Retroviral insertional mutagenesis will be used to create mutant murine erythroleukemia cells (MELC), after which clones of MELC that fail to commit to the terminal erythrodifferentiation program will be isolated. Analysis of the chromosomal DNA of such mutants should reveal common proviral DNA integration sites and thereby identify loci (genes) required for the switch from continued cell growth and division to commitment to differentiation accompanied by the accumulation of high levels of cell type-specific gene products followed ultimately by cell death. DNAse I hypersensitive sites will be identified in wild type MELC near the positions of retroviral DNA integrations in the mutant MELC, and regions of DNA sequence near or coincident with these sites will be examined for the ability to interact with nuclear DNA binding proteins in an attempt to examine, at the molecular level, the mechanisms of the control of expression of these genes, which themselves, participate in the process of commitment to terminal differentiation. Preliminary experiments described herein indicate this approach will be successful. It is proposed to study the function of the genes identified by this methodology to learn the process of commitment to terminal differentiation. It is further proposed to clone and study the genes for the nuclear DNA binding proteins that interact with (and control the expression of) such genes.
