Richard and Rhoda Goldman Distinguished Chair in the Biological Sciences & 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, the ability to survive transient loss of almost all intracellular water, is a remarkable trait found in rare species in all kingdoms of life. Studies of biological extremes like desiccation have greatly advanced our understanding of many biological processes. For example, the rapid division of viruses and bacteria was critical to the elucidation of DNA replication, and the shattering of chromosomes in Tetrahymena was critical to the elucidation of telomeres, the ends of chromosomes. With this in mind, we have initiated studies of desiccation tolerance, with the notion that studying extreme stress conditions will elucidate fundamental principles in water homeostasis, and more generally, cellular stress response. Furthermore, desiccation tolerance has broad applications in agriculture (drought resistance) and biomedicine (blood storage). 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. 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.
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 masking 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. Recently 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. We are currently investigating where hybrids form in the genome in the mutants and wild-type to understand the contribution of genome structure to hybrid formation as well as how hybrids are converted to DNA damage and eventually 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.
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 tolerance required expression of new genes. In addition, we showed that desiccation tolerance is regulated by the Tor1 Kinase and Ras GTPase, the general stress sensors in all eukaryotic cells and that the most important lethal stress of desiccation is likely protein misfolding and not salt, oxydation, DNA damage or osmotic stresses as previously proposed. The lab is currently building on these foundational studies and has exciting new observations identifying critical desiccation tolerant factors and connecting these factors with the propagation of amyloids, associated with a number of human diseases.
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.
Wahba, L., & Koshland, D. (2013). The rs of biology: R-loops and the regulation of regulators. Molecular Cell, 50(5), 611–612. doi:10.1016/j.molcel.2013.05.024
Wahba, L., Gore, S. K., & Koshland, D. (2013). The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability. eLife, 2(0), e00505–e00505. doi:10.7554/eLife.00505.027
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.
Welch, A. Z., Gibney, P. A., Botstein, D., & Koshland, D. E. (2013). TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae. Molecular biology of the cell, 24(2), 115–128. doi:10.1091/mbc.E12-07-0524
Calahan, D., Dunham, M., Desevo, C., and Koshland, D.E. (2011). Genetic Analysis of Desiccation Tolerance in Sachharomyces cerevisiae. Genetics 189, 507–519.
Photo Credit: Mark Hanson of Mark Joseph Studios
Last Updated 2013-07-17