Extrachromosomal Histone H2B Contributes to the Formation of the Abscission Site for Cell Division

Abstract

Histones are constitutive components of nucleosomes and key regulators of chromatin structure. We previously observed that an extrachromosomal histone H2B (ecH2B) localizes at the intercellular bridge (ICB) connecting the two daughter cells during cytokinesis independently of DNA and RNA. Here, we show that ecH2B binds and colocalizes with CHMP4B, a key component of the ESCRT-III machinery responsible for abscission, the final step of cell division. Abscission requires the formation of an abscission site at the ICB where the ESCRT-III complex organizes into narrowing cortical helices that drive the physical separation of sibling cells. ecH2B depletion does not prevent membrane cleavage rather results in abscission delay and accumulation of abnormally long and thin ICBs. In the absence of ecH2B, CHMP4B and other components of the fission machinery, such as IST1 and Spastin, are recruited to the ICB and localize at the midbody. However, in the late stage of abscission, these fission factors fail to re-localize at the periphery of the midbody and the abscission site fails to form. These results show that extrachromosomal activity of histone H2B is required in the formation of the abscission site and the proper localization of the fission machinery.

HIPK2 Is Required for Midbody Remnant Removal Through Autophagy-Mediated Degradation

“…HIPK2 has been shown to contribute to cytokinesis through two distinct targets, ecH2B, which contributes to the formation of the abscission site ( Monteonofrio et al, 2019 ), and Spastin, which severs microtubules for the final cut ( Pisciottani et al, 2019 ). Thus, we quantify MBRs also after depletion of Spastin and ecH2B obtained by transfecting HeLa cells with Spastin-specific siRNAs (siSpastin) and H2B-specific siRNAs (si-ecH2B), as reported ( Monteonofrio et al, 2019 ; Pisciottani et al, 2019 ). Alongside, for each condition, we also evaluated the induction of cytokinesis defects by analyzing the percentage of aberrant midbodies and binucleated cells, as we previously reported ( Rinaldo et al, 2012 ) ( Supplementary Table S1 ).…”

“…In details, cells were transfected with a mix of at least two stealth siRNAs at a final concentration of 40 nM using Lipofectamine RNAi MAX (Life Technologies). Depletion of ecH2B was obtained as in Monteonofrio et al, 2019. Briefly, cells were transfected with a mix of nine siRNAs each at 10 nM in a double pulse, the second one 24 h after the first one.…”

“… Cytokinesis defects were measured by IF in the indicated cells. DAPI was used to mark nuclei and β-tubulin immunostaining was used to identify midbody in telophase and cytoplasm in interphase, as described in Pisciottani et al, 2019 and Monteonofrio et al, 2019 . Aberrant midbodies are those filled with microtubules, elongated, and often associated with tubulin-labeled puncta, as previously described in Pisciottani et al, 2019 .…”

“…Mechanistically, HIPK2 regulates abscission with two apparently parallel mechanisms of action. HIPK2-mediated phosphorylation of the extrachromosomal histone H2B (ecH2B) at Ser14 is not required for its midbody localization but contributes to the formation of the abscission site and is essential for successful cytokinesis ( Rinaldo et al, 2012 ; Monteonofrio et al, 2019 ). HIPK2-mediated phosphorylation of the microtubule severing enzyme Spastin at Ser268 is required for its midbody localization to cut microtubules and the subsequent physical separation of the two daughter cells ( Pisciottani et al, 2019 ; Gatti et al, 2020 ).…”

An Alternative Splice Variant of HIPK2 with Intron Retention Contributes to Cytokinesis

“…One additional proliferation-associated role of HIPK2 has been reported in abscission, the final step of cytokinesis, in which the two daughter cells are physically separated [41][42][43]. We have shown that HIPK2 localizes at the intercellular bridge (ICB) connecting the daughter cells, in an Aurora B-dependent manner [44] and contributes to abscission by phosphorylating the extrachromosomal histone H2B at S14 [42,45] and the microtubule-severing enzyme spastin at S268 [46]. Loss of HIPK2 results in cytokinesis failure, the accumulation of tetra and polyploid cells, and the generation of aneuploidy, chromosomal instability, and increased tumorigenicity, supporting a caretaker function [47].…”