Colony stimulating factor-1 (CSF-1; M-CSF) is required for the growth and differentiation of macrophage progenitors as well as the survival of mature macrophages. In contrast to the known biological effects of CSF-1, little is known about the regulation of CSF-1 gene expression. The objective of this proposal is to identify regulatory mechanisms operating at the molecular and cellular level which modulate expression of the mouse colony stimulating factor-1 gene in a fibroblastic and in a monocyte/macrophage cell line. The goal of the first specific aim is to compare the regulation of CSF-1 expression in these two cell lines, under basal and stimulated conditions. Northern analysis of steady-state CSF-1 mRNA levels and nuclear run-on assays will be used to determine whether expression of the CSF-1 gene is under transcriptional or post- transcriptional control. The goal of the next three specific aims is to created a molecular map of the mouse CSF-1 gene promoter region. First, a physical map will be constructed of the 5' flanking region of the mouse CSF-1 gene. This will be accomplished by restriction enzyme mapping, subcloning and sequencing of a genomic fragment which includes sequences that flank the 5' end of the transcribed portion of the CSF-1 gene. Second, the transcriptional efficiency of the cloned regulatory regions will be analyzed in the fibroblastic and monocyte/macrophage cell lines. This will be accomplished by creating plasmid vectors that contain portions of the 5' flanking material adjacent to the chloramphenicol acetyltransferase gene, and using these vectors in transient transfection assays to evaluate the transcriptional efficiency of the regulatory region in the cell lines, under basal and stimulated conditions. Third, cis-acting regulatory elements in the 5' flanking region of the mouse CSF-1 gene will be identified. This will be accomplished by using subcloned fragments shown to contain regulatory and monocyte/macrophage cells in DNase 1 protection, gel retardation and methylation interference assays. These studies should help to identify some of the mechanisms and DNA sequences which are involved in regulating the expression of the CSF- 1 gene.
