Spindle pole bodies exploit the mitotic exit network in metaphase to drive their age-dependent segregation.

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud. Here, we show that the SPB component Nud1/centriolin acts through the mitotic exit network (MEN) to specify asymmetric SPB inheritance. In the absence of MEN signaling, Kar9 asymmetry is unstable and its preference for the old SPB is disrupted. Consistent with this, phosphorylation of Kar9 by the MEN kinases Dbf2 and Dbf20 is not required to break Kar9 symmetry but is instead required to maintain stable association of Kar9 with the old SPB throughout metaphase. We propose that MEN signaling links Kar9 regulation to SPB identity through biasing and stabilizing the age-insensitive, cyclin-B-dependent mechanism of symmetry breaking.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

Recombination-induced tag exchange to track old and new proteins.

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.

The mother enrichment program: a genetic system for facile replicative life span analysis in Saccharomyces cerevisiae.

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS--a median of 36 generations in the diploid MEP strain--while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.

Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes.

Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II. Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process. Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation. First, we identified Chd1 in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation. Secondly, we show through co-immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3. Thirdly, we demonstrate that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II. Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin. Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation.

Transcript elongation on a nucleoprotein template.

Chromatin forms a general, repeating barrier to elongation of transcripts by eukaryotic RNA polymerases. Recent studies of nucleosome structure and histone modifications reveal a set of likely mechanisms for control of elongation through chromatin. Genetic and biochemical studies of transcription have identified a set of accessory factors for transcript elongation by RNA polymerase II (Pol II) that appear to function in the context of chromatin. The C-terminal repeated domain (CTD) of Pol II may also play a role in regulating elongation through chromatin.

The Paf1 complex physically and functionally associates with transcription elongation factors in vivo.

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.