Chromatin in cell nuclei is formed by DNA associated with nucleosomes which consist of the core histone proteins (H2A, H2B, H3, H4). These play a crucial role in genome organization and are well-known regulators of a variety of biological processes occurring at the DNA template such as transcriptional activity. The histone subtype H3 exists in several varieties. Whereas most varieties are synthesized during S-phase of the cell cycle, the variant H3.3 is expressed throughout interphase and has been associated with transcriptionally active chromatin [1]. These differences suggest diverging roles of these variants in chromatin organization and function.

Here, we expressed histone H3 variants in normal and malignant cells and compared the nuclear expression patterns at different resolution levels using conventional epifluorescence and confocal microscopy, structured illumination imaging (SIM) and electron microscopy (Fig. 1).

We observed differing distribution patterns of the histone H3 variants within interphase cell nuclei (Fig. 2). In addition, we found distinct banding patterns after expression of histone H3.3 in mitotic chromosomes. Together this indicates differences in functions of these chromatin constituents in the interphase nucleus [2]. Surprisingly, the highly transcribed ribosomal genes within nucleoli were not labelled with histone H3.3 suggesting a difference in nucleosomal architecture or nucleosomal dynamics in active ribosomal genes to genes transcribed into mRNA molecules. In summary, we could demonstrate that histone H3 variants show diverging expression patterns in interphase nuclei and in mitotic chromosomes.

References: 1. K. Ahmad, S. Henikoff, Mol. Cell., 9, 2002; 2. L. Snyers et al., Nucleus, 5, 2014.

Figures:

Figure 1: Conventional epifluorescence imaging and electron microscopy. Hela cell nuclei expressing myc-tagged histone H3.3. Fluorescence left: DAPI (blue), middle: immunofluorescent detection (red), right overlay (DAPI displayed as green). Electron micrograph, post-embedding immunogold detection.

Figure 2: Structured illumination imaging (SIM). Hela cell nucleus expressing both histones H3.2 (green) and H3.3 (red); DNA staining by DAPI (B/W). The composite image and the scatter plot (right) demonstrate diverging localization patterns of histones H3.2 and H3.3.

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EMC Abstracts - https://emc-proceedings.com/abstract/revealing-chromatin-at-different-microscopy-resolutions/