RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis

Abstract

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

The lysine demethylase LSD1 is required for nuclear envelope formation at the end of mitosis

“…These marks are removed during mitotic exit by the phosphatase PP1, which is recruited to anaphase chromatin by its targeting cofactor RepoMan (also known as CDCA2) (Trinkle-Mulcahy et al, 2006) and mKI67 (Booth et al, 2014;Takagi et al, 2014). Although it has been recently proposed to be upstream of a histone modification cascade that promotes mitotic chromosome condensation (Wilkins et al, 2014), phosphorylation of histone H3 at S10 is dispensable for chromosome condensation (Hsu et al, 2000;MacCallum et al, 2002) and to date there is no evidence to suggest that the reversal of histone phosphorylation events are specifically required for chromatin decondensation at the end of mitosis (Magalska et al, 2014). Instead, chromatin remodelling downstream of histone dephosphorylation has been linked to nuclear envelope assembly by promoting the recruitment of LBR through heterochromatin protein 1β (HP1β) (Ye et al, 1997;Haraguchi et al, 2000;Fischle et al, 2005) and importin-β-bound nucleoporins through RepoMan (Vagnarelli et al, 2011).…”

“…In the presence of membranes, mitotic chromatin clusters not only decondense but also support the formation of a closed nuclear envelope containing NPCs that are competent for nuclear import (Magalska et al, 2014). Accordingly, the addition of membranes to the extracts results in larger nuclei that accommodate further chromatin decondensation (Philpott et al, 1991;Wright, 1999) also known as secondary decondensation or nuclear swelling ( Fig.…”

“…Sperm DNA decondensation is mechanistically distinct from the decondensation of highly compacted chromatin at the end of mitosis, and we have recently established a cell-free assay to specifically analyze the latter process (Magalska et al, 2014). To this end, we examined changes to the topology of mitotic chromatin clusters isolated from HeLa cells when incubated with Xenopus egg extracts.…”

“…After 120 min, samples were fixed, stained with DAPI and analysed by confocal microscopy. The smoothness of the chromatin boundary (light grey) and the homogeneity of DAPI staining (dark grey) were analysed as described previously (Magalska et al, 2014). The mean percentage decondensation is plotted for 10 chromatin substrates per condition in each of three independent experiments.…”

“…Isolation of mitotic clusters from HeLa cells, preparation of low-speed interphasic egg extracts and chromatin decondensation reactions were performed as described in detail previously (Magalska et al, 2014). Immunodepletion of LSD1 for chromatin decondensation was performed as for nuclear assembly reactions (above).…”