A set of precise programs of gene expression control many complex biological processes, such as cell growth, differentiation, and development. An elucidation of the mechanisms by which the normal controls can be aborted, as in various neoplasms and other diseases, will therefore require a basic understanding of the regulation of gene expression. One pivotal step at which regulation can occur is at the level of transcription of the gene, mediated for most genes by RNA polymerase II (pol II). Chromatin appears to play a major role in determining which genes are transcribed by pol II. The objective of this proposal is to elucidate some of the molecular bases for control of transcription by chromatin. The approach is to study the initiation of transcription from Simian Virus (SV40) minichromosomes in a cell-free transcription system, where the transcription factors and the template (both DNA sequence and chromatin components) can be independently altered and monitored. SV40 is an ideal system with which to study the effects of chromatin on RNA polymerase II transcription. Minichromosomes can be isolated in biochemical quantities from both infected and transfected cells, for analysis and alteration in vitro. In addition, some of the regulation of SV40 gene expression is likely to be dependent on the structure of the minichromosomes (regulation by the enhancer, and possibly T antigen). Such regulation has not yet been accurately reproduced with deproteinized DNA as a for in vitro transcription reactions. The feasibility of studying de novo initiation of transcription from both the SV40 early and late promoters on SV40 minichromosome templates in vitro has been demonstrated using either HeLa whole cell extracts or a reconstituted system of partially purified transcription factors. The specific objectives of this proposal are: 1) to characterize the population of SV40 minichromosomes which are competent to initiate transcription in vitro, 2) to determine which cellular transcription factors are stably complexed with the isolated minichromosomes, and whether these complexes form stable preinitiation complexes for transcription, 3) to determine the effect of the SV40 enhancer sequences on initiation of transcription at the SV40 promoters on minichromosomes, and 4) to examine whether the SV40 T antigen can stimulate initiation of transcription at the late SV40 promoter on minichromosomes.
