Heterochromatin Networks: Topology, Dynamics, and Function (a Working Hypothesis)

Abstract

Open systems can only exist by self-organization as pulsing structures exchanging matter and energy with the outer world. This review is an attempt to reveal the organizational principles of the heterochromatin supra-intra-chromosomal network in terms of nonlinear thermodynamics. The accessibility of the linear information of the genetic code is regulated by constitutive heterochromatin (CHR) creating the positional information in a system of coordinates. These features include scale-free splitting-fusing of CHR with the boundary constraints of the nucleolus and nuclear envelope. The analysis of both the literature and our own data suggests a radial-concentric network as the main structural organization principle of CHR regulating transcriptional pulsing. The dynamic CHR network is likely created together with nucleolus-associated chromatin domains, while the alveoli of this network, including springy splicing speckles, are the pulsing transcription hubs. CHR contributes to this regulation due to the silencing position variegation effect, stickiness, and flexible rigidity determined by the positioning of nucleosomes. The whole system acts in concert with the elastic nuclear actomyosin network which also emerges by self-organization during the transcriptional pulsing process. We hypothesize that the the transcriptional pulsing, in turn, adjusts its frequency/amplitudes specified by topologically associating domains to the replication timing code that determines epigenetic differentiation memory.

Keywords: chromatin organization; cytoskeleton; heterochromatin; networks; nucleolar boundary; physics of life; positional information; scale-free oscillations; transcriptional pulsing.

Figure 1

Visualization of the heterochromatin network in interphase cells: (a,b) Electron microscopic images of isolated rat thymocyte nuclei treated with 0.1% heparin/HBSS on supports; (a) 1 min; (b) 25 min (the nucleus fragment) by the method from [55]. PF fixation, uranyl acetate contrasting, tungsten oxide shadowing. (c) Human MCF7 cell, AO-DNA (after RNAse) fluorescent staining, deconvolution. (a–c) Large rosettes around nucleoli are encircled, heterochromatin clumps and “foots” inside them are arrowed, double alveoli are marked by double arrows. (d) Tissue imprint of chicken embryonic chondrocyte stained stoichiometrically for DNA; the method of image processing is described in [56]. (e) Mathematically skeletonized network of a DNA-stained chicken mesenchymal cell with overlaid dense chromocenters. (f) The relationship between the area of the dense chromocenters and the number of their network branches determined on chicken image-processed nuclei. Figure 1a,b republished from [52]. Figure 1d–f republished from [57].

Figure 2

Resolution of the heterochromatin network reconstructed from loci data of fluorescent molecules of an H3K9me3-stained breast cancer SKBr3 nucleus in a localization microscope. The images of an SkBr3 (breast cancer cell line) cell nucleus show: (a) A fluorescence wide-field image after heterochromatin staining with specific antibodies against H3K9me3 methylation sites. The inset shows a magnification of a region around small alveoli. (b) The same cell nucleus reconstructed from the localization data of the single fluorescence molecules as a density map. This means that point intensity relates to the number of neighbors. Due to soft focus filters, details of molecular arrangements are lost, but high (knots) and low (alveoli) density structures can be easily detected. Such image reconstructions based on density histograms are similar to conventional microscope images and allow a comparison to other techniques in many cases. The insert indicates that the shapes obtained by standard microscopy can be resolved into heterochromatin arrangements of different shape and molecular density. (c) Full pointillist localization image of all fluorescent molecules detecting H3K9me3 sites. Such images are obtained from data sets that are the basis for all quantitative structure and topology analyses using mathematical algorithms of statistics, geometry, and topology. The inset qualitatively indicates that the high-density knots revealed a structured folding. (d) Cluster image as a result of mathematical operations on the localization data set (visualized in (c)). The image only shows the areas that met a certain cluster parameter. The insert indicates that the dense heterochromatin regions consisted of clusters embedded in heterochromatin structures shown in (c).

Figure 3

The features of PADs and their network show a scale-free distribution in control MCF7 cells. (a,b) H3K9me3-positive PADs are clustered by centromeres, Nl—nucleolus; (c) the area and number of individual PADs clustered by centromeres and the negative exponential relationship between the area and number of individual PADs, with the dashed boundary of the largest likely static PADs; (d) preferential staining of heterochromatin by toluidine blue (method from [61]); (e) the filtered image of the same nucleus revealed the heterochromatin network with typical double alveoli (double-arrowed); (f) the negative exponential relationship between the area and the number of individual network alveoli with a set dashed boundary for the largest ones; (a–c) republished from [44].

Figure 4

Chromatin changing in a chicken embryonic femur growth plate during transcriptional activation (a,b) and suppression (c,d) in the transient tissue—hypertrophic chondrocytes of a 14-day chicken embryo femur growth plate; (e,f) the results of morphometry showing the relationship between DNA concentration (OD) in dynamic chromocenters depending on the stage and vectorial position between the interactively discriminated perinucleolar heterochromatin shell (qIOD ring) and nuclear border in the active (light circles) and ageing (filled circles) chondrocytes; squares—mesenchymal precursors; double circles—differentiated, transient to ageing cells. (f) The concerted condensation of small chromocenters (Y-axis) and DNA accumulation in the perinucleolar ring formed by NORs and attached chromocenters (X-axis) in ageing cells (filled rings), and much weaker dependence on NORs in transcriptionally active chondrocytes (light circles). The mesenchymal precursors (squares in (e,f)) are rather inert to these forces. (a,c) Scale bars = 5 µm. Republished from [38,57,68].

Figure 4

Chromatin changing in a chicken embryonic femur growth plate during transcriptional activation (a,b) and suppression (c,d) in the transient tissue—hypertrophic chondrocytes of a 14-day chicken embryo femur growth plate; (e,f) the results of morphometry showing the relationship between DNA concentration (OD) in dynamic chromocenters depending on the stage and vectorial position between the interactively discriminated perinucleolar heterochromatin shell (qIOD ring) and nuclear border in the active (light circles) and ageing (filled circles) chondrocytes; squares—mesenchymal precursors; double circles—differentiated, transient to ageing cells. (f) The concerted condensation of small chromocenters (Y-axis) and DNA accumulation in the perinucleolar ring formed by NORs and attached chromocenters (X-axis) in ageing cells (filled rings), and much weaker dependence on NORs in transcriptionally active chondrocytes (light circles). The mesenchymal precursors (squares in (e,f)) are rather inert to these forces. (a,c) Scale bars = 5 µm. Republished from [38,57,68].

Figure 5

Segregation of the heterochromatin (HR) compartment in SV-40 transformed hamster fibroblast cell line 4/21: (a) the undifferentiated growth state; (b) after 2 min with bleomycin (01%/HBSS)—the pattern of HR nucleation; (c) 20 min bleomycin treatment—no compartmentation after deep chromatin fragmentation; (d) 1% DMSO 5-day-induced typical differentiation pattern of HR distribution in cell nuclei of the same cell line; (a–d) republished from [53].

Figure 5

Segregation of the heterochromatin (HR) compartment in SV-40 transformed hamster fibroblast cell line 4/21: (a) the undifferentiated growth state; (b) after 2 min with bleomycin (01%/HBSS)—the pattern of HR nucleation; (c) 20 min bleomycin treatment—no compartmentation after deep chromatin fragmentation; (d) 1% DMSO 5-day-induced typical differentiation pattern of HR distribution in cell nuclei of the same cell line; (a–d) republished from [53].

Figure 6

Partial inhibition of rRNA and mRNA synthesis by AcD and alpha-amanitin revealed the hidden radial-concentric spatial relationship between centers of nucleolar synthesis, perinucleolar PADs, and speckles; this order was destroyed after full suppression of transcription: (a) Control: the granules of RPA1 fill the nucleoli, indicating active rRNA synthesis; (b) AcD 0.2 µM/mL, 1 h—RPA1 form rare fused granules, indicating suppressed rRNA synthesis; H3K9me3 PADs compact and encircle the nucleoli; (c) control: elongated speckles and PADs seem disorderly distributed in the cell nucleus; (d) AcD 0.2 µM/mL, 1 h—clumped speckles surround the compacted shells of perinucleolar PADs; (e) alpha-amanitin 2 µM/mL for 2 h suppressing Pol II—an example of swollen empty speckles radially circumventing the deteriorating perinucleolar ring of PADs; (f) AcD 2 µM/mL for 5 h suppressing both RNA syntheses with chaotically distributed disarranged PADs and empty speckles. Scale bars = 5 µm.

Figure 7

The radial-concentric nuclear order of the perinucleolar compacted PADs and speckles around them provoked in the course of transcription suppression by AcD treatment accompanied by gradual loss of lamin B stiffness: (a) After 1 h of AcD 0.2 µM/mL, a radial-concentric order of compacted perinucleolar PADs and the outer ring of compacted speckles was demonstrated in (b) by IF intensity measurements. (c) The radial-concentric model of the route-relay of the products of both rDNA and mRNA synthesis and splicing towards an arbitrary nuclear envelope (dashed). (d) Softening of nuclear lamin (evidenced by IF staining of lamin B1 (red)) testified as an increase of the proportion of cells with intranuclear lamin folds in the time course of suppression of RNA synthesis. Modified from [107].

Figure 7

The radial-concentric nuclear order of the perinucleolar compacted PADs and speckles around them provoked in the course of transcription suppression by AcD treatment accompanied by gradual loss of lamin B stiffness: (a) After 1 h of AcD 0.2 µM/mL, a radial-concentric order of compacted perinucleolar PADs and the outer ring of compacted speckles was demonstrated in (b) by IF intensity measurements. (c) The radial-concentric model of the route-relay of the products of both rDNA and mRNA synthesis and splicing towards an arbitrary nuclear envelope (dashed). (d) Softening of nuclear lamin (evidenced by IF staining of lamin B1 (red)) testified as an increase of the proportion of cells with intranuclear lamin folds in the time course of suppression of RNA synthesis. Modified from [107].

Figure 8

The speckles acting as radial spring pumps anchored between the nucleolar and perinuclear flexibly rigid heterochromatin shells and equipped by elastic actomyosin elements integrate the radial-concentric nuclear order for transcription pulsing. (a,b) EM thin sections of rat ascitic Zajdela hepatoma cells: (a) The links of the speckle (ICG) to the perinucleolar and perinuclear heterochromatin (arrows), Nl—nucleolus; (b) the view of the radial-concentric speckle substructure as revealed on shortly cold-PF-prefixed cells after short DNAse I treatment with 4.5 mM MgCl₂ in phosphate buffer/sucrose before dehydration and epoxy-embedment (conventional contrasting). ICG—speckle, HR—heterochromatin, Nl—nucleolus. Republished from [53]. (c) The representative image of the circular-radial self-organization of F-actin filaments (stained by phalloidin) of human foreskin fibroblasts, structured soon after seeding on isotropic substrate; from [108]. (d) Schematic of the functional transcriptional relay in its relationship with speckles and the concentric rings of the nucleolar and perinuclear heterochromatin shells, deduced from experiments with suppression of RNA synthesis and the literature on the participation of the nuclear cytoskeleton in it. Modified from [107].

Figure 8

The speckles acting as radial spring pumps anchored between the nucleolar and perinuclear flexibly rigid heterochromatin shells and equipped by elastic actomyosin elements integrate the radial-concentric nuclear order for transcription pulsing. (a,b) EM thin sections of rat ascitic Zajdela hepatoma cells: (a) The links of the speckle (ICG) to the perinucleolar and perinuclear heterochromatin (arrows), Nl—nucleolus; (b) the view of the radial-concentric speckle substructure as revealed on shortly cold-PF-prefixed cells after short DNAse I treatment with 4.5 mM MgCl₂ in phosphate buffer/sucrose before dehydration and epoxy-embedment (conventional contrasting). ICG—speckle, HR—heterochromatin, Nl—nucleolus. Republished from [53]. (c) The representative image of the circular-radial self-organization of F-actin filaments (stained by phalloidin) of human foreskin fibroblasts, structured soon after seeding on isotropic substrate; from [108]. (d) Schematic of the functional transcriptional relay in its relationship with speckles and the concentric rings of the nucleolar and perinuclear heterochromatin shells, deduced from experiments with suppression of RNA synthesis and the literature on the participation of the nuclear cytoskeleton in it. Modified from [107].

Figure 9

The topological relationship between PADs, NADs, and transcription hubs in MCF7 cells: (a) H3K9me3-labelled PADs are located near or inside the DAPI-poor alveoli decorated by H3K4me3-positive foci (enlarged on insert) which are (b) often occupied with the fibrillarin-marked nucleoli.

Figure 10

Replication timing and the tissue-specific “heterochromatin code” in a coordinate system for two different cell types (arbitrary hepatocytes and lymphocytes). (a) Hypothesis: The tissue-specific positional information is created by the latitude positions of silencing constitutive heterochromatin (CHR) clusters set (designed as red and blue balls) by replication timing upon the radial chromosome longitudes as the “3–4 D address” (to be specified by topology-associated domains (TADs)). (b) Principle sketch of the CHR clusters positions in the cell nucleus in a radial-concentric “geographic map” of coordinates (on 2D image) determined by Delaunay triangles. Nl—nucleolus.