The focus of this work is to define the regulatory mechanisms that govern the differential expression of myc-family genes. Myc-family oncoproteins (c-, N- and L-myc) are believed to function as regulators of normal development. Distinctive patterns and changes in expression of myc-family genes are correlated with critical developmental transitions in different cell lineages, suggesting that their differential expression may influence processes leading to cellular differentiation. The molecular mechanisms that govern the expression of myc-family genes are poorly understood. Nuclear run-on experiments indicate that transcriptional attenuation is an important mechanism in the regulation of myc-family gene expression with respect to tissue and developmental stage. The experiments outlined in this proposal are designed to (1) determine the mechanisms responsible for the tissue-specific and developmental stage-specific expression of the N-myc gene and (2) understand the physiological role of these regulatory mechanisms in normal development processes and in malignant transformation. The regulation of myc-family gene expression results from a complex regulatory strategy involving transcriptional initiation, attenuation and mRNA stability. In this proposal, we will focus primarily on the N-myc attenuator element because transcriptional attenuation appears to be the dominant mechanism regulating the tissue-specific and developmental stage-specific expression of the N-myc gene during mouse development. Regulatory sequences and factors that mediate attenuation will be identified by both biochemical and molecular genetic analysis. Candidate attenuator sequences identified by biochemical methods will be verified functionally in a transforming assay and gene-fusion experiments. Since myc gene expression studies using gene transfer into cultured cells and transgenic mice are subject to a number of artifacts, including chromosomal position effects and poor tissue-specific regulation, we will utilize gene targeting methods to mutate and analyze the attenuator sequence element in the endogenous N-myc gene. In situ sequence disruption will be performed first in a cell culture-based differentiation system and ultimately in the mouse germline. The biological impact of altered N-myc regulation will be examined. These studies will assess (1) the role of attenuation in the deregulation of myc expression in malignant transformation, (2) the significance of attenuation in the developmental expression of N-myc and (3) the significance of N-myc downregulation during cellular differentiation,
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