The overall goal of the research proposed in this application is to determine some of the molecular mechanisms responsible for the developmental and tissue specific regulation of gene expression at the transcriptional level. Enhancers are cis-acting regulatory elements which have recently been demonstrated to control tissue specific gene transcription in vivo. In order to clarify the role of enhancer sequences as controlling elements in cell differentiation, enhancer sequences derived from a developmentally regulated eukaryotic gene (such as immunoglobulin) will be placed upstream from a conveniently assayble structural protein gene, in an appropriate plasmid. This plasmid will be transfected into undifferentiated cell line of suitable lineage; the transfected cells will be differentiated, and subsequently will be assayed for enhancer-controlled induction of transcription during differentiation. To define the mechanism of action of such enhancers, an in vitro enhancer-driven transcriptional system will be developed. Various recombinant plasmids will be constructed to reproduce the in vivo observations, including orientation and position independence of enhancer function and decreased activity of core deletion mutants. Once such a system is functioning, the factor(s) responsible for enhancer function will be defined by fractionation of cell extracts used in the in vitro assay. Structural studies to define enhancer binding substances, such as DNAseI footprint assay and Western blotting, will also be done. These approaches should allow characterization of host cellular molecules that interact with enhancer regions, as well as allowing insight into the basic mechanism of enhancement. This knowledge will form the basis for a paradigm within which an analysis of differential gene activity in normal and disease states may be undertaken. In particular, the events involved in the exquisitely controlled coordinate expression of the sarcomeric protein genes during myogenesis, as well as the intracellular events involved in the response of muscle and myocardium to mechanical stress and endocrine imbalances would, at least in part, be amenable to experimental analysis.
| Weinberger, J; Baltimore, D; Sharp, P A (1986) Distinct factors bind to apparently homologous sequences in the immunoglobulin heavy-chain enhancer. Nature 322:846-8 |
