Kerstin Bystricky

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.

High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome

Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10(-2)-10(3) sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.

Activation of Estrogen-Responsive Genes Does Not Require Their Nuclear Co-Localization

The spatial organization of the genome in the nucleus plays a role in the regulation of gene expression. Whether co-regulated genes are subject to coordinated repositioning to a shared nuclear space is a matter of considerable interest and debate. We investigated the nuclear organization of estrogen receptor alpha (ERα) target genes in human breast epithelial and cancer cell lines, before and after transcriptional activation induced with estradiol. We find that, contrary to another report, the ERα target genes TFF1 and GREB1 are distributed in the nucleoplasm with no particular relationship to each other. The nuclear separation between these genes, as well as between the ERα target genes PGR and CTSD, was unchanged by hormone addition and transcriptional activation with no evidence for co-localization between alleles. Similarly, while the volume occupied by the chromosomes increased, the relative nuclear position of the respective chromosome territories was unaffected by hormone addition. Our results demonstrate that estradiol-induced ERα target genes are not required to co-localize in the nucleus.

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

ABSTRACT We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.

DNA dynamics during early double-strand break processing revealed by non-intrusive imaging of living cells.

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.

The Role of Histone Modifications and Variants in Regulating Gene Expression in Breast Cancer

The role of epigenetic phenomena in cancer biology is increasingly being recognized. Here we focus on the mechanisms and enzymes involved in regulating histone methylation and acetylation, and the modulation of histone variant expression and deposition. Implications of these epigenetic marks for tumor development, progression and invasiveness are discussed with a particular emphasis on breast cancer progression.

Ligands specify estrogen receptor alpha nuclear localization and degradation

BackgroundThe estrogen receptor alpha (ERα) is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate.ResultsA MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line) was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα.Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2), and pure antagonists (selective estrogen regulator disruptor; SERD), ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM), 4-hydroxytamoxifen (OHT) or RU39,411, diffuse nuclear staining persisted.Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus.ConclusionsOur results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on transcription. These findings provide a molecular basis for the selection of antiestrogen compounds issue from pharmacological studies aimed at improving treatment of breast cancer.

Auraptene Is an Inhibitor of Cholesterol Esterification and a Modulator of Estrogen Receptors

Auraptene is a prenyloxycoumarin from Citrus species with chemopreventive properties against colitis-related colon and breast cancers through a yet-undefined mechanism. To decipher its mechanism of action, we used a ligand-structure based approach. We established that auraptene fits with a pharmacophore involved in both the inhibition of acyl-CoA:cholesterol acyl transferase (ACAT) and the modulation of estrogen receptors (ERs). We confirmed experimentally that auraptene inhibits ACAT and binds to ERs in a concentration-dependent manner and that it inhibited ACAT in rat liver microsomes and in intact cancer cells of murine and human origins, with an IC50 value in the micromolar range. Auraptene bound to ERs with affinities of 7.8 μM for ERα and 7.9 μM for ERβ, stabilized ERs, and modulated their transcriptional activity via an ER-dependent reporter gene and endogenous genes. We further established that these effects correlated well with the control of growth and invasiveness of tumor cells. Our data shed light on the molecular mechanism underlying the anticancer and chemopreventive effects of auraptene.

Estrogen receptor alpha and the activating protein-1 complex cooperate during insulin-like growth factor-I-induced transcriptional activation of the pS2/TFF1 gene.

Insulin like growth factor I (IGF-I) displays estrogenic activity in breast cancer cells. This activity is strictly dependent on the presence of estrogen receptor α (ERα). However the precise molecular mechanisms involved in this process are still unclear. IGF-I treatment induces phosphorylation of the AF1 domain of ERα and activation of estrogen regulated genes. These genes are characterized by important differences in promoter architecture and response element composition. We show that promoter structure is crucial for IGF-I-induced transcription activation. We demonstrate that on a complex promoter such as the pS2/TFF1 promoter, which contains binding sites for ERα and for the activating protein-1 (AP1) complex, transcriptional activation by IGF-I requires both ERα and the AP1 complex. IGF-I is unable to stimulate transcription of an estrogen-regulated gene under the control of a minimal promoter containing only a binding site for ERα. We propose a molecular mechanism with stepwise assembly of the AP1 complex and ERα during transcription activation of pS2/TFF1 by IGF-I. IGF-I stimulation induces rapid phosphorylation and an increase in protein levels of the AP1 complex. Binding of the phosphorylated AP1 complex to the pS2/TFF1 promoter allows recruitment of the chromatin remodeling factor Brg1 followed by binding of ERα via its interaction with c-Jun.