Rap1 and Abf1 are General Regulatory Factors (GRFs) that function in transcriptional activation and repression, replication, and telomere structure in yeast. These two GRFs share an ability to create a local region of open chromatin which can facilitate binding of other activators to gene promoters and thus contribute to gene activation. However, these two GRFs also differ in some respects. Most dramatically, continued binding of Rap1 is required to facilitate ongoing transcriptional activation, whereas loss of Abf1 binding (using an abf1-1 ts mutant) does not result in decreased transcription. Thus, at least some promoters retain a "memory" of Abf1 binding. In this project, new insight into the mechanism by which Rap1 and Abf1 contribute to transcriptional activation and perturb local chromatin structure, and into the basis of this "memory effect," will be obtained using a combination of biochemical, genetic, and genomic approaches.
Gene activation depends on binding of specific transcription factors to gene promoters. In this project, two such factors, Abf1 and Rap1, that operate as "General Regulatory Factors" by assisting in activation of many genes in yeast will be investigated. The studies will include biochemical, genetic and genomic approaches to understanding the mechanism by which these two factors contribute to transcriptional activation, including their effects on the packaging of gene promoters into chromatin. The results of this work will provide new information that is likely to apply to all eukaryotes, as the transcription machinery is highly conserved. These investigations will include participation by undergraduates conducting summer research, and will yield genomic data that will be made available to the scientific community.
Intellectual Merit As one researcher has written, in eukaryotic cells "DNA is never all alone". That is because it is closely associated with histone proteins to form a more condensed structure, first observed by microscopy in the late 1800s, that we now refer to as chromatin. The basic unit of chromatin is a bead-like structure called the nucleosome, which contains 147 bp of DNA; a complete eukaryotic genome is packaged in many millions of nucleosomes. These nucleosomes create a potential problem for the cell, as the DNA needs to be contacted by other proteins for essential processes, in particular transcription, in which RNA polymerase copies DNA sequences into RNA molecules, including those that code for the cell’s proteins. This is true for all eukaryotes, from yeast to humans, and many studies have shown that chromatin and the cell’s means of coping with it are similar across this spectrum. Our lab has studied the problem of how proteins contact DNA that is packaged into chromatin to allow transcription in yeast, and has shown that one means by which access to DNA can be ensured is by binding of proteins that exclude nucleosomes from the vicinity of their binding sites. Our work during this grant period was aimed at determining the extent to which two such proteins found in yeast, Abf1 and Rap1, determine chromatin architecture across the entire genome. These proteins were already known to bind to several hundred genes each, and affect transcription of many of these. We used mutants in Abf1 and Rap1 that allowed us to compare genome-wide nucleosome maps when these proteins are bound, as in normal cells, to when they are not, in the mutants. We discovered that out of the approximately 6000 genes in yeast, about half show increased nucleosome occupancy at specific localized regions in their promoters when Abf1 binding is decreased, while about 20% show such an effect for Rap1. This means that these factors affect chromatin structure at a large proportion of promoters, which contain the control elements for transcriptional activation. It is likely that other proteins perform functions to "open" chromatin in mammalian cells, and our work provides a rationale and model for the study of such proteins. We also studied a strange "memory effect" exhibited by Abf1. At some genes, even though Abf1 must bind to the promoter for full activation of transcription, decreased Abf1 binding does not result in decreased transcription, while at other genes transcription does diminish as one would expect. We have found that the genes showing this "memory effect" also possess sequences making them inherently less likely to form nucleosomes, and that new nucleosomes do not form at their promoters when Abf1 binding decreases, in contrast to the genes that do show decreased transcription. These studies indicate that continuing gene transcription can depend on the propensity of a promoter to be packaged into nucleosomes. These findings provide insight into conceptually similar memory effects that are found in a variety of systems, including genes related to immunity, steroid hormone response, and genes expressed by human parasites. Finally, funding of this grant also allowed us to collaborate on published studies that led to the first high resolution determination of the location of nucleosomes across an entire eukaryotic genome—yeast—and on a comparison of nucleosome occupancy in evolutionarily related yeast species. The latter study has provided new insights into how evolution of nucleosome occupancy may affect transcriptional changes that occur during evolution and hence be involved in natural selection. Broader Impact In addition to the impact on researchers in related areas of our published research based on this grant, we have made our data on genome-wide nucleosome occupancy publicly available by depositing original data at the Genome Expression Omnibus (GEO) web site, and by making our processed data available at our lab web site. We have also supported and trained a graduate student and postdoctoral fellow funded by this project, and have hosted seven undergraduates who performed research on this project, including six female students and one minority student. Among these, the three students who have graduated are all now working towards post-graduate degrees in science.
