The long-term objectives of this project are to understand how chromatin structure is involved in gene regulation and how this""""""""epigenetic"""""""" information is faithfully passed on to daughter cells. Inappropriate gene expression may be an important factor in developmental abnormalities, cancer and other diseases. It has been known for some time that the activity of a transposed or inserted gene is dependent upon its chromosomal locus. Presumably, in many locations, adjacent inactive chromatin has a tendency to spread over the inserted gene and inactivate it, whereas in other locations the gene can be activated. Moreover, these states are somatically heritable, persisting after chromatin assembly. The recent demonstration that histone H1 can nucleate physiological nucleosome alignment (prerequisite for higher order structure formation) in vitro in a fully defined system, and alignment can then spread for several thousand base pairs, suggests that such a mechanism might be involved in chromosomal position effects and in gene regulation. A fortuitous signal that can nucleate the alignment of nucleosomes by histone H1 was first found in a region of plasmid pBR327. Subsequently, chromatin organizing signals were found in two introns of the chicken ovalbumin gene. These three signals will be precisely localized, and the molecular mechanism(s) by which they nucleate the formation of a nucleosome array through interaction with histone H1 will be determined. Additionally, a set of constructs containing the rat growth hormone gene with various combinations of introns deleted, which exhibited different average transcriptional activities in transgenic mice, will be examined in vitro to determine whether chromatin organizing signals are present in introns of this gene. Selected mouse and human DNA clones from known chromosomal loci will also be examined for the presence and characteristics of nucleosome alignment signals, as a rough survey of vertebrate genomes. Also, it has been possible to reproduce the anomalous (or compressed) """"""""active nucleosome"""""""" ladder observed for beta-globin gene chromatin in chick erythroid cells, using cloned chicken beta-globin DNA in an in vitro chromatin assembly system. It will be determined precisely how this ladder arises. Finally, the effects of nucleosome alignment signals on the chromatin assembled on replicating plasmids in transfected cells will be investigated, and a transient gene expression system will be used to test the idea that transcription factors compete with histones to turn on a gene. The ability to alter chromatin structure in defined ways through """"""""chromatin organizing signals"""""""" offers new promise for studying gene regulation.
