Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

A charge-dependent phase transition determines interphase chromatin organization

An emerging principle of cellular compartmentalization is that liquid unmixing results in formation of compartments by phase separation. We used electron spectroscopic Imaging (ESI), a transmission electron microscopy technology, to distinguish chromatin and nucleoplasmic phases of mammalian cell lines and their responses towards different environmental changes. We tested the hypothesis that charge-dependent phase separation mediated by the histone N-termini could explain the organization of chromatin. 3D images of nuclear chromatin with electron spectroscopic imaging (ESI) demonstrates that the amount of chromatin proximal to the interchromatin compartment (IC) differs between cell types, reflecting major differences in chromatin organization. These differences were lost when cells were treated overnight with a histone deacetylase inhibitor. We show that drastic, reversible changes in chromatin mixing or unmixing with the nucleoplasm/interchromatin space can be induced by modulating osmolarity of the medium or acetylation status of the chromatin. In vitro phase separation experiments demonstrated that chromatin separated from solution through a phase transition towards a more solid chromatin state.

106 STUDIES OF NUCLEAR ARCHITECTURE IN MAMMALIAN PRE-IMPLANTATION EMBRYOS AND EMBRYONIC STEM CELLS USING SUPER-RESOLUTION FLUORESCENCE MICROSCOPY

Three-dimensional (3-D) super-resolution fluorescence microscopy has allowed major progress in studies of the functional nuclear organization (Markaki et al. 2010 Cold Spring Harb. Symp. Quant. Biol. 75, 475–492; Markaki et al. 2012 Bioessays 34, 412–426). We have exploited these new possibilities to explore nuclear organization at different stages of bovine pre-implantation development (4-cell, 8-cell, 16-cell, morula, and blastocyst stage). In particular, we studied the topography of RNA polymerase II and the distribution of transcriptionally competent and noncompetent chromatin using antibodies against H3K4me3 and H3K27me3, respectively. For comparison, we have started analyses of mouse pre-implantation embryos and embryonic stem cells as well. Our results support the chromosome territory-interchromatin compartment (CT-IC) model (Cremer and Cremer 2010 Cold Spring Harb. Perspect. Biol. 2, a003889; Cremer et al. 2012 In: Epigenetic Regulation and Epigenomics 451–483). In all cell types, the nuclear space is occupied by chromosome territories (CTs; Koehler et al. 2009 Exp. Cell Res. 315, 2053–2063), the interchromatin compartment (IC), and one or several nucleoli. The CTs are built up from interconnected, megabase-sized chromatin domains (CDs). These ~1-Mbp CDs may consist of a series of ~100-kbp CDs (Cremer et al. 2000 Crit. Rev. Eukaryot. Gene Expr. 10, 179–212), which globally form a compact chromatin core surrounded by a layer of decondensed chromatin, called the perichromatin region. Current evidence supports the hypothesis that the perichromatin region represents the nuclear compartment, where transcription, co-transcriptional splicing, DNA-replication, and DNA-repair take place (Rouquette et al. 2010 Int. Rev. Cell Mol. Biol. 282, 1–90). The IC provides a contiguous, crowded compartment, which starts with channels at nuclear pores and pervades the chromatin compartment both between and within CTs. Small-scale chromatin loops of the perichromatin region can protrude into the interior of IC channels allowing direct contacts between CDs in cis and trans. At other sites the IC expands to wider, chromatin-free lacunas with splicing speckles and nuclear bodies. This model is in line with a fractal higher-order chromatin arrangement at all levels from CTs, chromosome arms and bands to ~1 Mbp CDs organized as fractal globules (Mirny 2011 Chromosome Res. 19, 37–51). This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).

Large-scale Chromatin Organization and the Localization of Proteins Involved in Gene Expression in Human Cells

Compartmentalization of the interphase nucleus is an important element in the regulation of gene expression. Here we investigated the functional organization of the interphase nucleus of HeLa cells and primary human fibroblasts. The spatial distribution of proteins involved in transcription (TFIIH and RNA polymerase II) and RNA processing and packaging (hnRNP-U) were analyzed in relation to chromosome territories and large-scale chromatin organization. We present evidence that these proteins are present predominantly in the interchromatin space, inside and between chromosome territories, and are largely excluded by domains of condensed chromatin. We show that they are present throughout the active and inactive X-chromosome territories in primary female fibroblasts, indicating that these proteins can freely diffuse throughout the interchromatin compartment in the interphase nucleus. Furthermore, we established that the in vivo spatial distribution of condensed chromatin in the interphase nucleus does not depend on ongoing transcription. Our data support a conceptually simple model for the functional organization of interphase nuclei.