The study of gene regulation is a prerequisite for understanding how cells respond appropriately to a changing environment, how they implement developmental programs, and how a defect in gene regulation can result in carcinogenesis. For many years it was thought that gene regulation involved only transcription factors and their interactions with DNA; changes in the chromatin structure of a gene were considered to be the passive consequence of the binding of these factors. However, it is now clear that chromatin is central to gene regulation. The structural subunit of chromatin is the nucleosome, which is composed of ~147 bp of DNA wrapped ~1.7 times around a central octamer composed of two molecules each of the four core histones (H2A, H2B, H3 and H4). Nucleosomes are regularly spaced along the DNA, like beads on a string.
Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and transcription. These events must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent this chromatin block, eukaryotic cells possess ATP-dependent chromatin remodelling machines and nucleosome modifying complexes. The former (e.g. the SWI/SNF complex) use ATP to drive conformational changes in nucleosomes and to move nucleosomes along DNA. The latter contain enzymatic activities which modify the histones post-translationally to alter their DNA-binding properties and to mark them for recognition by other complexes, which have activating or repressive roles (the basis of the "histone code"). Nucleosome-modifying enzymes include histone acetylases (HATs), deacetylases (HDACs), methylases and kinases. Thus, the cell possesses complex systems for manipulation of the repressive properties of chromatin to maximum effect. Furthermore, multiple connections between chromatin and disease are apparent.
We are exploiting massively parallel sequencing technologies to create genome-wide nucleosome maps (MNase-seq) and other chromatin components (ChIP-seq) in budding yeast and mouse cells. In vivo, active gene promoters and enhancers are occupied by sequence-specific transcription factors and associated proteins. Such nucleosome-depleted regions (NDRs) are flanked by arrays of regularly spaced nucleosomes that are phased relative to the NDR. In yeast, the first (+1) nucleosome is usually located directly over the transcription start site. What happens to nucleosomes on genes when they are activated? We find that the chromatin structure of the most heavily transcribed genes is heavily disrupted: some nucleosomes are lost, others are stripped of one or both of their H2A-H2B dimers, and those that remain are out of position. Furthermore, RNA polymerase II complexes queue up on these genes, apparently waiting to terminate. We are actively exploring the implications of this fascinating observation.
Our major focus is on the regulatory functions of various ATP-dependent chromatin remodeling machines found in yeast and man, particularly RSC, SWI/SNF, ISW1, ISW2 and CHD1. They are of great interest because mutations in genes encoding remodeler subunits (e.g., the hSNF5 subunit of SWI/SNF) are strongly associated with various cancers, particularly pediatric cancers. These complexes move nucleosomes, thereby regulating access to DNA. We are addressing the roles of SWI/SNF and the related essential RSC complex in nucleosome phasing. We find that RSC depletion results in global re-positioning of nucleosomes: both upstream and downstream nucleosomal arrays shift toward the NDR, resulting in a narrower NDR. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighbouring NDRs. We propose a modified barrier model, in which a stable complex located at the NDR directs bidirectional phasing. Current studies are aimed at identifying barrier complexes, which we propose play a critical role in organizing chromatin. We are also examining the roles of the ISW1, ISW2 and CHD1 nucleosome spacing enzymes. Our studies indicate that these three enzymes space nucleosomes differently in vivo, which may determine the degree of chromatin folding by the linker histone (H1), by controlling the average linker DNA length. We are also examining the links between the ATP-dependent remodelers and histone modifications. Currently, we are developing a new quantitative measure of DNA accessibility in yeast and mouse cells to test the hypothesis that chromatin does indeed regulate access to DNA in cells.