We have recently isolated a transforming gene from the DNAS of a patient with chronic myeloid leukemia using the NIH3T3 cell focus formation assay. The sequences responsible for transformation have been cloned and characterized. The transforming gene has been identified as fibroblast growth factor 4 (FGF4). Expression of FGF4 is restricted to undifferentiated embryonic stem and embryonal carcinoma cells and is not expressed in somatic tissue. The coding sequences of the FGF4 gene (located on chromosome 11) have been fused to RNA processing signals from an unidentified gene on chromosome 15. Transformation by FGF4 in this and previously described studies is the result of deregulated expression rather than mutation of the coding sequences. It is therefore of value to investigate the mechanism of regulation of FGF4 expression. Paradoxically, expression of endogenous FGF4 is suppressed in NIH3T3 cells yet an exogenously added copy of the normal gene is expressed and capable of inducing transformation. The goals of the proposed study are threefold. Firstly we wish to further previous studies and investigate the mechanism of local control of FGF4 expression. Specifically, to date the promoter has been shown to be inactive in all cell lines tested unless linked to an activating 3' enhancer sequence. Study of functional promoter domains has thus proven difficult. Using a transient reporter assay system (luciferase) which is more sensitive than the system (CAT) used previously by others, we have identified active promoter domains within the 5' flanking sequence of FGF4 and have shown the promoter to be active in both embryonal carcinoma cells (F9) which are permissive for FGF4 expression and HeLa cells which are not. We wish to further these studies and characterize functional domains both within the promoter and the enhancer sequence and to investigate how such sequences might interact to control local expression of FGF4. Secondly, based on previous data demonstrating that cosmid clones harboring the FGF4 gene are transforming while the endogenous gene is silent, we suggest the possibility that FGF4 expression may also be regulated from a distant locus. We will investigate this possibility by the transfer into NIH3T3 cells of YAC clones containing the FGF4 gene. These clones will therefore contain substantially greater sequence flanking the FGF4 gene than clones previously described. If transfer of YACs produce cell colonies of normal rather than transformed morphology, it may be inferred that a putative suppressor locus has been co-transferred and this locus will then be further characterized. Thirdly, little is known about the genes which are activated in response to FGF4 induced transformation of NIH3T3 cells. We will attempt to identify and characterize these genes by utilizing a recent and novel PCR based method which allows the identification and cloning of cDNA from differentially expressed genes.
