The research in my lab focuses on understanding how cells regulate epigenetic processes. Our research examines the role of the cell cycle and DNA replication in assembly or maintenance of chromatin structures and the effects of these structures on DNA replication. We are interested in how the cell restricts heterochromatin to specific genomic loci, why heterochromatin formation is regulated by the cell cycle, how transcription of genes is prevented in silenced regions, and whether epigenetic processes are influenced by or influence events including DNA damage and the initiation of DNA replication. We are intrigued by how epigenetic states are maintained throughout the cell cycle and are duplicated and inherited each time the chromosome itself is replicated and the cell divides. We investigate how environmental factors perturb epigenetic processes and can contribute to inappropriate gene expression, developmental defects, tumorogenesis and other catastrophic disorders.
Our research explores the interface between epigenetic processes, histone modifications, chromatin assembly, DNA replication and the cell cycle, Our laboratory combines molecular biology, biochemical and quantitative microscopy-based approaches with mammalian cell culture and the power of yeast genetics to understand the impact of genetic and external factors on epigenetic gene regulation.
Silencing in Saccharomyces cerevisiae
We use silent chromatin in S. cerevisiae as a model for understanding how epigenetically regulated chromatin structures are established, maintained and inherited. Silenced chromatin in budding yeast, is akin to heterochromatin in organisms such as maize, flies, and mammals. S. cerevisiae uses epigenetically inherited chromosomal structures to regulate a variety of cellular activities including controlling cell-type specific gene expression, modulating ribosomal RNA levels, and preserving telomere structure and stability. Silenced regions in S. cerevisiae that are regulated, in part, by the Silent Information Regulator, or Sir, proteins include the silent mating-type loci, HML and HMR, the rDNA locus and the telomeres.
To mediate silencing at a given site on a chromosome, an organism must first have a way to recruit the proteins that compose silenced chromatin to that locus. In yeast, regulatory sites known as silencers flank the silent mating-type loci. Silencers contain binding sites for the Origin Recognition Complex (ORC), and the transcriptional regulators Rap1p and Abf1p. In addition, the Sir proteins, Sir1p, Sir2p, Sir3p and Sir4p, are structural components of silenced chromatin in yeast. Unlike ORC, Rap1p and Abf1p, however, the Sir proteins do not bind to DNA site-specifically. Instead, Sir proteins associate with silencers through protein-protein interactions between each other, proteins bound at silencers, and histones H3 and H4. Once initiated, silencing spreads along the chromosome over several kilobases of DNA and inactivates gene expression. Once established, the transcriptionally inactive state of this region of the chromosome is maintained throughout the cell cycle and can be stably inherited in subsequent generations. Many proteins involved in silencing in yeast have homologs in a wide variety of organisms, including humans where several are involved in development and differentiation or have been implicated in cancer.
Epigenetic Processes and Replication-Coupled Chromatin Assembly
We are investigating how a network of chromatin assembly factors, histone-modifying enzymes and replication factors interact to assemble appropriately modified nucleosomes during DNA replication and thereby promote epigenetic processes and genome integrity. We have been characterizing how silencing is affected by the DNA polymerase processivity factor PCNA through pathways involving several factors including the chromatin assembly factors Asf1p and CAF-1, the PCNA loading complex RF-C and the histone acetyltransferase Rtt109p. As nucleosomal DNA serves as the foundation upon which silent chromatin is built, perturbations in replication-coupled chromatin assembly can alter the efficiency and location of silent chromatin formation as well as lead to defects in maintaining and inheriting epigenetic states. Repair of DNA damage is also often compromised in mutants with defects linked to histone modifications and replication-coupled chromatin assembly pathways.
Defining the Composition of Chromatin
The simplest structural unit of chromatin, the nucleosome, can exist in a variety of configurations depending on the histone variants and post-translational modifications present. The composition of individual nucleosomes influences chromatin structure and function and provides signals to the cellular machinery to promote gene activation or repression. These signals tend to be dynamic and can vary as a function of the cell cycle or growth conditions. Yet, our understanding of the patterns found within individual nucleosomes and presented to the cellular machinery is limited. Population-based approaches widely used to characterize chromatin composition have been valuable in identifying and mapping individual modifications within histones, but have been limited in describing which modifications are combined within the same nucleosomes. As a powerful complement to standard approaches, we are utilizing single molecule strategies in vitro and in single mammalian and yeast cells to probe epigenetic processes that regulate transcriptional states, responses to DNA damage, centromere function and differentiation in several collaborative projects.