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.

The 7SK small nuclear RNA (snRNA) regulates RNA polymerase II transcription elongation by controlling the protein kinase activity of the positive transcription elongation factor b (P-TEFb). In cooperation with HEXIM1, the 7SK snRNA sequesters P-TEFb into the kinase-inactive 7SK/HEXIM1/P-TEFb small nuclear ribonucleoprotein (snRNP), and thereby, controls the nuclear level of active P-TEFb. Here, we report that a fraction of HeLa 7SK snRNA that is not involved in 7SK/HEXIM1/P-TEFb formation, specifically interacts with RNA helicase A (RHA), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), A2/B1, R and Q proteins. Inhibition of cellular transcription induces disassembly of 7SK/HEXIM1/P-TEFb and at the same time, increases the level of 7SK snRNPs containing RHA, hnRNP A1, A2/B1, R and Q. Removal of transcription inhibitors restores the original levels of the 7SK/HEXIM1/P-TEFb and '7SK/hnRNP' complexes. 7SK/HEXIM1/P-TEFb snRNPs containing mutant 7SK RNAs lacking the capacity for binding hnRNP A1, A2, R and Q are resistant to stressinduced disassembly, indicating that recruitment of the novel 7SK snRNP proteins is essential for disruption of 7SK/HEXIM1/P-TEFb. Thus, we propose that the nuclear level of active P-TEFb is controlled by dynamic and reversible remodelling of 7SK snRNP.

Summary Mating type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, while MATα cells use HMR. The Recombination Enhancer (RE), located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating type-specific, and possibly RE-dependent chromosome folding. Here we use Hi-C, 5C, and live cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating type-specific conformational difference in the left arm. Deletion of a 1 kb subregion within the RE, which is not necessary during switching, abolished mating type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching, and a separate region involved in modulating chromosome conformation.

Inherently dynamic, chromosomes adopt many different conformations in response to DNA metabolism. Models of chromosome organization in the yeast nucleus obtained from genome-wide chromosome conformation data or biophysical simulations provide important insights into the average behavior but fail to reveal features from dynamic or transient events that are only visible in a fraction of cells at any given moment. We developed a method to determine chromosome conformation from relative positions of three fluorescently tagged DNA in living cells imaged in 3D. Cell type specific chromosome folding properties could be assigned based on positional combinations between three loci on yeast chromosome 3. We determined that the shorter left arm of chromosome 3 is extended in MATα cells, but can be crumpled in MAT a cells. Furthermore, we implemented a new mathematical model that provides for the first time an estimate of the relative physical constraint of three linked loci related to cellular identity. Variations in this estimate allowed us to predict functional consequences from chromatin structural alterations in asf1 and recombination enhancer deletion mutant cells. The computational method is applicable to identify and characterize dynamic chromosome conformations in any cell type.

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Spatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock. One Sentence Summary:Unequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.Main Text: Eukaryotic genomes are highly organised in three dimensions ( 1 , 2 ) and this spatial organisation has to be maintained in order to achieve the correct gene expression profiles ( 3 -6 ) . The 3D organisation of the genome is thus central to many aspects of cell biology and has been intensely investigated during normal growth ( 7 -9 ) , differentiation ( 10 -12 ) , cell division ( 13 ) , senescence ( 14 ) , and disease ( 5 , 6 , 15 ) , and has been shown to arise independently of transcription ( 16 ) . In the budding yeast Saccharomyces cerevisiae , target genes of most transcription factors are enriched in specific regions along the chromosome in one dimension ( 17 ) , or in the genome in three dimensions ( 18 ) . A central question in the field is by which mechanisms this 3D organisation is achieved.Any mechanism that organises genome structure has to do so in a highly dynamic and crowded nucleoplasm ( 19 , 20 ) . The prevalent view is that 3D genome organisation comes about despite the known intrinsic fluctuations of the chromatin fibre. Most studies focus on stable interactions between DNA-bound proteins that connect two chromatin loci ( 9 , 21 -27 ) . Here, we propose and validate a fundamentally different mechanism: The mobility of the chromatin fibre is not uniform, but heterogeneous, along its length, as a result of the unequal distribution of protein binding along the genome. This leads to thermodynamically driven self-organisation, which we observe experimentally, and which we show to have important functional implications.Determination and characterisation of chromatin states. In order to analyse the global effects of protein binding on spatial organisation of yeast chromosomes, and incorporate these data into pe...

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