One of the unique aspects of regulations of gene expression during development is the extent of genome inactivation that accompanies cell type-specific patterns of gene expression. The large majority of specialized genes are repressed in any given cell type; additionally, the stringency of this gene inactivation suggests that it is achieved by an unusually effective mechanism. A special structural state of chromatin (identified by its resistance to DNAse) characterizes inactive sequences. We propose to use extremely sensitive assays for gene expression to examine whether the stringency of gene inactivation is due to this DNAse I resistant chromatin conformation. As a model system we will examine integrated retrovirus (MTV) sequences which in difference cell clones exist in different chromatin structures. We will test by cell fusion experiments whether trans-acting regulatory factors can specify states of chromatin structures. Specifically, growth hormone is actively expressed by the pituitary derived GH3 cell line and stringently repressed in the liver derived HTC cell line. These two rat cell lines carry allelic variants of the growth hormone gene which can be distinguished by hybridization analysis because of a restriction enzyme site polymorphism. Thus, the DNAase I sensitivity of these two genes can be separately analyzed at various times following cell fusion. This assay should allow us to begin a molecular analysis of the functioning of cell type specific regulatory factors. Analysis of chromatin structgure offers a general approach to the study of development which differs substantially from direct analysis of gene expression and may provide new insights into the specification and maintenance of cell type specific patterns of gene expression. These biochemical approaches to estimation of the stability of differentiative states could add to our understanding of the transformation processes by which cancer cells arise.
NIH ExPorter
NIH ExPorter