Chromatin conformation regulates gene expression and thus, constant remodeling of chromatin structure is essential to guarantee proper cell function. To gain insight into the spatiotemporal ...organization of the genome, we use high-density photoactivated localization microscopy and deep learning to obtain temporally resolved super-resolution images of chromatin in living cells. In combination with high-resolution dense motion reconstruction, we find elongated ~45- to 90-nm-wide chromatin "blobs." A computational chromatin model suggests that these blobs are dynamically associating chromatin fragments in close physical and genomic proximity and adopt topologically associated domain-like interactions in the time-average limit. Experimentally, we found that chromatin exhibits a spatiotemporal correlation over ~4 μm in space and tens of seconds in time, while chromatin dynamics are correlated over ~6 μm and last 40 s. Notably, chromatin structure and dynamics are closely related, which may constitute a mechanism to grant access to regions with high local chromatin concentration.
H2A.Z association with specific genomic loci is thought to contribute to a chromatin structure that promotes transcription activation. Acetylation of H2A.Z at promoters of oncogenes has been linked ...to tumorigenesis. The mechanism is unknown. Here, we show that in triple negative breast cancer cells, H2A.Z bound to the promoter of the constitutively, weakly expressed cyclin D1 oncogene (CCND1), a key regulator of cellular proliferation. Depleting the pool of H2A.Z stimulated transcription of CCND1 in the absence of its cognate transcription factor, the estrogen receptor (ER). During activation of CCND1, H2A.Z was released from the transcription start site (TSS) and downstream enhancer (enh2) sequences. Concurrently, acetylation of H2A.Z, H3 and H4 at the TSS was increased but only H2A.Z was acetylated at enh2. Acetylation of H2A.Z required the Tip60 acetyltransferase to be associated with the activated CCND1 on both TSS and enh2 sites. Depletion of Tip60 prevented CCND1 activation. Chromosome conformation capture experiments (3C) revealed specific contacts between the TSS and enh2 chromatin regions. These results suggest that release of a histone H2A.Z-mediated repression loop activates CCND1 for transcription. Our findings open new avenues for controlling and understanding aberrant gene expression associated with tumorigenesis.
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.
In breast cancer, approximately one-third of tumors express neither the estrogen receptor (ERalpha) nor estrogen-regulated genes such as the progesterone receptor gene (PR). Our study provides new ...insights into the mechanism allowing hormone-activated expression of ERalpha target genes silenced in ERalpha-negative mammary tumor cells. In cell lines derived from ERalpha-negative MDA-MB231 cells, stable expression of different levels of ERalpha from a transgene did not result in transcription of PR. A quantitative comparative analysis demonstrates that inhibiting DNA methyltransferases using 5-aza-2'-deoxycytidine or specific disruption of DNMT1 by small interfering RNAs and treatment with the histone-deacetylase inhibitor trichostatin A enabled ERalpha-mediated hormone-dependent expression of endogenous PR. We show that demethylation of a CpG island located in the first exon of PR was a prerequisite for ERalpha binding to these regulatory sequences. Although not a general requirement, DNA demethylation is also necessary for derepression of a subset of ERalpha target genes involved in tumorigenesis. PR transcription did not subsist 4 days after removal of the DNA methyltransferase blocking agents, suggesting that hormone-induced expression of ERalpha target genes in ERalpha-negative tumor cells is transient. Our observations support a model where an epigenetic mark confers stable silencing by precluding ERalpha access to promoters.
We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin ...higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53
S when maximally folded in 1
mM or 100
mM MgCl
2. The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14–19.
Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase ...chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of ≈110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.
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.
We present a novel Lab-on-Chip technology for 3D particle tracking in living cells based on V-shaped micro-mirrors, which are used to observe fluorescent specimens from multiple vantage points, ...providing simultaneous stereo-images that can be recombined for 3D reconstruction. Our technology can be readily used with standard fluorescence microscopes, and we apply it to study chromatin dynamics using yeast strains with one or two GFP-tagged gene loci. Using an Andor EMCCD camera, loci are followed in 3D with inter-frame intervals of up to 10 ms and with an error of 27 nm per axis, yielding quantitative information on their dynamics with exquisite temporal spatial resolution.
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