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

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

Mating type specific chromosome conformation in Saccharomyces cerevisiae

Budding yeast switch their mating type by a gene conversion event at the MAT locus which uses either of two silent loci (HML or HMR) on opposite ends of chromosome three as a template. InMATa cells the left arm of Chr. Ill is “activated” which allows for the preferential recombination ofHML with the MAT locus. The left arm is otherwise “repressed” for recombination in MATα cells which then prefer to use HMR, on the right arm, as a template for gene conversion. We set out to analyze the potential role of chromosome conformation in this “activation”/”repression” phenomenon observed on the left arm of Chr. III. We used Chromosome Conformation Capture Carbon Copy (5C) to comprehensively analyze the conformation of chromosomes III, V, and XII in the two mating types. Our data reveals that the yeast genome is organized in a unique way compared to other species. We have found that global nuclearorganization such ascentromereclustering, telomere tethering to the periphery, and sequestration of the rDNA array into the nucleolus affect both the specific conformations of each chromosome but also the interactions between these chromosomes. Our analysis indicates that the overall architecture for these 3 chromosomes is very similar between the two mating types. Interestingly, a mating type specific difference in conformation of the left arm of Chr. Ill was identified. Furthermore, the 5C data was used, in conjunction with the Integrative Modeling Platform (IMP), to generate three dimensional models of Chr. III in both mating types. This method provides a more intuitive way of viewing 5C data and reveals that, in general, Chr. Ill has a more crumpled conformation in MATacells than in MATα. However, this crumpling is most evident on the left arm of the chromosome. Thus the phenomenon of “activation”/”repression” of the left arm of Chr. III which is associated with mating type-specific switching preference is, in fact, associated with a difference in the innate conformation of Chr. Ill between the two mating types. This difference in structure between mating types will be used as a phenotype to analyze the effect of cis and trans acting factors that play a role in switching preference through alteration of chromosome conformation

Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

Challenges and guidelines toward 4D nucleome data and model standards

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models

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

Role of the chromosome three organization in the directionality of the mating type switch in S. cerevisiae

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Genome duality

A report on 'Higher Order Genome Architecture', the third meeting of the Marie Curie Conferences and Training Courses (MC-GARD), Edinburgh, UK, 1-5 April 2009