Professor of Genetics, Genomics and Development*
**And Affiliate, Division of Cell and Developmental Biology
Higher Order Chromosome Structure is important for the integrity, transmission and expression of chromosomes. The molecular basis for the formation, maintenance and disassembly of higher order chromosome organization are major unsolved problems in cell biology.
The Maintenance of Genome Integrity (chromosome number and integrity) is critical to prevent cell inviability, inherited disorders and disease. A major threat to genome integrity is breaks in the DNA that can be misrepaired to cause gross chromosomal rearrangements (GCR) such as translocations, internal deletions or terminal truncations. While the cause of DSBs and their repair have been studied intensively, major new discoveries will be required to explain the massive rearrangement of chromosomes associated with cancer.
Desiccation Tolerance is a remarkable trait found in rare species in all kingdoms of life. Desiccation tolerance has significant biomedical potential and will likely provide novel insights into the role of different cell biological processes in water homeostasis. While the molecular basis of desiccation tolerance has been correlated with many factors and processes, none of them have been shown to have a causative role in tolerance.
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.
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 currently investigating how RNA:DNA hybrids lead to GCR.
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 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.
Guacci, V., and Koshland, D. (2012). Cohesin-independent segregation of sister chromatids in budding yeast. Mol Biol Cell 23, 729–739.
Onn, I., and Koshland, D. (2011). In vitro assembly of physiological cohesin/DNA complexes. Proc Natl Acad Sci USA 108, 12198–12205.
Heidinger-Pauli, J.M., Mert, O., Davenport, C., Guacci, V., and Koshland, D. (2010a). Systematic reduction of cohesin differentially affects chromosome segregation, condensation, and DNA repair. Curr Biol 20, 957–963. Heidinger-Pauli, J.M., Onn, I., and Koshland, D. (2010b).
Genetic evidence that the acetylation of the Smc3p subunit of cohesin modulates its ATP-bound state to promote cohesion establishment in Saccharomyces cerevisiae. Genetics 185, 1249–1256.
Heidinger-Pauli, J.M., Unal, E., and Koshland, D. (2009). Distinct targets of the Eco1 acetyltransferase modulate cohesion in S phase and in response to DNA damage. Mol Cell 34, 311–321.
Onn, I., Heidinger-Pauli, J.M., Guacci, V., Unal, E., and Koshland, D.E. (2008). Sister chromatid cohesion: a simple concept with a complex reality. Annu. Rev. Cell Dev. Biol. 24, 105–129.
Heidinger-Pauli, J.M., Unal, E., Guacci, V., and Koshland, D. (2008). The kleisin subunit of cohesin dictates damage-induced cohesion. Mol Cell 31, 47–56.
Unal, E., Heidinger-Pauli, J.M., Kim, W., Guacci, V., Onn, I., Gygi, S.P., and Koshland, D.E. (2008). A molecular determinant for the establishment of sister chromatid cohesion. Science 321, 566–569.
Tan, F.J., Hoang, M.L., and Koshland, D. (2012). DNA resection at chromosome breaks promotes genome stability by constraining non-allelic homologous recombination. PLoS Genet 8, e1002633.
Wahba, L., Amon, J.D., Koshland, D., and Vuica-Ross, M. (2011). RNase H and Multiple RNA Biogenesis Factors Cooperate to Prevent RNA:DNA Hybrids from Generating Genome Instability. Mol Cell 44, 978–988.
Hoang, M.L., Tan, F.J., Lai, D.C., Celniker, S.E., Hoskins, R.A., Dunham, M.J., Zheng, Y., and Koshland, D. (2010). Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination. PLoS Genet 6, e1001228.
Calahan, D., Dunham, M., Desevo, C., and Koshland, D.E. (2011). Genetic Analysis of Desiccation Tolerance in Sachharomyces cerevisiae. Genetics 189, 507–519.
Last Updated 2012-08-06