Alterations in the chromatin packaging, driven by transcriptional activity, revealed by AFM.

Background: The gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed.
Methods: Nuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis.
Results: The malignant nuclei under the study were stable to deformation and assembled of 100-300 nm beads-like units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20-60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data.
Conclusions: Self-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei.
General Significance: We demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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