The TFIIH complex is required to establish and maintain mitotic chromosome structure
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This paper reports the surprising observation that the general transcription factor TFIIH, but not transcription, is required for chromosome condensation in frog egg extracts. TFIIH may act by facilitating condensin localization and function. This opens up a lot of interesting new questions and lines of research that promise to add significantly to the field of chromosome biology. It will now be interesting to directly test the mechanism of action, and to examine whether this role of TFIIH extends to somatic cells and other animals.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Cell Biology (eLife)
Abstract
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 rapidly and reversibly induces a complete loss of chromosome structure and prevents the enrichment of condensins I and II, but not topoisomerase II, on chromatin. In addition, inhibiting TFIIH prevents condensation of both mouse and Xenopus nuclei in Xenopus egg extracts, which suggests an evolutionarily conserved mechanism of TFIIH action. 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 the chromatin environment to facilitate condensin loading and condensin-dependent loop extrusion.
Article activity feed
-
-
Evaluation Summary:
This paper reports the surprising observation that the general transcription factor TFIIH, but not transcription, is required for chromosome condensation in frog egg extracts. TFIIH may act by facilitating condensin localization and function. This opens up a lot of interesting new questions and lines of research that promise to add significantly to the field of chromosome biology. It will now be interesting to directly test the mechanism of action, and to examine whether this role of TFIIH extends to somatic cells and other animals.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
The authors show that the general transcription factor complex TFIIH is required for chromosome condensation in Xenopus egg extracts. Inhibition of the ATPase activity of the TFIIH subunit XPB or depletion of XPB both strongly impaired chromosome condensation. Inhibition showed a discernible effect after 5 min, even when chromosomes were already condensed. This loss of condensation was associated with a loss of condensin, but not topo II from chromosomes, Interestingly, both the condensation defect and the loss of condensin localization could be counteracted by slightly reducing the histone concentration in the egg extract. Based on this and the known DNA unwinding activity of TFIIH, the authors propose that TFIIH may act by promoting nucleosome-free regions for condensin to bind DNA and create DNA loops. …
Reviewer #1 (Public Review):
The authors show that the general transcription factor complex TFIIH is required for chromosome condensation in Xenopus egg extracts. Inhibition of the ATPase activity of the TFIIH subunit XPB or depletion of XPB both strongly impaired chromosome condensation. Inhibition showed a discernible effect after 5 min, even when chromosomes were already condensed. This loss of condensation was associated with a loss of condensin, but not topo II from chromosomes, Interestingly, both the condensation defect and the loss of condensin localization could be counteracted by slightly reducing the histone concentration in the egg extract. Based on this and the known DNA unwinding activity of TFIIH, the authors propose that TFIIH may act by promoting nucleosome-free regions for condensin to bind DNA and create DNA loops. Although an interaction between TFIIH and condensin was detected, whether this interaction is functionally important remains unclear.
The experiments are well-conducted and clearly and logically presented, but could be strengthened by adding a quantification of the phenotypes observed.
The strength of the paper lies in the identification of a novel contributor to chromosome condensation, which is a fundamental process in cell division that has been much studied and is still little understood.
Weaknesses are that it remains unexplored how exactly TFIIH promotes condensation, and that it remains unclear whether TFIIH plays a role in condensation in somatic cells or other species. The authors show that the role of TFIIH in promoting condensation can be observed both by using Xenopus sperm and using mouse sperm, which suggests - but is far from proving - that it might have a conserved role.
-
Reviewer #2 (Public Review):
The authors of this paper provide exciting and convincing evidence that the transcription machinery is involved in mitotic condensation in the frog egg extract system. This opens up a lot of interesting new questions and lines of research that promise to add significantly to the field of chromosome biology. Weaknesses in the work are minor and primarily have to do with lack of image data quantification. A few of the conclusions are a bit too speculative based on the experiments performed, but the overall quality of this work is very high.
-
Reviewer #3 (Public Review):
How DNA is compacted into chromosomes during mitotic and meiotic cell division is a question of great interest. Over the years, the prowess of Xenopus egg extracts to condense sperm DNA into mitotic chromatid structures resulted in the identification and characterisation of protein complexes that contribute to this function. However, the precise contributions of Condensin I, Condensin II, and DNA topoisomerase remain unclear, especially in the context of DNA substrates with different histone compositions.
In this study, the Kelly lab used Xenopus egg extracts to investigate a possible link between transcription and the structure of mitotic chromatids. They discover that mitotic chromatid formation is impossible in the presence of triptolide (TPL), a small compound that covalently inhibits XPB, a subunit of …
Reviewer #3 (Public Review):
How DNA is compacted into chromosomes during mitotic and meiotic cell division is a question of great interest. Over the years, the prowess of Xenopus egg extracts to condense sperm DNA into mitotic chromatid structures resulted in the identification and characterisation of protein complexes that contribute to this function. However, the precise contributions of Condensin I, Condensin II, and DNA topoisomerase remain unclear, especially in the context of DNA substrates with different histone compositions.
In this study, the Kelly lab used Xenopus egg extracts to investigate a possible link between transcription and the structure of mitotic chromatids. They discover that mitotic chromatid formation is impossible in the presence of triptolide (TPL), a small compound that covalently inhibits XPB, a subunit of the transcription initiation TFIIH complex. Mitotic chromatids form normally following exposure to other transcription inhibitors, RNAase treatment, or ERCC1 depletion, demonstrating convincingly that TPL effects are transcription- and nucleotide excision repair independent. This is a surprising and interesting finding.
In figure 2, the authors demonstrate that TPL addition results in a rapid (5-10 minutes) decondensation of chromosomes. This effect is reversible since a ten-fold dilution of TPL restores chromosome condensation, underscoring that maintaining condensed chromosomes is an active process (see point 2 below).
Although TPL-treated decondensed chromosomes lack XPB and Condensins (Fig 3), TPL exposure results in chromosome decondensation before XPB and Condensin levels are reduced (Fig 4).
Following the effects of TPL addition over time is insightful. It is namely intriguing to see that chromatids already decondense in the first 5-10 minutes following TPL exposure, XPB levels go down later, and total Condensin levels do even increase in this period. A direct interaction between XPB and Condensin, suggested based on data shown in Fig3C, requires more evidence.
Interesting insight comes from an experiment in which the authors reduce the levels of H3/H4 on chromatin mouse sperm DNA through immunodepletion. Following a mild (24%) reduction of H3/H4 levels, chromosome condensation was no longer inhibited in the presence of TPL. It would be good if this effect was quantified beyond the representative images shown in panel A. XPB localisation to chromatin with reduced H3/H4 levels was still abolished by TPL, but Condensin levels were not. The ability to uncouple XPB and Condensin localisation is exciting.
-
-