scholarly journals The TFIIH Complex is Required to Establish and Maintain Mitotic Chromosome Structure

2021 ◽  
Author(s):  
Julian Haase ◽  
Richard Chen ◽  
Mary Kate Bonner ◽  
Lisa M Miller Jenkins ◽  
Alexander E Kelly

Condensins compact chromosomes to promote their equal segregation during mitosis, but the mechanism of condensin engagement with and action on chromatin is incompletely understood. Here, we show that the general transcription factor TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. Inhibiting the DNA-dependent ATPase activity of the TFIIH complex subunit XPB prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, TFIIH inhibition reversibly induces a complete loss of chromosome structure within minutes, prior to the loss of condensins from chromatin. Reducing nucleosome density through partial histone depletion restores chromosome structure and condensin enrichment in the absence of TFIIH activity. We propose that the TFIIH complex promotes mitotic chromosome condensation by dynamically altering chromatin structure to facilitate condensin loading and condensin-dependent loop extrusion.

1993 ◽  
Vol 120 (3) ◽  
pp. 601-612 ◽  
Author(s):  
T Hirano ◽  
T J Mitchison

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.


2003 ◽  
Vol 163 (3) ◽  
pp. 477-487 ◽  
Author(s):  
Yoshiaki Azuma ◽  
Alexei Arnaoutov ◽  
Mary Dasso

We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase–anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.


Science ◽  
2017 ◽  
Vol 356 (6344) ◽  
pp. 1284-1287 ◽  
Author(s):  
Keishi Shintomi ◽  
Fukashi Inoue ◽  
Hiroshi Watanabe ◽  
Keita Ohsumi ◽  
Miho Ohsugi ◽  
...  

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