Chromosome Structure

Higher order chromosome structure is important for transcription, chromosome segregation and DNA repair. Higher order chromosome structure is mediated by a conserved set of protein complexes called SMC for structural maintenance of chromosomes. These SMC complexes provide a powerful platform to understand both the biological function and molecular basis for different types of higher order chromosome structure. SMC complexes facilitate higher order organization of chromosomes by their ability to tether together two different regions of DNA, either within the same chromosome or between different chromosomes My laboratory has shown that different types of higher order chromosome organization are coordinated because the function of SMC complexes are highly interdependent. Different SMC complexes interact in at least three distinct ways to mediate proper chromosome morphogenesis in mitosis, meiosis and DNA repair. They regulate each other's assembly on chromosomes, regulate each other's disassembly from chromosomes and cooperate to build other chromosomal structures like synaptonemal complex. However, what is the molecular basis for the tethering activity of SMC complexes and how this activity is modulated by regulatory modifications of the complex or auxillary factors remain unanswered.

To address these two questions we have studied a prototypic SMC complex, cohesin. The tethering activity of cohesin is used for sister chromatid cohesion, chromosome condensation, DNA repair and regulation of gene expression, including studies implicating a potential role in stem cell gene expression. We have shown that cohesins bind chromatin and then become cohesive. We also have shown that this conversion to the cohesive state is regulated by complex post-translational modifications in response to cell cycle and DNA damage. Our understanding of cohesin regulation allows us to lock cohesin in either the non-cohesive or cohesive-competent state, providing critical new tools to facilitate poweful genetic suppressor studies, to assess specificity of in vitro activities as well as to generate sufficient quantities of active complex for in depth biochemical and structure studies. Our current genetic, molecular, biochemical and imaging studies of cohesin and its regulation have revealed novel roles for cohesin ATPase function, cohesin acetylation and cohesin oligomerization that dramatically reshape current models for the molecular mechanism by which cohesin modulates chromosome structure. In collaboration with the Meyer lab, we are leveraging the genetic tools developed to study cohesin in budding yeast to interogate in metazoans the roles of its tethering and oligomerization activities in sister chromatid cohesion and gene expression.

Genome Integrity

Dissecting the molecular basis for chromosome integrity is challenging because changes in chromosome integrity like GCRs (Gross Chromosomal Rearrangement) are usually rare and often either lethal or genetically silent.To identify novel processes that prevent GCR, we developed a high throughput assay to follow GCR using a yeast artificial chromosome (YAC). Our studies identified novel proteins required to promote chromosome integrity. As one example, we find that factors required for general mRNA metabolism play an active role in masking newly formed RNA molecules to prevent them from hybridizing back to DNA. When RNA-DNA hybrids do form, two evolutionarily conserved RNases H acts as a surveillance mechanism for their removal, thereby safeguarding the genome. We have shown that hybrids form in trans (that is RNA hybridizes to homologous DNA sequences distinct from their site of synthesis), catalyzed by the homologous DNA repair machinery. Fundamental questions remain unaswered including: 1) where do hybrids form in the genome?; 2) why are there two RNAses H and what are their roles in hybrid removal; 3) what type of DNA damage is induced by hybrids, and 4) what are the repair mechanisms of hybrid induced damage? To address these questions, we have developed new genetic and molecular reagents in yeast including a novel genome-wide mapping methods of hybrids. These tools have defined causal elements for hybrid formation, revealed distinct spacial functions of the RNAses H, and implicated hybrids not only in damge formation but also modulating DNA repair.

Stress Biology through the Lens of Desiccation Tolerance

Studies of biological extremes have transformed biology, for example the study of telomeres in tetrahymena, the giant axons of squids, or the identfication thermo-resistant polymerases in thermophiles. With this precedent in mind, we study desiccation tolerance in yeast as mechanism to provide novel insights into water homeostasis and stress biology. Two fundamental questions about desiccation tolerance remain unanswered. What is the stress(es) that kill desiccation sensitive cells and how do tolerant cells mitigate these stresses to survive. While the rare trait of desiccation has been studied for many years, almost all studies have succeeded in establishing only correlations (for example the presence of specific small molecules like special sugars or peptides) without any means of testing causality. Indeed, desiccation tolerance has been correlated with a number of stress response pathways including, heat shock, osmotic, DNA damage or high-salt. We have estalbished budding yeast as a genetic model to study desiccation tolerance and demonstrated that the lethal stresses of desiccation are likely protein aggregation and membrane damage, and not salt, oxydation, DNA damage or osmotic stresses as previously proposed. Hugo Tapia has shown that the small sugar trehalose plays a profound causal role in desiccation tolerance in part by mitigating these stresses. These exciting new observations identify critical desiccation tolerant factors and connect them with the propagation of amyloids, associated with a number of human diseases. We are currently investigating the role of small intrinsically disordered proteins (IDP) as stress protectors. Intrinsically disordered proteins and domains represent a huge unexplored proportion of the proteome. Thus the study of IDPs in desiccation tolerance provides a powerful tool to unlock the mysteries of their biological and molecular functions.