We have used retroviral insertion to create a mutant embryonal carcinoma (EC) cell line, NRI-6, that is unique in its morphological, adhesive, tumorigenic and differentiative properties. Genetic analyses of mutant, hybrid and revertant cell lines indicates that there is only a single retroviral insertion site which we have mapped to the proximal portion of the mouse X chromosome. A mouse mammary gland expressed sequence taq (EST) has greater than 97 percent homology to a region within the >18kb of insertion site flanking genomic DNA that we have sequenced. Utilizing this EST for molecular studies, we have identified two transcripts expressed in parental, but not in mutant, cells. The predominant transcript, -2.3kb, contains two exons, the second of which is disrupted by the insertion. Expression analyses indicates that this transcript is widely expressed both temporally and spatially. We hypothesize that loss of this transcript is the underlying basis for the NRI-6 I mutation and that this transcript plays a critical role in both embryonic development and adult homeostasis. We have also searched for other genes that might act downstream of the insertion site locus to regulate specific phenotypes associated with the mutation (i.e., downstream effector genes). We have found that the nuclear receptors RARI3 and y are expressed at higher basal levels in mutant cells as compared to parental and that inhibition of histone deacetylation increases parental levels to those characteristic of mutant cells. We have also found that histone deacetylase inhibition differentially affects other key parameters of mutant vs parental cell biology. We hypothesize that mutant cells have less histone deacetylase activity associated with certain promoters (specifically RARB and y) than do parental cells and that the consequent increased expression of these nuclear receptors accounts, at least in part, for the observed mutant retinoid hypersensitivity. Finally, we have isolated and analyzed another putative downstream effector gene called MyoR that is highly expressed in mutant cells relative to parental or revertant. This gene encodes a novel basic helix-loop-helix (bLHLH) transcription factor that had been proposed to function as a repressor of embryonic skeletal muscle myogenesis. However, we have found that is expressed in very early stage embryos (3 5dpc I blastocyst) and that the ECIES cells which express MyoR neither differentiate into skeletal muscle nor express the obligate myogenic transcription factors (i.e., MyoD, myf5). These results have led us to hypothesize that MyoR plays a broader, more fundamental role in early embryogenesis than was previously suspected. In this proposal, we will evaluate the three hypothesis proffered above. We will isolate, clone, sequence and translate the full length insertion locus transcript, determine its genomic structure and regulation and examine the function of the protein product(s) (Specific Aim I). We will continue our analyses of the mutant cell retinoid hypersensitivity, focusing our efforts on the role of histone acetylation, specifically as regards the RARB and y promoters. (Specific Aim II). Finally, we will study the regulation of the MyoR gene and examine its expression and role in early embryogenesis and in the NRI-6 mutation [Specific Aim III]. These studies will contribute significantly to our understanding of stem cell biology, embryonic development and cancer.
