Florence Hediger

Nuclear pore association confers optimal expression levels for an inducible yeast gene.

The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3′ untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics

We present a new, robust, computational procedure for tracking fluorescent markers in time-lapse microscopy. The algorithm is optimized for finding the time-trajectory of single particles in very noisy dynamic (two- or three-dimensional) image sequences. It proceeds in three steps. First, the images are aligned to compensate for the movement of the biological structure under investigation. Second, the particles signature is enhanced by applying a Mexican hat filter, which we show to be the optimal detector of a Gaussian-like spot in 1//spl omega//sup 2/ noise. Finally, the optimal trajectory of the particle is extracted by applying a dynamic programming optimization procedure. We have used this software, which is implemented as a Java plug-in for the public-domain ImageJ software, to track the movement of chromosomal loci within nuclei of budding yeast cells. Besides reducing trajectory analysis time by several 100-fold, we achieve high reproducibility and accuracy of tracking. The application of the method to yeast chromatin dynamics reveals different classes of constraints on mobility of telomeres, reflecting differences in nuclear envelope association. The generic nature of the software allows application to a variety of similar biological imaging tasks that require the extraction and quantitation of a moving particles trajectory.

Functional Targeting of DNA Damage to a Nuclear Pore-Associated SUMO-Dependent Ubiquitin Ligase

Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)–dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.

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.

Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control.

The positioning of chromosomal domains in interphase nuclei is thought to facilitate transcriptional repression in yeast. It has been reported that two large coiled-coil proteins of the nuclear envelope, myosin-like proteins 1 and 2, play direct roles in anchoring yeast telomeres to the nuclear periphery, thereby creating a subcompartment enriched for Sir proteins. We have created strains containing complete deletions of mlp1 and mlp2 genes, as well as the double null strain, and find no evidence for the disruption of telomere anchoring at the nuclear periphery in these cells. We also detect no disruption of telomere-associated gene silencing. We confirm, on the other hand, that mlp mutants are particularly sensitive to DNA-damaging agents, such as bleomycin. Moreover, we show that rather than having short telomeres as in yKu-deficient strains, the mlp1 mlp2 strains have extended telomeres, resembling phenotypes of mutations in rif1. Whereas the mlp1 mlp2 mutations act on a pathway of telomere length regulation different from that of yKu70, the effects of the tel1 deletion are epistatic to the mlp mutations, suggesting that the Mlp proteins restrict telomere length in wild-type cells by influencing the Rif-Tel1 pathway of telomerase regulation.

Nuclear organization and silencing: putting things in their place

The positioning of a gene within the nucleus is thought to help regulate its transcriptional state. An example is yeast telomeres, which have a propensity to cluster at the nuclear periphery and suppress subtelomeric genes. With a membrane anchoring technique, new data indicate that there may be a second class of perinuclear silencing sites, which require pore-associated myosin-like proteins to establish repression.

The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast

Double-strand breaks (DSB) in yeast lead to the formation of repair foci and induce a checkpoint response that requires both the ATR-related kinase Mec1 and its target, Rad53. By combining high-resolution confocal microscopy and chromatin-immunoprecipitation assays, we analysed the genetic requirements for and the kinetics of Mec1 recruitment to an irreparable HO-endonuclease-induced DSB. Coincident with the formation of a 3′ overhang, the Mec1-Ddc2 (Lcd1) complex is recruited into a single focus that colocalises with the DSB site and precipitates with single-strand DNA (ssDNA). The absence of Rad24 impaired cut-site resection, Mec1 recruitment and focus formation, whereas, in the absence of yKu70, both ssDNA accumulation and Mec1 recruitment was accelerated. By contrast, mutation of the N-terminus of the large RPA subunit blocked Mec1 focus formation without affecting DSB processing, arguing for a direct involvement of RPA in Mec1-Ddc2 recruitment. Conversely, loss of Rad51 enhanced Mec1 focus formation independently of ssDNA formation, suggesting that Rad51 might compete for the interaction of RPA with Mec1-Ddc2. In all cases, Mec1 focus formation correlated with checkpoint activation. These observations led to a model that links end-processing and competition between different ssDNA-binding factors with Mec1-Ddc2 focus formation and checkpoint activation.

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.

Subtelomeric factors antagonize telomere anchoring and Tel1-independent telomere length regulation

Yeast telomeres are anchored at the nuclear envelope (NE) through redundant pathways that require the telomere‐binding factors yKu and Sir4. Significant variation is observed in the efficiency with which different telomeres are anchored, however, suggesting that other forces influence this interaction. Here, we show that subtelomeric elements and the insulator factors that bind them antagonize the association of telomeres with the NE. This is detectable when the redundancy in anchoring pathways is compromised. Remarkably, these same conditions lead to a reduction in steady‐state telomere length in the absence of the ATM‐kinase homologue Tel1. Both the delocalization of telomeres and reduction in telomere length can be induced by targeting of Tbf1 or Reb1, or the viral transactivator VP16, to a site 23 kb away from the TG repeat. This correlation suggests that telomere anchoring and a Tel1‐independent pathway of telomere length regulation are linked, lending a functional significance to the association of yeast telomeres with the NE.