Fiber-Like Organization as a Basic Principle for Euchromatin Higher-Order Structure

Abstract

A detailed understanding of the principles of the structural organization of genetic material is of great importance for elucidating the mechanisms of differential regulation of genes in development. Modern ideas about the spatial organization of the genome are based on a microscopic analysis of chromatin structure and molecular data on DNA-DNA contact analysis using Chromatin conformation capture (3C) technology, ranging from the "polymer melt" model to a hierarchical folding concept. Heterogeneity of chromatin structure depending on its functional state and cell cycle progression brings another layer of complexity to the interpretation of structural data and requires selective labeling of various transcriptional states under nondestructive conditions. Here, we use a modified approach for replication timing-based metabolic labeling of transcriptionally active chromatin for ultrastructural analysis. The method allows pre-embedding labeling of optimally structurally preserved chromatin, thus making it compatible with various 3D-TEM techniques including electron tomography. By using variable pulse duration, we demonstrate that euchromatic genomic regions adopt a fiber-like higher-order structure of about 200 nm in diameter (chromonema), thus providing support for a hierarchical folding model of chromatin organization as well as the idea of transcription and replication occurring on a highly structured chromatin template.

Keywords: electron tomography; euchromatin; higher-order chromatin folding; replication; transcription.

FIGURE 1

Comparison of labeling efficiency and probe penetration into EdU-labeled cells. (A) 10 min formaldehyde fixation, detection with AlexaFluor-azide. (B) 1 h glutaraldehyde fixation, detection with biotin-azide–streptavidin-AlexaFluor. Bar, 10 μm. (C,D) DAPI staining. Bar 10 μm.

FIGURE 2

EdU pulse-labeled HT1080 cells after biotin-azide streptavidin-Nanogold detection and embedding demonstrate clear replication patterns en bloc under a transmitted light microscope, enabling selection of cells with early-S patterns (arrow) for sectioning. Bar, 10 μm.

FIGURE 3

Raw zero-tilt image of a 250-nm-thick section of HT1080 cell in early S-phase labeled with 2 h pulse of EdU with subsequent detection with biotin-azide–streptavidin-Nanogold (A). Segments of labeled fiber-like chromatin structures (arrows) are randomly distributed throughout nuclear interior. Bar, 1 μm (A) and 0.5 μm (B).

FIGURE 4

Tomographic slice of HT1080 cell nucleus labeled with 2 h pulse of EdU and detected with biotin-azide and streptavidin-Nanogold (A) and main steps of image analysis. (B–F) (B) Segmentation of Ag-enhanced Au particles. (C) 3D density calculation for different radii. The plot demonstrates total number of clusters depending on density calculation radius, green lines indicating the thresholds used for further calculations. (D) 3D density map. (E) Thresholded 3D density map. (F) 3D Distance map; Bar, 500 nm. (G) Histograms of local thickness distribution, calculated for two clustering thresholds show modal radii of higher-order chromatin fibers between 75 and 90 nm.