Chromatin plays a critical role in regulating the expression of eukaryotic genes. To a first approximation, it exists in two forms: as an unfolded, 10-nm-like filament, comprised of arrays of nucleosomes in a beads-on-a-string configuration, or as a compacted 30-nm-like fiber. The former is characteristic of transcriptionally active euchromatin, while the latter is characteristic of silent chromatin (heterochromatin), which represses transcription in a regional rather than promoter-specific fashion. How silent chromatin inhibits gene expression is not well understood, although the traditional view is that it does so by sterically hindering access of positive regulators of transcription to the underlying DNA. In the budding yeast, Saccharomyces cerevisiae, silent chromatin is formed by Silent Information Regulator (SIR) proteins. Previous work has revealed, very unexpectedly, that silent chromatin is permissive to the binding of both gene-specific activators and general transcription factors (GTFs). Indeed, an initiation-competent form of RNA polymerase II (Pol II) is present at hyperrepressed promoters. By contrast, occupancy of downstream factors, including enzymes that cap the nascent mRNA and factors that facilitate Pol II elongation, is virtually abolished, resulting in Pol II stalling at the gene's 5' end. How this differential accessibility is established is unknown, but is central to understanding the mechanism of transcriptional silencing. In this project, two important issues concerning the fundamental mechanisms by which silent chromatin regulates gene transcription will be investigated: 1. How do Sir proteins silence basal gene transcription? This question will be addressed by (i) Testing the hypothesis that differential factor accessibility in silent chromatin reflects the temporal order of factor recruitment to a gene during the transcription cycle; (ii) Characterizing the role of key downstream factors in triggering silencing through mutational and functional analyses; and (iii) Evaluating the role of factor deacetylation in triggering silencing. 2. How does gene activation take place in silent chromatin? This will be investigated by (i) Characterizing the dynamic alterations of nucleosomes during gene activation in silent chromatin; (ii) Determining the extent of spread of Sir proteins at transgenes subject to different degrees of silencing; and (iii) Comparing the coactivator requirements of euchromatic and heterochromatic genes. Benefits of this project, beyond the anticipated scientific discoveries, include the training of two full-time Ph.D. graduate students and two undergraduates.
The growth and development of all living things depend on the regulation of their genes. Genes are arranged on chromosomes present within the nucleus of every cell of a eukaryote (an organism whose cells contain nuclei). Each chromosome is comprised of DNA and proteins, a complex termed "chromatin." The most abundant DNA-binding proteins are called "histones." Histones not only package the DNA into repeating subunits (termed nucleosomes) that resemble beads on a string, but also directly participate in the regulation of gene expression. One way that histones, and the bead-like structures that they form, regulate genes is by preventing the genes from being copied into RNA by an enzyme known as RNA polymerase. The copying of DNA into RNA –"transcription" – represents the initial step in gene expression. The RNA transcribed from a single gene serves as a messenger in a pathway culminating in the synthesis of a specific protein whose chemical nature is encoded by the gene. A common perception is that a specific type of chromatin – termed "heterochromatin" – prevents gene expression by blocking access of proteins (including RNA polymerase) to the DNA, thereby preventing the act of transcription. In part I of this project, we tested the validity of this idea in a model eukaryote – baker’s yeast (brewer’s yeast). The chromosomes in yeast, like those in more complex organisms including humans, are comprised of linear arrays of nucleosomes. The parts of yeast chromosomes that are heterochromatic – by virtue of the presence of a special class of DNA-binding proteins termed "SIRs" – include their ends ("telomeres") and regions containing genes specifying gender ("mating-type"). Our work has shown that the genes found within these regions are not repressed simply as a consequence of the SIR-containing chromatin preventing the access of RNA polymerase (and associated proteins) to the DNA. Rather, this specialized chromatin, which bears structural and functional similarities to the heterochromatin found in mammalian cells, represses gene expression by using a three-pronged strategy: [1] by partially hindering access of RNA polymerase to promoters (the regions of genes that act as genetic on and off switches); [2] by preventing the RNA polymerase that does succeed in gaining access to the promoter from transcribing the adjacent stretch of DNA; and [3] by stripping off a loosely attached subunit from the RNA polymerase molecule, effectively locking polymerase into an initiation-incompetent state. The combination of these three mechanisms can explain why RNA polymerase, although present at silent gene promoters, is irreversibly stalled under normal growth conditions. A second common perception in the field is that distinctive (and reversible) chemical changes on nucleosomes act as signposts that direct protein complexes to the local region of the chromosome containing the gene that is to be expressed. (These protein complexes, along with RNA polymerase, are necessary for transcription.) The chemical modifications are found on the histones, and the idea has been termed the "histone code." In part II of this project, we asked whether histone modifications were necessary to permit the transcription of heterochromatic genes that are activated in response to external signals. We examined two genes, one that responds to high temperatures and a second that responds to the presence of toxic drugs. We found that activation of both genes took place without any of the changes in chemical makeup typically seen. Absence of detectable chromatin modifications, despite a substantial increase in transcription, is unprecedented for any gene. The importance of our findings is that they place limitations on the universality of the "histone code," since they demonstrate that this pathway is not operative for heterochromatic yeast genes. The same might be true for heterochromatic genes in other organisms, including humans. Our findings raise the possibility that an entirely different mechanism of gene expression exists that can bypass the need for chemical signposts on nucleosomes altogether to ensure the precise gene regulation necessary for growth and development.
