Research

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. 
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.

We have also studied the SMC complex, cohesin, in detail as a prototype to understand the molecular mechanism of how a Smc complex tethers two strands of chromatin to achieve higher order chromosome folding. Cohesin tethers together the two replicated copies of a chromosome to facilitate chromosome segregation. Cohesin also protects cells from DNA damage both at the site of the lesion and genome wide. Most recently cohesin has been shown to help regulate transcription in metazoans, 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 assess specificity of in vitro activities as well as to generate sufficient quantities of active complex for in depth biochemical and structure studies. We are addressing fundamental questions including: How do Smc molecules tether chromatin? How do they know how to tether in within or between chromatin strands? How is there position on the genome or activity modulated to mediate DNA repair, chromosome structure and gene regulation?





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. Factors that prevent GCRs have been identified in budding yeast from a candidate list using an elegant but labor-intensive genetic assay. While extremely important, these studies have likely missed processes that protect cells from GCR because the candidate list included mostly DNA repair and replication factors. To identify novel processes that prevent GCR, we developed a high throughput assay to follow GCR using a yeast artificial chromosome (YAC). We used this assay to screen mutant yeast deleted for each of 5000 nonessential genes. 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 sequestering newly formed RNA molecules to prevent them from hybridizing back to DNA. When RNA:DNA hybrids do form, RNase H acts as a surveillance mechanism for their removal, thereby safeguarding the genome. We are addressing the following questions: What are cis determinants in RNA and DNA that enhance or inhibit the formation of RNA hybrids? How do proteins change the distribution of hybrids in the genome? How do the anti-hybrid protection systems work? Are some hybrids good?
How do hybrids cause DSBs?

Genome evolution both in speciation and cancer often involves changes in chromosome number and GCRs. GCRs occur frequently through non-allelic homologous recombination (NAHR) between repetitive elements at distinct chromosomal positions. We would like to understand the mechanism of NAHR and the contribution of GCRs to genome and organism fitness in the context of purebred and hybrid genomes. Yeast is uniquely suited to analyze the mechanism of NAHR because of its sophisticated genetics and the knowledge of the position and sequence of every repeat in the large repetitive family of Ty retrotranspons. In addition, because of its short generation time, yeast is ideally suited to study the impact of GCR on genome fitness through chemostat evolution experiments. Our analyses of NAHR in purebreds and hybrid yeast has already generated surprising observations that challenge widely accepted models for homologous recombination, suggesting exciting alternative mechanisms how homologous recombination shapes genome.

Desiccation Tolerance

Desiccation tolerance has been correlated with a number of stress response pathways including, heat shock, osmotic, DNA damage or high-salt. However, we showed through targeted mutational analysis that all of these pathways are dispensable for desiccation tolerance, either because of underlying redundancies or the role of novel unknown stress responses. Given that these prior correlations were uninformative, we screened the Yeast Knockout Collection for desiccation sensitive mutants. We observed extreme desiccation sensitivity (10,000 fold reduction in viability) only when respiration is compromised. Experiments exploiting temporal inactivation of respiration show that desiccation tolerance does not require respiration at the time of desiccation but rather respiration prior to desiccation. We hypothesized that entry into the respiration state induces gene products or metabolites that protect cells from desiccation. Indeed we have identified loss of function second-site suppressors that restore nearly wild-type levels of desiccation tolerance to respiration defective knockout cells. One second site suppressor is a transcriptional repressor implying that desiccation tolerance is controlled at least in part by a transcriptional program. The identification of a transcriptional program sets the stage for the elucidation of the actual effectors desiccation tolerance. We are currently addressing the following questions: What are the lethal stresses? How does the cell protect against these stresses?