Mitotic post-translational modifications of histones promote chromatin compaction in vitro

Abstract

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.

Effects of altering histone post-translational modifications on mitotic chromosome structure and mechanics

“…Furthermore, histone acetylation is drastically decreased in mitosis meaning that that the effect of increased histone acetylation via HDACi may be negligible for metaphase chromosomes. While a decrease in acetylation in mitosis coincides with a higher degree of compaction (Zhiteneva et al, 2017), it appears that the increased acetylation of histones caused by our treatments with HDACis does not have an intrinsic effect on metaphase chromosome stiffness.…”

“…Our original hypothesis had been that HDACi-induced histone hyperacetylation would weaken mitotic chromosomes. This hypothesis was based on the observations that histone acetylation is normally reduced in mitosis (Doenecke, 2014), and is thought to intrinsically affect nucleosome packing (Zhiteneva et al, 2017). Furthermore, we expected to see weaker mitotic chromosomes since interphase hyperacetylated chromatin is decompacted (Doenecke, 2014) and hyperacetylating chromatin weakens the chromatin-dependent stiffness of interphase nuclei Stephens et al, 2018).…”

“…Histone PTMs may also intrinsically affect mitotic chromosome organization (Vagnarelli, 2012;Zhiteneva et al, 2017). Recent experiments suggest that nucleosomes reconstituted using core histones from mitotic cells have a greater propensity to aggregate, compared to nucleosomes assembled using core histones from interphase cells (Zhiteneva et al, 2017). This suggests that histone PTMs and their changes in mitosis may intrinsically affect mitotic compaction through nucleosome-nucleosome interactions.…”

A pathway for mitotic chromosome formation

“…Our simulations also show that to achieve agreement with Hi-C data, chromatin should also be condensed (computationally analogous to poor solvent conditions) forming densely packed chromatin loops within mitotic chromosomes analogous to the dense packing of chromatin observed in mitotic chromosomes by electron microscopy (46, 78, 79). The molecular basis for this condensation is not known but may involve mitosis-specific chromatin modifications (80, 81) or active motor proteins such as KIF4A (82, 83). …”

Chromosome disentanglement driven via optimal compaction of loop-extruded brush structures

“…SMC activity is crucial for the dense core in metaphase chromosomes. The high osmotic pressure from close packing of nucleosomes in the core is possibly stabilized by additional mechanisms such as nucleosome-nucleosome attraction in mitosis [71], and a higher concentration of chromatin-crosslinking proteins (including Topo II) inside the axial core. A recent proposal, supported by Hi-C studies, posits that SMC complexes drive a helical brush axis during metaphase [25].…”