficient to generate pausing at this site. Elongation of c-myc transcription is regulated in normal cells, and Dr. Groudine's working hypothesis is that two distinct types of RNA polymerase II elongation complexes, which differ in their ability to read through downstream arrest signals, are assembled at the c-myc promoter. Evidence suggests that transcriptional activators and enhancers can alter gene expression, at least in part, by influencing the elongation stage of transcription; they appear, therefore, to be capable of enhancing formation of elongation-efficient elongation complexes. In human Burkett's lymphoma (BL) and murine plasmacytoma cells, which are characterized by translocations that juxtapose c-myc and immunoglobulin (ig) sequences, the c-myc elongation block is abrogated, resulting in constitutive synthesis of c-myc RNA. Dr. Groudine and his colleagues have discovered a novel locus control region (LCR) in the Ig heavy chain locus, which becomes linked in cis to c-myc as a consequence of these translocations. Preliminary functional analysis of this element supports a model in which deregulation of c-myc in BL and plasmacytomas results from cis effects imposed on c-myc by this LCR. The goals of this proposal are to determine the molecular basis of promoter-proximal pausing and the composition of RNA polymerase II transcription complexes that differ in elongation efficiency, as well as to further elucidate the mechanisms by which the newly discovered 3'Calpha IgH LCR deregulates c-myc expression. Specifically, Dr. Groudine proposes to: (1) determine the role of the transcribed template, including its chromatin structure, in promoter proximal pausing; (2) determine the subunit composition of elongation efficient and elongation deficient transcription complexes, and identify and isolate factors that affect transcriptional elongation; (3) determine the cis components of the 3' Calpha LCR involved in the deregulation of c-myc expression in cell culture models, and isolate factors that interact with these sequences; and (4) determine the function of the 3' Calpha LCR in vivo by transgenic analyses of c-myc and reporter genes linked to the LCR, and in its native chromosomal position using novel homologous recombination strategies.
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