Unveiling nuclear chromatin distribution using IsoConcentraChromJ: A flourescence imaging plugin for IsoRegional and IsoVolumetric based ratios analysis

Abstract

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods.

Fig 1. Summary of the visualizing and processing images for a representative nucleus of both pre-adipocytes and mature adipocytes.

Input Image (left), Sequential ROI Filtering (middle), and Splitting Channels (right) for Genomic DNA and Epigenetic Marker (Acetylated Histones) in 3D Imaging. Scale bar: 6μm.

Fig 2. Filtering procedure for a pre-adipocyte nucleus.

Detection of Genomic DNA (Channel 1) with Synchronous Edge Selection on Epigenetic Acetylated Histones Marker (Channel 2). Scale bar: 6μm.

Fig 3. Area regions.

Visualization of the area regions; Representative Areas Highlighted in Red Bold for Full, Intermediate, and Center Regions. (Top) Full Area (I) of two channels, Genomic DNA and Epigenetic Marker (Acetylated Histones) respectively. (Middle) splitting percentage dialog for selecting the percentage of splitting for the intermediate and center area. (Bottom) intermediate area of the two channels selected using imageJ Plugin and it represents 66.66% of the full area (II) and center area, where it represents 33.33% of the full area (III) selected using the ImageJ plugin. Scale bar: 6μm.

Fig 4. 2D spatial distribution of total chromatin and euchromatin in pre-adipocytes and mature adipocytes.

The ratios represented in percentages. (A) B1/B, B2/B, and B3/B Ratios: Chromatin organization across peripheral, intermediate, and central regions within the nucleus relative to total chromatin; (B) A/B Ratio: Total euchromatin relative to total chromatin in Pre-adipocytes and Mature Adipocytes; (C) A1/B, A2/B, and A3/B Ratios: H3K9Ac-associated euchromatin in Pre-adipocytes and Mature Adipocytes Across Nuclear Chromatin Regions with respect to total chromatin; (D) Isoconcentric Distribution of euchromatin Regions Relative to Total euchromatin Within Nuclei, A1/A, A2/A, and A3/A Ratios: Isoconcentric organization of euchromatin across the peripheral, intermediate, and central nuclear regions, respectively, in Pre-adipocytes and Mature Adipocytes.

Fig 5. 3D spatial organization of chromatin and euchromatin dynamics during adipocyte maturation.

The ratios represented in percentages. (A) B1/B, B2/B, and B3/B Ratios: Chromatin organization across peripheral, intermediate, and central regions within the nucleus relative to total chromatin; (B) A/B Ratio: Total euchromatin relative to total chromatin in Pre-adipocytes and Mature Adipocytes; (C) A1/B, A2/B, and A3/B Ratios: H3K9Ac-associated euchromatin in Pre-adipocytes and Mature Adipocytes Across Nuclear Chromatin Regions with respect to total chromatin; (D) Isoconcentric Distribution of euchromatin Regions Relative to Total euchromatin Within Nuclei, A1/A, A2/A, and A3/A Ratios: Isoconcentric organization of euchromatin across the peripheral, intermediate, and central nuclear regions, respectively, in Pre-adipocytes and Mature Adipocytes.