Abstract
Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
Highlights
Chromatin, which fills the nucleus, is a repository of information, but it is a physical element that provides structure, mechanical rigidity, shape, and function to the nucleus
Using these novel CRISPR-derived Heterochromatin Protein 1α (HP1α)-AID-Superfolder Green Fluorescent Protein (sfGFP) cells, we find that the transcriptional profile 127 and chromatin organization are largely unchanged by rapid degradation of HP1α
We find that depletion of HP1α 450 reduced mitotic chromosome doubling force by approximately 40%, from 262 ± 50 pN in control 451 cells to 148 ± 12 pN in auxin-treated cells (16 pN/μm) (p = 0.03, Figure 5E), indicating that HP1α significantly contributes to mitotic chromosome mechanics. 454 We investigated whether histone methylation and the HP1α protein separately govern 455 chromosome mechanics during mitosis, as they do during interphase
Summary
Chromatin, which fills the nucleus, is a repository of information, but it is a physical element that provides structure, mechanical rigidity, shape, and function to the nucleus. HP1α homodimerization and/or higher-order oligomerization could directly impact mechanics through physical bridging of two chromatin fibers, resulting in crosslinking of DNA or H3K9me2,3-marked nucleosomes (Canzio et al, 2011; Cheutin et al, 2003; Machida et al, 2018). Consistent with this possibility, chromatin crosslinks have been suggested to be a key element of chromatin organization and mechanics (Banigan et al, 2017; Belaghzal et al., 2021; Lionetti et al, 2020; Stephens et al, 2017). It is critical to determine the role of HP1α in controlling chromatin mechanics during both interphase and mitosis, as well as the functions of HP1α-mediated chromatin mechanics
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