Frank R. Neumann

Live Imaging of Telomeres: yKu and Sir Proteins Define Redundant Telomere-Anchoring Pathways in Yeast

BACKGROUND The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle. RESULTS We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms. CONCLUSIONS Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP‐tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312‐aa domain of Sir4 (Sir4PAD) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4PAD‐mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku‐ and Esc1‐dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

Sir-Mediated Repression Can Occur Independently of Chromosomal and Subnuclear Contexts

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.

Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination.

Chromatin in the interphase nucleus moves in a constrained random walk. Despite extensive study, the molecular causes of such movement and its impact on DNA-based reactions are unclear. Using high-precision live fluorescence microscopy in budding yeast, we quantified the movement of tagged chromosomal loci to which transcriptional activators or nucleosome remodeling complexes were targeted. We found that local binding of the transcriptional activator VP16, but not of the Gal4 acidic domain, enhances chromatin mobility. The increase in movement did not correlate strictly with RNA polymerase II (PolII) elongation, but could be phenocopied by targeting the INO80 remodeler to the locus. Enhanced chromatin mobility required Ino80s ATPase activity. Consistently, the INO80-dependent remodeling of nucleosomes upon transcriptional activation of the endogenous PHO5 promoter enhanced chromatin movement locally. Finally, increased mobility at a double-strand break was also shown to depend in part on the INO80 complex. This correlated with increased rates of spontaneous gene conversion. We propose that local chromatin remodeling and nucleosome eviction increase large-scale chromatin movements by enhancing the flexibility of the chromatin fiber.

Methods for visualizing chromatin dynamics in living yeast.

Publisher Summary This chapter describes the techniques for the visualization of chromatin in living cells (primarily in budding yeast), while pointing out pitfalls and artifacts that can arise during live cell imaging. It also presents analytical tools that have been developed for the quantitation of data generated by digital imaging. These tools allow defining a new field of quantitative analysis: the dynamic behavior of DNA in real time. To visualize the laci repressor in yeast, its gene is fused in frame to sequences encoding a nuclear localization signal, and the S65T derivative of natural green fluorescent protein (GFP), which has a red-shifted excitation spectrum and higher emission intensity. Fusions to optimized forms of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) can be used. The detection of chromatin movement can be monitored by new tracking algorithm that uses dynamic programming to extract the optimal spatiotemporal trajectory of the particle. The automated image analysis software consists of three components: alignment phase, preprocessing phase, and tracking phase. Chromatin mobility is nearly identical in the three organisms studied in detail to date. Notably, tagged sites along yeast chromosomes, sites on the X chromosome in Drosophila spermatocytes, and various insertions at random positions on human chromosomes show similar dynamics.

Long time behaviour of diffusing particles in constrained geometries; application to chromatin motion

Inspired by experiments that use single-particle tracking to measure the regions of confinement of selected chromosomal regions within cell nuclei, we have developed an analytical approach that takes into account various possible positions and shapes of the confinement regions. We show, in particular, that confinement of a particle into a subregion that is entirely enclosed within a spherical volume can lead to a higher limit of the mean radial square displacement value than the one associated with a particle that can explore the entire spherical volume. Finally, we apply the theory to analyse the motion of extrachromosomal chromatin rings within nuclei of living yeast.

Nucleosome Remodeling Complexes Increase Subnuclear Dynamics Of Chromatin In Yeast

We have described the mobility of internal loci on yeast interphase chromosomes by live fluorescence microscopy with high precision. Based on single particle tracking and mathematical simulations of random walks in a confined volume, we could define the constraints exerted by the chromatin fiber. We find that local recruitment of the transcriptional activator VP16 and components of the Ino80 chromatin remodeling complex significantly increases diffusion rate, large rapid steps and the radius of constraint of a given locus. Importantly, the Ino80 induced mobility increase is largely dependent on its ATPase activity. Inhibition of transcription did not alter chromatin mobility and we find no correlation between transcriptional elongation and increased mobility. In analogy to experiments in mammalian cells, this indicates that transcription per se does not directly influence chromatin dynamics. These are the first mechanistic indications that chromatin remodelers can indeed alter mobility of the chromatin fiber - a process can facilitate chromatin contacts to distinct subnuclear regions.To examine the function of increased chromatin mobility, we have monitored the recombination rate of substrates to which Ino80 is targeted. We find that Ino80 enhances the spontaneous rate of gene conversion. This is not achieved by targeting the VP16 transactivation domain. The mode of Ino80 action at DSBs and at collapsed replication forks will be discussed both with respect to the physical characteristics of the chromatin fiber, and with respect to local nucleosome eviction.

Chapter 46 – Tracking Individual Chromosomes with Intergrated Arrays of lacop Sites and GFP-laci Repressor: Analyzing Position and Dynamics of Chromosomal Loci in Saccharomyces cerevisiae

Publisher Summary This article describes a technique for analyzing position and dynamics of chromosomal loci in saccharomyces cerevisiae. The visualization of specific DNA sequences in living cells, achieved through the integration of lac operator arrays and expression of a GFP-lac repressor fusion, has provided new tools to examine how the nucleus is organized and how basic events such as sister chromatid separation occur. Plasmids or integrations of repetitive arrays are difficult to propagate in both bacteria and yeast due to recombination induced excision events. Insert a multimerized lac op array into the chromosome by standard transformation using a linearized construct that integrates by homologous recombination. By comparing the motion of two tagged loci, one can calculate the average movement without concern for nuclear drift. Depletion of glucose or growth of alternative carbon sources can alter chronetin dynamics. Wash cells once before observation to avoid YPD auto-fluorescence. Cells sealed in this way are in a closed environment in which the depletion of oxygen and production of carbon dioxide bubbles can influence growth and impair visualization.