Dataset Information

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.

ABSTRACT: The chromatin structure of DNA determines genome compaction and activity in the nucleus. On the basis of in vitro structures and electron microscopy (EM) studies, the hierarchical model is that 11-nanometer DNA-nucleosome polymers fold into 30- and subsequently into 120- and 300- to 700-nanometer fibers and mitotic chromosomes. To visualize chromatin in situ, we identified a fluorescent dye that stains DNA with an osmiophilic polymer and selectively enhances its contrast in EM. Using ChromEMT (ChromEM tomography), we reveal the ultrastructure and three-dimensional (3D) organization of individual chromatin polymers, megabase domains, and mitotic chromosomes. We show that chromatin is a disordered 5- to 24-nanometer-diameter curvilinear chain that is packed together at different 3D concentration distributions in interphase and mitosis. Chromatin chains have many different particle arrangements and bend at various lengths to achieve structural compaction and high packing densities.

SUBMITTER: Ou HD

PROVIDER: S-EPMC5646685 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Publications

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.

Ou Horng D HD Phan Sébastien S Deerinck Thomas J TJ Thor Andrea A Ellisman Mark H MH O'Shea Clodagh C CC

Science (New York, N.Y.) 20170701 6349

The chromatin structure of DNA determines genome compaction and activity in the nucleus. On the basis of in vitro structures and electron microscopy (EM) studies, the hierarchical model is that 11-nanometer DNA-nucleosome polymers fold into 30- and subsequently into 120- and 300- to 700-nanometer fibers and mitotic chromosomes. To visualize chromatin in situ, we identified a fluorescent dye that stains DNA with an osmiophilic polymer and selectively enhances its contrast in EM. Using ChromEMT (C ...[more]

PMID: 28751582

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Project description:BackgroundThe transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Methodology/principal findingsUsing confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Conclusions/significanceOur results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress.

Dataset Information

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.

Publications

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.

Similar Datasets

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