1. Cell Biology
  2. Chromosomes and Gene Expression

The TFIIH complex is required to establish and maintain mitotic chromosome structure

  1. Julian Haase
  2. Richard Chen
  3. Wesley M Parker
  4. Mary Kate Bonner
  5. Lisa M Jenkins
  6. Alexander E Kelly  Is a corresponding author
  1. Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, United States
  2. Laboratory of Cell Biology, National Cancer Institute, NIH, United States
https://doi.org/10.7554/eLife.75475
Share this article
Cite this article
  1. Julian Haase
  2. Richard Chen
  3. Wesley M Parker
  4. Mary Kate Bonner
  5. Lisa M Jenkins
  6. Alexander E Kelly
(2022)
The TFIIH complex is required to establish and maintain mitotic chromosome structure
eLife 11:e75475.
https://doi.org/10.7554/eLife.75475
  2. Download BibTeX
  3. Download .RIS

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.

Editor's evaluation

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 proposed mechanism of action, and to examine whether this role of TFIIH extends to somatic cells and other animals.

https://doi.org/10.7554/eLife.75475.sa0

Introduction

Entry into mitosis triggers dramatic structural changes in chromosomes that are required for their equal segregation (Zhou and Heald, 2020). A process known as chromosome condensation compacts sister chromatids into rod-like structures to promote individualization and prevent DNA damage during division. Sister chromatids form linear rod-like structures and separate from each other during prophase, and then shorten and thicken during prometaphase, ultimately forming fully condensed metaphase chromosomes. Electron microscope analyses of isolated mitotic chromosomes suggested that DNA is organized into loops around a protein scaffold that is concentrated along the central axis of each chromatid (Earnshaw and Laemmli, 1983; Paulson and Laemmli, 1977; Paulson et al., 2021). In agreement with this, recent studies using chromosome conformation capture and modeling indicate that a sequential helical arrangement of loops is formed around a central axis (Naumova et al., 2013; Gibcus et al., 2018; Elbatsh, 2019), although alternative models of loop arrangement have been proposed (Chu et al., 2020). How these elements of chromosome structure are established and maintained during chromosome segregation remains a major question in biology.

The evolutionary conserved five-subunit condensin complex is an ATPase that serves as the primary driver of mitotic chromosome condensation (Kinoshita and Hirano, 2017). Many eukaryotes express two different forms of the condensin complex, condensin I and II (Hirano et al., 1997; Ono et al., 2003). Condensin I and II share two structural maintenance of chromosomes (SMC) subunits (SMC2 and SMC4) that form a V-shaped dimer with two ATP-binding head domains, but have distinct regulatory subunits (CAP-H/H2, CAP-D2/D3, and CAP-G/G2). Condensin I is only loaded onto chromosomes during mitosis, whereas condensin II is present on chromatin throughout the cell cycle (Hirota et al., 2004; Ono et al., 2004; Walther et al., 2018). During mitosis, both condensin complexes are concentrated along the central axis of individual chromatids, but have been demonstrated to have distinct roles in maintaining the lateral and axial compaction of chromatids (Green et al., 2012; Samejima et al., 2012; Bakhrebah et al., 2015). A recent study in chicken DT40 cells proposed that condensin II initially forms large DNA loops, which are then subdivided into smaller loops by condensin I (Gibcus et al., 2018). However, in Xenopus egg extracts, condensin II is not essential for the formation of condensed chromatids but remains important for optimal chromosome morphology and proper centromere function (Shintomi and Hirano, 2011; Bernad et al., 2011). Topoisomerase IIα (topo II) is an ATP-dependent DNA strand-passing enzyme that is required to individualize and resolve chromatids that is also involved in chromosome condensation (Kinoshita and Hirano, 2017). Like the condensins, topo II is localized to the mitotic chromatid axis, and current models suggest it acts to control the axial compaction of chromosomes through the regulation of condensin-mediated loops (Nielsen et al., 2020; Shintomi and Hirano, 2021; Paulson et al., 2021).

Multiple factors promote the association of condensin with chromatin (Robellet et al., 2017), including but not limited to, histones (Ball et al., 2002; Liu et al., 2010; Tada et al., 2011), nucleosome-free regions (Piazza et al., 2014; Toselli-Mollereau et al., 2016), single-stranded DNA (Sakai et al., 2003; Sutani et al., 2015), and supercoiling (Kimura et al., 1998; Kim et al., 2021). Tuning condensin levels on chromatin is important for proper chromosome condensation, as prior studies have shown that altering condensin levels leads to gross changes in the axial and lateral compaction of chromosomes, as well as the alteration of the size of extruded DNA loops (Shintomi and Hirano, 2011; Thadani et al., 2018; Elbatsh, 2019; Choppakatla et al., 2021; Goloborodko et al., 2016a; Fitz-James et al., 2020; Goloborodko et al., 2016b). However, how condensin levels and activity on chromosomes are regulated remains an outstanding question. One conserved feature of condensins across multiple eukaryotes is their enrichment at sites of RNA polymerase II (Pol II)-dependent transcription (Bernard and Vanoosthuyse, 2015; D’Ambrosio et al., 2008; Schmidt et al., 2009; Nakazawa et al., 2015; Sutani et al., 2015; Kim et al., 2016; Kranz et al., 2013; Kim et al., 2013; Dowen et al., 2013). This suggests a potentially conserved role for transcription or the transcriptional machinery in regulating condensin loading and function during mitosis. However, the role of transcription in mitotic chromosome condensation is not well understood (Robellet et al., 2017). Here, we show that the TFIIH complex, part of the transcriptional pre-initiation complex, is continuously required for chromosome condensation by promoting condensin loading and function on chromatin.

Results

The TFIIH complex is required for chromosome condensation in Xenopus egg extracts

To examine possible relationships between transcription-associated activities and mitotic chromosome condensation, we used cell-free Xenopus M phase egg extracts, a system in which condensin function is well characterized and in which zygotic transcription is inactive (Kinoshita et al., 2015; Hirano and Mitchison, 1994; Barrows and Long, 2019; Chen et al., 2019; Amodeo et al., 2015; Jukam et al., 2017). As previously demonstrated (Hirano and Mitchison, 1993; Shintomi et al., 2015), sperm nuclei incubated with a high-speed supernatant (HSS) of egg extracts first swell during the exchange of protamines for maternal histones, assemble into a diffuse ‘cloud’ of chromatin, and gradually form clusters of condensed single-chromatid structures (Figure 1A). As expected, depletion of condensins I and II with anti-CAP-E antibodies caused a severe loss of condensation (Figure 1B and Figure 1—figure supplement 1A), and this effect could be quantified using a previously described ‘condensation parameter’ (Figure 1C; Maddox et al., 2006). We then asked if various inhibitors of the Pol II-dependent transcriptional machinery affect chromosome condensation in egg extracts when added at the beginning stages of the condensation process (t = 25’), after protamine exchange and ‘cloud’ formation (Figure 1A; Shintomi et al., 2015). Inhibitors of RNA polymerase II elongation, such as α-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) had no effect on chromosome condensation (Figure 1B and C), but induced the loss of the actively elongating (S2ph) form of RNA polymerase II at centromeres as expected (Figure 1—figure supplement 2A and B; Grenfell et al., 2016; Blower, 2016; Perea-Resa et al., 2020). In addition, condensation was not altered by pretreatment of extracts with RNase A to remove endogenous RNAs (Figure 1—figure supplement 1B and C and Figure 1—figure supplement 3A) or by pretreatment with RNase H to remove RNA-DNA hybrids (Figure 1—figure supplement 1C and Figure 1—figure supplement 3A; Thakur and Henikoff, 2020). These findings, combined with the fact that very little transcription occurs in Xenopus embryos before the maternal-to-zygotic transition (Jukam et al., 2017; Chen et al., 2019), indicates that active transcription is not required for chromosome condensation.

Figure 1 with 3 supplements see all
Download asset Open asset
The TFIIH complex is required for chromosome condensation in Xenopus egg extracts.

(A) Top: Schematic of chromatid assembly in M-phase high-speed supernatant (HSS) extract. Bottom: Representative fluorescence images of fixed samples from a chromatid assembly reaction taken at indicated timepoints after sperm addition. Chromatin was stained with Hoechst. (B) Representative fluorescence images of chromatid assembly at steady state (180 min after sperm addition) in the presence of indicated inhibitors. Each drug or DMSO control was added at 25 min to exclude effects on the protamine-histone exchange process. Final inhibitor concentrations: Triptolide (50 μM), DRB (100 μM), and a-amanitin (54 μM). See Figure 1—figure supplement 1A for CAP-E depletion. Mean condensation parameters for each condition are indicated in lower left corner of each image. (C) Scatter plot of the percentage of pixels below a threshold of 35% of image maximum fluorescence intensity (the condensation parameter), which measures the progressive change in the fluorescence intensity distribution that occurs during condensation, for each condition. Error bars represent SD, and the mean values are indicated. n = 20 structures for each condition. Two biological replicates were performed, quantified structures are from a single experiment. (D) Schematic of the TFIIH complex. TFIIH core complex is green, CAK subcomplex in red. (E) Western blot for XPB, p62, and Tubulin in IgG or XPB-depleted extracts. (F) TFIIH complex members that interact with XPB in M-phase HSS extract, as identified by mass spectrometry. Purifications were performed in the absence of chromatin. PSMs = peptide spectrum matches. (G) Representative fluorescence images of chromatid assembly 90 min after sperm addition in IgG or XPB depleted extracts. Chromatin was stained with Hoechst. (H) Western blot for XPF, ERCC1, and Tubulin in IgG or ERCC1-depleted extracts. (I) Representative fluorescence images of chromatid assembly 180 min after sperm addition in IgG or ERCC1-depleted extracts. Chromatin was stained with Hoechst.

Figure 1—source data 1

Source data for Figure 1E and H.

https://cdn.elifesciences.org/articles/75475/elife-75475-fig1-data1-v2.zip

In contrast, treatment of extracts with the transcription initiation inhibitor triptolide completely blocked chromosome condensation, resulting in decondensed chromatin masses similar to structures observed upon condensin depletion (Figure 1B and C; Ono et al., 2003; Shintomi and Hirano, 2011). Triptolide is an inhibitor of the multi-subunit TFIIH complex (Figure 1D), which as part of the pre-initiation complex is required for Pol II-dependent transcription initiation (Titov et al., 2011; Compe and Egly, 2012). Triptolide covalently binds to the XPB subunit of TFIIH and blocks its DNA-dependent ATPase (Titov et al., 2011). The ATPase activity of XPB is required for the DNA translocase function of TFIIH, which in collaboration with the pre-initiation complex, is essential to twist and open DNA to allow RNA polymerase II to productively engage the template strand to initiate transcription (Fishburn et al., 2015; Tomko et al., 2017; Kim et al., 2000; Grünberg et al., 2012; Nogales and Greber, 2019; Schilbach et al., 2021). In agreement with these described functions of TFIIH, triptolide prevented the accumulation of both paused (S5ph) and actively elongating (S2ph) forms of Pol II at centromeres (Figure 1—figure supplement 2A and B).

To rule out off-target effects of triptolide, we also examined chromosome condensation in extracts depleted of XPB ( > 95%) using anti-XPB antibodies (Figure 1E and Figure 1—figure supplement 1D). XPB depletion also depleted the TFIIH subunit p62 (Figure 1E), and mass spectrometry analysis of proteins co-depleted with XPB identified strong enrichment of other core TFIIH complex members (Figure 1F). As shown in Figure 1G, XPB depletion completely abrogated chromosome condensation (Figure 1—figure supplement 3B), thus confirming that XPB is the physiological target of triptolide and demonstrating an unprecedented role for the TFIIH complex in chromosome condensation.

The TFIIH complex is involved in processes other than transcription initiation that could also potentially affect chromosome condensation, including the nucleotide excision repair (NER) pathway (Compe and Egly, 2012). Consistent with a previous report (Ito et al., 2007), we identified the NER endonuclease XPG as a strong interactor with the TFIIH complex (Figure 1F). During NER, XPG and ERCC1-XPF, another NER endonuclease, act together to resect DNA lesions after the XPB and XPD subunits of TFIIH have unwound DNA around the lesion (Kolesnikova et al., 2019; Compe and Egly, 2012). To address whether the NER pathway is required for condensation, we immunodepleted ERCC1 from egg extracts, which prevents NER (Klein Douwel et al., 2017). Depletion of the ERCC1/XPF complex did not perturb chromosome condensation (Figure 1H and I, Figure 1—figure supplement 1E, and Figure 1—figure supplement 3C), indicating that the TFIIH complex does not act through the NER pathway to promote chromosome condensation.

TFIIH also associates with the three protein CAK complex (CDK-Activating Kinase) (Figure 1F), which contains the kinase CDK7. CDK7 directly phosphorylates the Pol II C-terminus at Ser5 (Pol II S5ph), and is required for the release of Pol II from the pre-initiation complex to promote transcriptional pausing and other transcriptional events (Rimel and Taatjes, 2018; Glover-Cutter et al., 2009; Akhtar et al., 2009; Ebmeier et al., 2017). As expected, inhibition of CDK7 with THZ1 (Kwiatkowski et al., 2014) induced the loss of the paused and elongating forms of Pol II from centromeres (Figure 1—figure supplement 2A and B). However, THZ1 treatment had no effect on condensation (Figure 1—figure supplement 1F and Figure 1—figure supplement 3D). Furthermore, inhibition of CDK7 did not rescue the condensation defects observed in triptolide-treated extracts, ruling out abnormal upregulation of CDK7 activity upon inhibition of XPB (Figure 1—figure supplement 1F and Figure 1—figure supplement 3D). CDK7, as part of the labile CAK complex, is also required for the initial activation of CDK1 to promote entry into mitosis (Larochelle et al., 2007), and thus we tested the possibility that triptolide exerts its effects on condensation through down-regulation of CDK1 activity. However, triptolide did not affect CDK1 activity or M phase maintenance in HSS extracts, as indicated by unaltered CDK1 activation loop phosphorylation (T161ph) and histone H3T3ph levels (Kelly et al., 2010; Figure 1—figure supplement 1G). Thus, the ATPase activity of the TFIIH complex is required for condensation in a manner that is independent of TFIIH activities in NER, active transcription, and the cell cycle.

The TFIIH complex is required for the dynamic maintenance of mitotic chromosome structure

Chromosome condensation must be actively maintained during mitosis (Kinoshita et al., 2015), but prior work has suggested that condensation establishment and maintenance may involve distinct mechanisms (Nielsen et al., 2020; Hirano and Mitchison, 1993). To ask if the TFIIH complex is required for the maintenance of chromosome condensation as well as its establishment, we added triptolide at various timepoints during the condensation process: at 25 min after addition of sperm to extracts, at condensation initiation; at 60 min, when chromosomes were partially condensed; and at 180 min, when chromosomes were in a steady state of full condensation (Figure 2A). In DMSO-treated controls, condensation increased steadily during these times until reaching a plateau at 180 min (Figure 2A, and Figure 2—figure supplement 1A). However, addition of triptolide at any of these times during the condensation process, including at 180 min, induced the rapid (less than 5 min) loss of discernable individual chromatids and a diffuse DNA staining pattern, similar to the structures observed upon condensin depletion (Figure 1B), that persisted throughout the rest of the timecourse (Figure 2A, and Figure 2—figure supplement 1A). Thus, TFIIH activity is required for maintaining chromosome condensation as well as its establishment.

Figure 2 with 2 supplements see all
Download asset Open asset
Continuous TFIIH activity is required to maintain chromosome structure.

(A) Representative fluorescence images of chromatid assembly reactions sampled at indicated timepoints. DMSO or triptolide (TPL) were added at indicated times, immediately after sample retrieval for imaging. Chromatin was stained with Hoechst. Red boxes indicate representative images of decondensed chromosomes after triptolide addition. Mean condensation parameters for each condition are indicated in lower left corner of each image, see Figure 2—figure supplement 1A for more details. (B) Representative fluorescence images of chromatid masses fixed and spun onto coverslips 215 min after sperm addition, subjected to the indicated manipulations. Nuclei were incubated in extracts for 180 min to allow for the formation of fully condensed chromatids, followed by addition of DMSO or triptolide. After 30 min of incubation with DMSO/triptolide, reactions were diluted 10-fold with extract containing DMSO or triptolide, and further incubated for 5 min before taking samples for imaging. See Figure 2—figure supplement 2 for additional controls. Chromatin was stained with Hoechst. (C) Scatter plots of the percentage of pixels below a threshold of 35% of image maximum fluorescence intensity (the condensation parameter), for each condition in (B). Error bars represent SD, and the mean values are indicated. n = 20 structures for each condition. Two biological replicates were performed, quantified structures are from a single experiment. (D) Schematic illustrating the effects of triptolide addition and dilution on chromosome structure maintenance and reformation.

To test if the triptolide-induced loss of mitotic chromosome structure is reversible, we allowed chromosomes to completely condense for 180 min, treated with triptolide for 30 min to induce loss of chromosome structure, and then diluted 10-fold with extracts containing triptolide or DMSO (Figure 2B). Dilution with DMSO-treated extracts resulted in the rapid regain (within 5 min) of chromatid structure, while chromosomes remained uncondensed upon dilution with triptolide-treated extract (Figure 2B and C, Figure 2—figure supplement 2A and B). These results demonstrate that the effects of TFIIH inhibition on chromosome condensation can be reversed rapidly (Figure 2D), suggesting that the TFIIH complex acts continually in dynamic processes required for condensation.

TFIIH is required for the enrichment of condensins on mitotic chromatin

The proper assembly of mitotic chromosomes in egg extracts requires the activities of condensins I and II and Topoisomerase IIα (topo II), which localize along condensed chromatids on a central axis (Samejima et al., 2012; Ono et al., 2003; Hirano and Mitchison, 1993; Shintomi et al., 2015; Earnshaw and Heck, 1985). To test the hypothesis that TFIIH is involved in this localization, we used immunofluorescence to measure levels of XPB, condensin I/II, and topo II on chromatin in the presence or absence of triptolide (added at t = 25’, imaged at t = 180’). In control extracts, the XPB was present in a diffuse pattern over the chromatid mass (Figure 3A), with discrete puncta of XPB visible along the entire length of isolated chromatids (Figure 3A (inset)). Condensins I and II and topo II were each present on the central axis of each chromatid, as previously reported (Ono et al., 2003; Maeshima and Laemmli, 2003; Figure 3A). Triptolide treatment severely reduced XPB levels on chromatid clusters (Figure 3B; 21% of control), indicating that inhibition of the XPB ATPase affects its association with chromatin. Strikingly, triptolide also caused significant reductions in the levels of CAP-E (37% of control) and CAP-C (31% of control), which are subunits of both condensin I and II, and also reduced levels of the condensin II-specific subunit CAP-D3 (Figure 3B; 25% of control). The condensin that remained on chromosomes was present in a diffuse pattern with no defined axis (Figure 3A). Since the amount of condensin I in extracts is three- to fivefold higher than that of condensin II, and since condensin I is the major driver of chromosome condensation in egg extracts (Ono et al., 2003; Shintomi and Hirano, 2011), we conclude that triptolide prevents the proper loading and enrichment of both condensin I and condensin II. Conversely, triptolide had no significant effect on topo II levels (Figure 3A and B; 93% of control), although topo II was no longer concentrated on a central axis. A similar effect on topo II localization is seen upon condensin depletion from egg extracts (Shintomi et al., 2017). Similarly, SUPT16H, a member of the FACT complex implicated in both transcription (Zhou et al., 2020) and chromosome condensation (Shintomi et al., 2015), was present at similar levels in the presence or absence of triptolide (Figure 3A and B; 113% of control). CAP-C levels also decreased significantly (61% of control) when fully condensed chromosomes were treated with triptolide at t = 180’ for 35 min (Figure 2—figure supplement 2C). These results demonstrate that the TFIIH complex is required for the enrichment of condensin I/II on chromosomes during condensation establishment and maintenance. They also suggest that the TFIIH complex specifically regulates condensins and not topo II, since TFIIH inhibition alters condensin but not topo II levels on chromatin (Figure 3B) and causes defects in condensation that are similar to condensin depletion but not topo II inhibition (Ono et al., 2003; Shintomi et al., 2017; Hirano and Mitchison, 1993).

Figure 3 with 1 supplement see all
Download asset Open asset
The TFIIH complex is required for the enrichment of condensins on chromosomes.

(A) Representative immunofluorescence images of Xenopus sperm nuclei incubated with DMSO or triptolide (TPL) treated extracts for 180 min. Chromatids were labeled with Hoechst and anti-XPB, anti-CAP-D3 (condensin II), anti-CAP-C (condensin I & II), anti-CAP-E (condensin I and II), anti-SUPT16H (FACT complex), or anti-Topo II antibodies. Images of individual chromatids are shown in insets. (B) Quantification of fluorescence intensity of indicated proteins from experiment in (A), normalized to DMSO. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001), NS indicates no statistical significance. A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment.

In reciprocal experiments, we found that XPB localization to chromatin was maintained following the depletion of condensins (Figure 3—figure supplement 1A). Since both TFIIH and condensin levels on chromatin decreased in response to triptolide (Figure 3B), we hypothesized that TFIIH might promote recruitment of condensin complexes to chromosomes through physical interaction. We found that the condensin II subunit CAP-D3 co-immunoprecipitated with XPB from high-speed supernatants of egg extracts (Figure 3—figure supplement 1B). However, XPB was not co-immunoprecipitated with anti-CAP-E antibodies, even though we observed robust interactions with other condensin I and II subunits including CAP-D3 (Figure 3—figure supplement 1C). These results suggest that any interactions between TFIIH and condensins are weak, and not necessarily direct. Furthermore, TFIIH only partially co-localized with condensins on single chromatids, and the TFIIH complex did not demonstrate the stereotypical axial staining pattern observed for condensins, which argues against a scaffolding role for TFIIH (Figure 3—figure supplement 1D). Altogether, these results show that the TFIIH complex acts upstream of condensin enrichment and weakly associates with the CAP-D3 subunit of condensin II, but indicate that the main role of TFIIH in condensin enrichment is not through direct recruitment.

TFIIH inhibition perturbs chromosome condensation before condensin and TFIIH are lost from chromosomes

To further investigate how TFIIH promotes condensation and the enrichment of condensins on chromatin, we determined the kinetics of condensin enrichment with and without TFIIH inhibition. Specifically, we added triptolide 25 min after addition of sperm nuclei to egg extracts and monitored chromatin morphology and condensin levels on chromatin over time (Figure 4A). As expected, the addition of triptolide led to rapid decondensation (Figures 2A and 4B, and Figure 4—figure supplement 1A), and an amorphous cloud of chromatin without discernable chromatids was seen within five minutes of drug addition. DMSO-treated extracts progressed in a stereotypical fashion, with increasing condensation over time (Figure 4B and Figure 4—figure supplement 1A). In contrast, total condensin levels, as monitored by CAP-E staining, increased for the first 10 min after triptolide addition, remaining similar to controls within the first 20 min before decreasing to 35% of control over the next 135 min (Figure 4B and C, and Figure 4—figure supplement 2A). Similarly, condensin II and XPB remained at control levels for 10 min after triptolide addition before decreasing relative to controls (Figure 4—figure supplement 2B and C). These results indicate that TFIIH plays an immediate role in promoting condensation, and is also required for the ongoing enrichment of condensins on chromosomes that occurs throughout the condensation process.

Figure 4 with 2 supplements see all
Download asset Open asset
Triptolide perturbs condensation prior to its effects on condensin levels.

(A) Schematic of assay to test the timing of triptolide-induced defects in condensation and condensin levels. After a twenty-five minute incubation, either DMSO or triptolide (TPL) was added, and samples were taken at indicated times and processed for immunofluorescence. (B) Representative immunofluorescence images of DMSO or triptolide treated extracts at indicated timepoints. Chromatids were labeled with Hoechst and anti-CAP-E (condensin I and II). Note that condensation is already lost in in the first timepoint after triptolide addition, as in Figure 2A. Mean condensation parameters for each condition are indicated in lower left corner of each image. (C) Quantification of fluorescence intensity of CAP-E from experiment in (B), normalized to the 180 min DMSO-treated sample. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001). A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment.

Artificial formation of nucleosome-free regions bypasses the requirement for TFIIH in chromosome condensation

Several lines of evidence suggest that nucleosomes are a barrier to condensin loading and function on chromosomes. In Xenopus egg extracts, condensins display a strong preference for binding to DNA over nucleosomes (Zierhut et al., 2014; Shintomi et al., 2017; Choppakatla et al., 2021). The creation of nucleosome-free regions has been proposed to be important for chromosome condensation in fission yeast (Toselli-Mollereau et al., 2016), and double-stranded DNA has been shown to preferentially activate the ATPase activity of budding yeast condensin (Piazza et al., 2014). The TFIIH complex, which acts as a DNA translocase and facilitates changes to chromatin, might promote condensin binding and condensation by altering the chromatin environment to allow the exposure of DNA. To test this suggestion, we artificially introduced nucleosome-free regions on chromosomes by using anti-H4K12ac antibodies to partially deplete histones H3 and H4 from egg extracts (Zierhut et al., 2014) and examined the effects of triptolide on chromosome condensation. Since Xenopus sperm retain paternally derived histones H3 and H4 on chromatin (Shintomi et al., 2015), we used mouse sperm, which have previously been shown to condense in a condensin-dependent manner in Xenopus egg extracts (Shintomi et al., 2017) but which contain very low levels of histones H3 and H4 (Brykczynska et al., 2010). We found that triptolide inhibits the condensation of mouse sperm nuclei incubated in Xenopus egg extracts (Figure 5A and B, Figure 5—figure supplement 1A, and Figure 5—figure supplement 2A), indicating that TFIIH activity is required for condensation regardless of chromatin source. Control depletions with IgG did not perturb condensation, nor did it alter the inhibitory effects of triptolide (Figure 5A and B, Figure 5—figure supplement 1B, and Figure 5—figure supplement 2A). Remarkably, depletion of total histone H3 levels in extracts by only 24%, which reduced histone H3 levels on chromosomes by 38% (Figure 5B and C, and Figure 5—figure supplement 1B), rescued chromosome condensation in the presence of triptolide (Figure 5A and Figure 5—figure supplement 2A). Importantly, chromosomes were resistant to decondensation ten minutes after TFIIH inhibition, suggesting a direct effect of histone H3/H4 depletion (Figure 5A, and Figure 5—figure supplement 2A). Triptolide treatment caused substantial reductions in both XPB and condensin I/II subunit CAP-E on mouse chromosomes (respectively to 23% and 40% of control levels, Figure 5B and C), as it did with Xenopus chromosomes (Figure 3A and B). However, partial depletion of histones H3 and H4 resulted in a substantial increase in CAP-E levels (to 79% of control levels), and CAP-E labeled the chromatid axis as in controls (Figure 5B and C). In contrast, histone H3/H4 depletion did not suppress the triptolide-induced loss of XPB levels from chromosomes (Figure 5B and C and Figure 5—figure supplement 1B), indicating that histone depletion does not affect the inhibition of TFIIH by triptolide. The finding that TFIIH function in condensation can be bypassed by partial histone depletion strongly suggests that the main role of TFIIH in condensation is to alter the chromatin environment, possibly by regulating nucleosome composition and/or inhibitory proteins, which in turn promotes condensin function and enrichment on chromatin.

Figure 5 with 2 supplements see all
Download asset Open asset
Partial histone H3/H4 depletion rescues chromosome condensation in the absence of TFIIH activity.

(A) Representative fluorescence images of mouse sperm nuclei incubated with ∆IgG or ∆H4K12ac extracts and spun onto coverslips at indicated timepoints. DMSO or Triptolide were added at t = 25’, immediately after sample acquisition for imaging. Chromatin was stained with Hoechst. Mean condensation parameters for each condition are indicated in lower left corner of each image. (B) Representative immunofluorescence images of mouse sperm nuclei incubated with ∆IgG or ∆H4K12ac extracts for 180 min. DMSO or triptolide were added at t = 25’. Chromatids were labeled with anti-XPB, anti-CAP-E (condensin I and II), or anti-histone H3, and co-stained with Hoechst. (C) Quantification of fluorescent intensity of indicated proteins from (B), normalized to DMSO. n = 50 structures for each condition. Error bars represent SD, and asterisks indicate a statistically significant difference (*, p < 0.001). A.U., arbitrary units. Two biological replicates were performed, quantified structures are from a single experiment. (D) Model depicting the role of the TFIIH complex in regulating condensin dynamics. Our data are consistent with a model in which TFIIH alters the chromatin environment to allow condensins to load and extrude loops of DNA.

Discussion

Roles of the TFIIH complex in chromosome condensation

The prevailing model of chromosome condensation posits that condensins extrude loops of chromatin in an ATP-dependent manner to promote the formation of multiple intra-chromatid contacts (Davidson and Peters, 2021). Nucleosomes may form a barrier to both condensin loading and loop extrusion, since condensins bind more strongly to nucleosome-free DNA than to nucleosomes (Zierhut et al., 2014; Shintomi et al., 2017; Toselli-Mollereau et al., 2016; Choppakatla et al., 2021) and the binding to free DNA activates condensin ATPase activity (Piazza et al., 2014). In vitro experiments have reported that condensin can extrude loops of chromatinized DNA, but nucleosomes were present in these studies at low densities sufficient to expose naked DNA and permit condensin loading (Kong et al., 2020). Because mitotic chromatin in vivo is more compact and nucleosome dense (Ou et al., 2017; Grigoryev et al., 2016), it may need to be modified before condensin can load and extrude loops. Here, we have shown that mitotic chromosome condensation and condensin loading in Xenopus egg extracts require the TFIIH complex, and that TFIIH’s roles in these processes can be bypassed by reducing nucleosome density. These findings provide further support for the idea that nucleosomes inhibit condensin function, and suggest that the TFIIH complex regulates the exposure of nucleosome-free DNA where condensin can load and function (Figure 5D). Chromosome condensation depends on condensin loading as well as condensin-mediated loop extrusion, and previous experiments have suggested that these events can be separated (Goloborodko et al., 2016a; Kong et al., 2020; Kinoshita et al., 2015; Thadani et al., 2018; Ganji et al., 2018). Our results suggest that TFIIH activity promotes both steps, since TFIIH inhibition rapidly stalls the condensation process before it impacts levels of condensins on chromatin (Figure 4 and Figure 4—figure supplement 2), although it remains unclear whether TFIIH regulates these processes directly, or indirectly through removal of inhibitory proteins (see below). Since histone depletion restores both condensation and condensin loading in the absence of TFIIH activity (Figure 5A–C), it is likely that TFIIH-dependent changes to the chromatin environment are also required for condensin-mediated loop extrusion and chromatid formation. These changes could promote loop extrusion by creating additional binding sites necessary for intra-chromatid contacts and condensin processivity (Goloborodko et al., 2016b; Goloborodko et al., 2016a), or through regulation of condensin turnover (Kinoshita et al., 2015; Thadani et al., 2018). However, it is possible that TFIIH also promotes condensation in a condensin-independent manner.

During transcription initiation, the XPB subunit of TFIIH acts as a DNA translocase to help ‘melt’ promoter DNA to allow for Pol II to engage the template strand (Nogales and Greber, 2019; Aibara et al., 2021). Our studies with triptolide, which inhibits the ATPase activity of XPB required for translocation (Titov et al., 2011; Kappenberger et al., 2020; Tomko et al., 2017), suggest that the DNA translocase function of TFIIH is important to promote condensation. TFIIH is also important for recruiting factors that promote transcription, including the histone acetyltransferase KAT2A which controls histone H3K9 acetylation (Sandoz et al., 2019), a modification that is associated with increased nucleosome turnover (Aygün et al., 2013; Zentner and Henikoff, 2013). We speculate that the TFIIH complex regulates transient cycles of DNA opening and closing at Pol II-dependent promoters that do not result in transcription but help to maintain nucleosome-free regions required for condensin loading and function (Gilchrist et al., 2010; Figure 5D). This process may be part of or similar to mitotic transcriptional bookmarking observed in other cell types (Palozola et al., 2019). Importantly, we found that the condensation of both mouse and Xenopus sperm nuclei in Xenopus egg extracts require TFIIH activity (Figure 5—figure supplement 1A). This suggests a common fundamental mechanism that regulates condensin function. Interestingly, the TFIIH complex is present on chromosomes during mitosis in Drosophila embryos prior to the activation of transcription at the mid-blastula transition (Cruz-Becerra et al., 2018). Furthermore, in Drosophila, TFIIH interacts with the chromatin remodeling SRCAP complex (Herrera-Cruz et al., 2012), and mutation of the p52 TFIIH subunit causes chromosome condensation defects in mitotic neuroblasts (Fregoso et al., 2007). Thus, it is likely that the role of TFIIH in mitotic chromosome condensation during embryogenesis is conserved. However, the chromatin landscape during embryogenesis is quite distinct from that of somatic cells (Jukam et al., 2017; Ke et al., 2017; Du et al., 2017), and therefore factors other than or in addition to TFIIH may regulate condensin function depending on cell type. In addition, the TFIIH complex has been reported to act independently of Pol II in various non-transcriptional processes (Mizuki et al., 2007; Compe and Egly, 2016), and therefore it is possible that TFIIH promotes a condensation-competent chromatin environment on its own or in association with unknown factors independent of transcription.

Seminal work from the Hirano lab demonstrated that mitotic chromatid structure can be reconstituted in vitro using Xenopus sperm and a limited set of purified factors that include core histones, histone chaperones (nucleoplasmin, Nap1 and FACT), condensin I, and topo II (Shintomi et al., 2015; Shintomi and Hirano, 2021). The lack of TFIIH dependence in this reconstituted system could be explained by either incomplete or highly dynamic chromatinization, which would expose DNA and eliminate the need for TFIIH (Shintomi et al., 2015; Barrows and Long, 2019). Interestingly, in vitro chromatid reconstitution in this system specifically requires the use of an egg-specific histone (Shintomi et al., 2015; Shechter et al., 2009), histone H2A.X-F (also known as H2A.X.3), and it was suggested that this variant is a better substrate of the FACT complex chaperone than is the canonical histone H2A, which is also present in egg extracts (Shintomi et al., 2015; Wang et al., 2014). Since the FACT complex promotes both nucleosome assembly and disassembly (Zhou et al., 2020), it may act in the reconstituted system to provide the nucleosome dynamics required for condensation that TFIIH provides in egg extracts. Since the FACT histone chaperone acts in concert with Pol II during transcription (Petrenko et al., 2019), and helps promote the loading of the condensin-related cohesin complex (Garcia-Luis et al., 2019), it will be important to investigate contributions of FACT to TFIIH-dependent condensation in egg extracts and in other systems. It is also possible that TFIIH regulates the access of other non-nucleosomal factors, not present in the reconstituted system, that either impede condensin loading or serve to restrict nucleosome mobility.

Although the regulation of nucleosome occupancy may be an important factor in condensin loading and function on chromatin, it is also possible that TFIIH promotes other changes necessary for condensation. Previous work has demonstrated that changes in DNA supercoiling and/or the creation of single-stranded DNA are important for condensin loading (Sutani et al., 2015; Kim et al., 2021). Indeed, TFIIH complex can act in all three processes: it helps to maintain nucleosome-free regions (Gilchrist et al., 2010), its DNA translocase activity exposes ssDNA during transcription initiation (Aibara et al., 2021; Tomko et al., 2017; Fishburn et al., 2015), and it promotes DNA supercoiling (Aibara et al., 2021; Tomko et al., 2017). TFIIH may also be involved in the removal of other non-nucleosomal barriers to condensin function, such as histone H1 or Pol II itself (Choppakatla et al., 2021; Brandão et al., 2019; Rivosecchi et al., 2020; Pradhan et al., 2021). Understanding how TFIIH alters the chromatin environment to promote condensin function and condensation should be the focus of future efforts.

Maintenance of the condensed chromosome state is a dynamic process regulated by TFIIH

The mechanisms that maintain chromosome condensation are not well understood (Paulson et al., 2021). Our data show that continuous TFIIH activity is needed both to load condensin and to promote its function during the establishment and maintenance of chromosome condensation. In line with this, multiple studies have reported that the inactivation of condensin or its acute depletion from fully condensed chromosomes results in the rapid loss of condensation (Hirano and Mitchison, 1994; Piskadlo et al., 2017; Samejima et al., 2018; Nakazawa et al., 2011). Furthermore, continual ATP hydrolysis by condensin is required to maintain condensed chromosomes (Kinoshita et al., 2015; Thadani et al., 2018; Elbatsh, 2019). Thus, maintenance of mitotic chromosome structure is a dynamic and continuous process, and our data suggest that condensin access to DNA continues to be a critical step even after condensation is achieved. As the folding of chromatids is entropically unfavorable (Goloborodko et al., 2016b; Vasquez et al., 2016), it is worth noting that all the major factors required to promote condensation, including the XPB subunit of TFIIH, are ATPases (Paulson et al., 2021). Thus, our findings provide further support to the conclusion that mitotic chromosome assembly and maintenance are energy-dependent bidirectional processes opposed by nucleosomes and entropic forces (Shintomi and Hirano, 2021; Hirano, 2014).

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-XPB (rabbit polyclonal)Novus BiologicalsCat# NB100-61060, RRID:AB_925377IF (1:200), WB (1:1000)IP (0.24 µg antibody per µl beads and 0.24 µg antibody per µl extract)Immunodepletion (0.24 µg Antibody per µl beads and 1.44 µg Antibody per µl extract)
AntibodyAnti-XPB (mouse monoclonal)MilliporeCat# MABE1123IF (1:50)
AntibodyAnti-p62 (rabbit polyclonal)AbcamCat# ab232982WB (1:400)
AntibodyAnti-XPF (rabbit polyclonal)Gift of Puck Knipscheer, Hubrecht Institute Klein Douwel et al., 2014CustomWB (1:10,000)
AntibodyAnti-ERCC1 (rabbit polyclonal)Gift of Puck Knipscheer, Hubrecht Institute Klein Douwel et al., 2014CustomWB (1:10,000)Immunodepletion (0.24 µg antibody per µl of beads and 1.44 µg of antibody per µl of extract)
AntibodyAnti-CAP-E Serum (rabbit polyclonal)This studyHL7914Immunodepletion (24 µl of serum per µl extract and 3 µl of serum per µl of beads)IP (3 µl of serum per µl HSS and 3 µl of serum per µl of beads)Refer to ‘Antibody Production’ section in Methods
AntibodyAnti-CAP-D3 (rabbit polyclonal)Gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011CustomIF (1:100)WB (1:400)
AntibodyAnti-CAP-C (rabbit polyclonal)Gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011CustomIF (1:100)WB (1:500)
AntibodyAnti-CAP-E (rabbit polyclonal)Gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011CustomIF (1:100)WB (1:1,000)
AntibodyAnti-CAP-G (rabbit polyclonal)Gift of Susannah Rankin, Oklahoma Medical Research FoundationOMRF191WB (1:5,000)
AntibodyAnti-Topo II (rabbit polyclonal)Gift of Yoshi Azuma, University of Kansas Pandey et al., 2020CustomIF (1:200)
AntibodyAnti-Histone H3 (rabbit polyclonal)AbcamCat# ab1791, RRID:AB_302613IF (1:400)WB (1:500)
AntibodyAnti-Histone (T3ph) (rabbit monoclonal)AbcamCat# ab78351, RRID:AB_1566301WB (1:10,000)
AntibodyAnti-histone H4K12ac (mouse monoclonal)Gift of Hiroshi Kimura,Tokyo Institute of Technology Hayashi-Takanaka et al., 2015CustomImmunodepletion (0.24 µg antibody per µl beads and 4.15 µg antibody per µl extract)
AntibodyAnti-α-Tubulin (mouse, monoclonal)MilliporeSigmaCat# T9026, RRID:AB_477593WB (1:20,000)
AntibodyAnti-CDK1T116Phos (rabbit polyclonal)Cell Signaling TechnologyCat# 9,114 S, RRID:AB_2074652WB (1:500)
AntibodyAnti-SUPT16H (rabbit polyclonal)AbcamCat# ab204343IF (1:100)
AntibodyAlexa Fluor 488 anti-rabbit IgG (donkey polyclonal)Jackson ImmunoResearchCat# 711-545-152, RRID:AB_2313584IF (1:400)
AntibodyAlexa Fluor 647 anti-rabbit IgG (goat polyclonal)Jackson ImmunoResearchCat# 111-605-144, RRID:AB_2338078IF (1:400)
AntibodyAlexa Fluor 488 anti-mouse IgG (goat polyclonal)Jackson ImmunoResearchCat#115-545-166, RRID:AB_2338852IF (1:400)
AntibodyIRDye 800 CW anti-rabbit IgG (H + L) (goat polyclonal)LI-COR BiosciencesCat# 926–32211, RRID:AB_621843WB (1:17,500)
AntibodyIRDye 680 LT anti-mouse IgG (H + L) (goat polyclonal)LI-COR BiosciencesCat# 926–68020, RRID:AB_10706161WB (1:17,500)
AntibodyIgG (rabbit polyclonal)MilliporeSigmaCat# I5006, RRID:AB_1163659Immunodepletion (used as control at 0.24 µg antibody per µl of beads)
Peptide, recombinant proteinxCAP-E C-terminal peptideVivitideCustomNH2-CSKTKERRNRMEVDK-COOH
OtherProtein A DynabeadsThermo Fisher ScientificCat# 10,002DImmunodepletion and Immunoprecipitation
Chemical compound, drugBS3 (bis(sulfosuccinimidyl) suberate)Thermo Fisher ScientificCat# A392664 mM
Chemical compound, drugTriptolideTocris or MilliporeSigmaTocris Cat# 3,253MilliporeSigma Cat# T365250 µM
Chemical compound, drugα-amanitinTocrisCat# 4,02554.4 µM
Chemical compound, drugActinomycin DTocrisCat# 1,2290.05 µM, 0.4 µM, 4.0 µM
Chemical compound, drugTHZ1CaymanCat# 9002215–130 µM
Chemical compound, drugDRBCaymanCat# 10010302100 µM
Chemical compound, drugRNase AThermo Fisher ScientificCat# EN0531100 µg/ml
Chemical compound, drugRNase HThermo Fisher ScientificCat# EN02010.2 Units/µl
OtherHoechst StainThermo Fisher ScientificCat# H35690.5 µg/ml
OtherBio-Rad Trans-Blot NitrocelluloseBio-RadCat# 170–4159Western blot
Biological sample (Xenopus laevis)Xenopus laevis, 9 + cm mature females, pigmentedNASCOCat# LM00535MFemale, adult frogs
Biological sample (Xenopus laevis)Xenopus laevis, 7.5–9 cm mature males, pigmentedNASCOCat# LM00715MXMale, adult frogs
Software, algorithmFiji v2.1.0/1.53 fFijiRRID: SCR_002285Image analysis and quantification
Software, algorithmGraphPad Prism v9GraphPadRRID: SCR_002798Graph creation and statistical analysis
Software, algorithmNIS Elements AR 4.20.02Nikon InstrumentsRRID: SCR_014329Capture and process chromatin images
Software, algorithmLicor Image Studio v3.1LI-COR BiosciencesRRID: SCR_013715Capture and process Western blot images
Software, algorithmAdobe Illustrator CC v25.4.3AdobeRRID: SCR_010279Organize and prepare figures

Preparation of Xenopus egg extracts

Request a detailed protocol

Metaphase-arrested Xenopus laevis egg extract was prepared as previously described (Haase et al., 2017). This low-speed supernatant (LSS) extract was used to prepare mitotic high-speed supernatant (HSS) extract as previously described (Maresca and Heald, 2006). Briefly, LSS extracts were supplemented with energy mix (7.5 mM creatine phosphate, 1 mM ATP, 1 mM MgCl2, 0.01 mM EGTA), 50 mM sucrose, and sea urchin cyclin Δ90 (Groen et al., 2011). Extracts were incubated at 21 °C for 15 min then spun for 2 hr at 200,000 x g at 4 °C. Post spin, the middle cytosolic layer was removed and spun once more for 30 min. The resulting supernatant was removed and aliquoted, and stored at –80 °C.

Sperm nuclei preparation

Request a detailed protocol

Xenopus sperm nuclei were prepared as described previously (Maresca and Heald, 2006). Mouse (Mus musculus) sperm nuclei were prepared as described previously (Shintomi et al., 2017), with the following modifications. Sperm were isolated from the cauda epididymis of ~6-month-old C57BL/6 J mice. To detach sperm tails from the nuclei-containing sperm heads, isolated sperm were resuspended in 2 ml of tissue-culture grade trypsin/EDTA (Thermo) and incubated for 5 min in a 37 °C water bath. Digestion was halted by resuspension of the mixture in 1 X PBS containing 10 X LPC (10 mg/mL each of leupeptin, pepstatin, and chymostatin). To isolate sperm heads from tails, the digestion mixture (resuspended in 1 X MH buffer [20 mM HEPES-KOH, pH 7.7, 2 mM MgCl2] + 1.8 M sucrose) was layered onto a sucrose step gradient (containing 2.05 and 2.2 M sucrose, in 1 X MH buffer) and centrifuged for 45 min at 93,000 x g at 2 °C. After washing the interfaces with 1 X MH buffer, the pellet containing sperm heads was resuspended in 1 X MH +250 mM sucrose. Sperm nuclei were demembranated as described previously (Shintomi et al., 2017), and protamines were reduced in a solution of 1 X MH, 250 mM sucrose, 0.4% BSA, 1 X LPC, and 50 mM DTT for 2 hr in a room temperature water bath. Sperm were washed, resuspended, and stored as described.

Chromatid assembly reactions and drug treatments

Request a detailed protocol

To monitor the assembly of chromatids, HSS egg extracts were first diluted with an equal volume of XBE5 buffer (10 mM HEPES-KOH [pH 7.7], 100 mM KCl, 5 mM EGTA, 5 mM MgCl2, 50 mM sucrose) and supplemented with energy mix. Sperm nuclei isolated from Xenopus or mouse were then added to the diluted HSS extracts at a final concentration of 2000 sperm/μl, and incubated for the indicated times at 21 °C. Samples were taken at indicated timepoints for analysis via microscopy and western blot.

For inhibition experiments, chromatin assembly reactions were allowed to proceed for 25 min to allow for protamine exchange with maternal histones, and then drugs or an equal volume of DMSO were added and the extracts were further incubated at 21 °C for the indicated times. The final concentration of inhibitors was: 54.4 μM α-amanitin (Tocris), 50 μM triptolide (TPL; Tocris or Sigma), 30 μM THZ1 (Cayman), and 100 μM DRB (Cayman).

RNase treatments

Request a detailed protocol

To determine the role of RNA in chromosome condensation, HSS extracts were either mock-treated (RNase-free water) or treated with RNase A (Thermo Fisher Scientific) at a final concentration of 100 μg/ml and incubated at 21 °C for 30 min prior to the addition of sperm. Sperm nuclei were then added to a final concentration of 2000 sperm/μl and the reaction was further incubated at 21 °C for up to 180 min in the presence of active RNase A. Samples were taken at indicated timepoints and processed for microscopy analysis. To analyze total RNA levels, RNA was isolated from 100 μl of HSS extracts according to the RNeasy kit ‘RNA clean up’ protocol (Qiagen), precipitated in the presence of glycogen (MilliporeSigma), and resuspended in 2 μl of RNase-free water. Samples were then diluted with RNA loading buffer (95% formamide, 10 mM EDTA pH 8, 0.025% SDS, bromophenol blue) and run on a 1% agarose gel in 0.5 X TBE, and visualized with SYBR safe DNA gel stain (Thermo Fisher Scientific). Note that total RNA levels in HSS extracts are low compared to LSS extracts due to the lower concentration of ribosomal RNAs (Groen et al., 2011).

To degrade RNA:DNA hybrids, HSS extracts were treated with RNase H (Thermo Fisher Scientific) or heat-inactivated RNase H (65 °C for 20 min) at a final concentration of 0.2 U/μl. Sperm nuclei were then added to a final concentration of 2000 sperm/μl and the reaction was incubated at 21 °C for up to 180 min. Samples were taken at indicated timepoints and processed for microscopy analysis.

Antibody production

Request a detailed protocol

CAP-E antibodies were raised against a C-terminal peptide of Xenopus laevis CAP-E (CSKTKERRNRMEVDK; synthesized by Vivitde) (Hirano et al., 1997). The peptide was then coupled to keyhole limpet hemocyanin protein and used to immunize rabbits (Labcorp).

Immunodepletion of HSS extracts

Request a detailed protocol

Control rabbit IgG (MilliporeSigma, I5006), anti-XPB (Novus Biologicals, NB100-61060), anti-ERCC1 (a kind gift of Puck Knipscheer, Hubrecht Institute, Klein Douwel et al., 2017), or anti-histone H4K12ac (a kind gift of Hiroshi Kimura, Tokyo Institute of Technology, Zierhut et al., 2014) were bound and crosslinked to Protein-A Dynabeads as previously described (Haase et al., 2017). HSS extract was incubated with antibody-coupled beads at 4 °C for 30 min with occasional mixing, followed by two additional rounds of depletion. For CAP-E depletion, CAP-E serum or pre-immune serum (“Mock”) were bound to Protein-A Dynabeads without crosslinking, and HSS extract was incubated with antibody-coupled beads at 4 °C for 45 min, followed by one additional round of depletion. The volume ratio of beads to extract for each depletion were as follows: CAP-E serum (4:1), anti-XPB (2:1), anti-ERCC1 (2:1), and anti-H4K12ac (5.76:1). After depletion, the extract was used for chromatid assembly reactions as indicated.

Immunoprecipitations

Request a detailed protocol

To provide samples for the analysis of XPB interaction partners by mass spec, XPB antibody (Novus Biologicals, NB100-61060) was incubated with Protein-A Dynabeads (Thermo Fisher Scientific, 10001D) and then crosslinked with BS3 (Thermo Fisher Scientific, A39266) at a ratio of 0.24 μg of antibody per μl of beads. A total of 300 μl of HSS extract was then incubated with an equal volume of anti-XPB beads for 60 minutes at 4 °C, with occasional mixing. Anti-XPB beads were then captured and washed four times with wash buffer (1 X PBS, 0.01% Tween-20), and boiled in 2 X sample buffer for 5 min at 95 °C.

For XPB and CAP-E immunoprecipitations, XPB antibody (Novus Biologicals, NB100-61060), control IgG antibody (MilliporeSigma, I5006), anti-CAP-E serum, or pre-immune (“Mock”) serum were incubated with Protein-A Dynabeads at a ratio of 0.24 μg XPB or IgG antibody per μl of beads or 3 μl anti-CAP-E or pre-immune serum per μl of beads, and subsequently crosslinked with BS3. A total of 75 μl of HSS extract was then incubated with an equal volume of antibody-bound beads for 60 min at 4 °C, with occasional mixing. Antibody-bound beads were then captured and washed four times with wash buffer (1 X PBS, 0.01% Tween-20), and boiled in 2 X sample buffer for 10 min at 70 °C.

Mass spectrometry

Request a detailed protocol

The immunoprecipitated proteins were separated by gel electrophoresis. Each lane was divided into 12 fractions and the proteins were in-gel digested with trypsin (Thermo Fisher Scientific) at 37 °C for 16 hr, as described (Shevchenko et al., 2006). Dried peptides were separated on a 75 µm x 15 cm, 2 µm Acclaim PepMap reverse phase column (Thermo) at 300 nL/min using an UltiMate 3000 RSLCnano HPLC (Thermo Fisher Scientific). Peptides were eluted into a Thermo Orbitrap Fusion mass spectrometer using a linear gradient from 96% mobile phase A (0.1% formic acid in water) to 55% mobile phase B (0.1% formic acid in acetonitrile) over 30 min. Parent full-scan mass spectra were collected in the Orbitrap mass analyzer set to acquire data at 120,000 FWHM resolution; ions were then isolated in the quadrupole mass filter, fragmented within the HCD cell (HCD normalized energy 32%, stepped ±3%), and the product ions were analyzed in the ion trap. Proteome Discoverer 2.4 (Thermo Fisher Scientific) was used to search the data against Xenopus laevis proteins from the PHROG database (Wühr et al., 2014; Presler et al., 2017) using SequestHT. The search was limited to tryptic peptides, with maximally two missed cleavages allowed. Cysteine carbamidomethylation was set as a fixed modification, with methionine oxidation and serine/threonine phosphorylation set as variable modifications. The precursor mass tolerance was 10 ppm, and the fragment mass tolerance was 0.6 Da. The Percolator node was used to score and rank peptide matches using a 1% false discovery rate.

Western blots

Request a detailed protocol

Primary antibodies were diluted in Licor blocking solution with a final Tween-20 concentration of 0.1%. The following antibodies and antibody dilutions were used: anti-CDK1T161ph (9114S, 1:500, Cell Signaling Technology), anti-XPB (NB100-61060, 1:1000, Novus Biologicals), anti-p62 (ab232982, 1:400, Abcam), anti-α-Tubulin (DM1, 1:20,000, MilliporeSigma), anti-H3 (ab1791, 1:500, Abcam), anti-histone H3T3ph (ab78351, 1:10,000, Abcam), anti-XPF (a kind gift of Puck Knipscheer, Hubrecht Institute, Klein Douwel et al., 2017, 1:10,000), anti-ERCC1 (a kind gift of Puck Knipscheer, Hubrecht Institute, Klein Douwel et al., 2017, 1:10,000), anti-CAP-E (a kind gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011, 1:1000), anti-CAP-C (a kind gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011, 1:500), anti-CAP-D3 (a kind gift of Alexei Arnaoutov and Mary Dasso, NICHD Bernad et al., 2011, 1:400), and anti-CAP-G (a kind gift of Susannah Rankin, OMRF, 1:5000). Secondary antibodies (Licor) were used at a 1:17,500 dilution (goat anti-rabbit 800 nm and goat anti-mouse 680 nm), and membranes were scanned on the Odyssey imaging system (Licor).

Microscopy and fluorescence quantification

Request a detailed protocol

To immunostain chromatin structures, extracts samples were fixed by diluting ten-fold with KMH buffer (20 mM Hepes-KOH pH 7.7, 100 mM KCl, 2.5 mM MgCl2) containing 4% formaldehyde and 0.1% Triton X-100, and incubated for 15 min at room temperature. To isolate chromatids/nuclei, the fixed reactions were layered onto cushions of KMH +30% glycerol in a 24-well plate, which contained poly-L-lysine–coated coverslips (No. 1) at the bottom of each well, and then spun at 4,614 x g for 12.5 min at 18 °C in a centrifuge (Eppendorf 5810 R). Cushion was removed and the coverslips were washed with TBS-TX twice for 5 minutes each, then blocked with Abdil (TBS +0.1% Tween20 +2% BSA + 0.1% sodium azide) for 30 minutes at 4 °C, then incubated in primary at 4 °C overnight unless otherwise noted. Chromatids/nuclei were then washed three times with AbDil and then incubated with secondary antibody for 2 hr. All washes and antibody dilutions were carried out with AbDil buffer. Chromatids/nuclei were stained with Hoechst 33258 prior to mounting in 80% glycerol +PBS medium. The following antibodies were used at the indicated dilutions: XPB (Millipore, MABE1123) 1:50, XPB (Novus Biologicals NB100-61060) 1:200, CAP-D3 (a kind gift of Alexei Arnaoutov and Mary Dasso, NICHD, Bernad et al., 2011) 1:100, CAP-C (Bernad et al., 2011) 1:100, CAP-E (Bernad et al., 2011) 1:100, Topo II (a kind gift of Yoshi Azuma, University of Kansas, Pandey et al., 2020) 1:200, SUPT16H (Abcam, ab204343) 1:100, histone H3 (Abcam, ab1791) 1:400. Alexa 488 (anti-rabbit, Jackson Immunoresearch), Alexa 488 (anti-mouse, Jackson Immunoresearch), and Alexa 647 (anti-rabbit, Jackson Immunoresearch) conjugated secondary antibodies were used at a 1:400 dilution for immunofluorescence detection.

All immunofluorescence was imaged with 0.2 μm step size using an Eclipse Ti (Nikon) comprised of a Nikon Plan Apo x100/1.45, oil immersion objective, a PlanApo x40/0.95 objective, and a Hamamatsu Orca-Flash 4.0 camera. Images were captured and processed using NIS Elements AR 4.20.02 software (Nikon), and analyzed in Fiji ImageJ. The acquired Z-sections of 0.2 μm each were converted to a maximum projection using NIS Elements and Fiji. Intensity of chromatin bound proteins was measured using Fiji by centering 9 × 9 and 13 × 13 pixel regions along multiple points of individualized chromatids and averaging the maximum intensity of each sample point to determine average intensity per chromatid. In cases where condensation was inhibited, chromatin masses were sampled to determine an average intensity. To correct for background fluorescence, the difference in intensities between the two regions was determined, and then made proportionate to the smaller region. This background value was then subtracted from the smaller region to determine chromatin bound protein intensity with background correction as previously reported (Hoffman et al., 2001).

Condensation parameter

Request a detailed protocol

To quantify condensation of chromatin, 20 chromatin structures per condition were sampled with a 38 by 50 pixel region. Pixel intensities were recorded and binned. To measure the degree of condensation, the percentage of pixels below a threshold of 35% of the maximum image intensity were determined (Maddox et al., 2006). This percentage serves as the condensation parameter, and represents the changes in distribution of fluorescence intensity that result from condensation.

Statistical analysis

Request a detailed protocol

All analyses were performed with a minimum number of 50 chromatids, or chromatin masses in cases where condensation was not achieved. This sample size was chosen to ensure a high ( > 90%) theoretical statistical power in order to generate reliable P values. All graphs and statistical analysis were prepared with GraphPad Prism. Fluorescence values from experimental conditions were compared to control conditions using an ordinary one-way ANOVA with Tukey’s multiple comparison tests to determine significance. All graphs show the mean with error bars representing the S.D. unless otherwise indicated. At least two biological replicates (using two different preparations of egg extracts) were performed for each experiment and each confirmed similar behavior amongst the replicates.

Data availability

The original files of the full raw unedited gels and blots and figures with the uncropped gels and blots with the relevant bands clearly labelled have been provided as Source Data files.

References

Decision letter

  1. Silke Hauf
    Reviewing Editor; Virginia Tech, United States
  2. Jessica K Tyler
    Senior Editor; Weill Cornell Medicine, United States
  3. Rebecca Heald
    Reviewer; UC Berkeley, United States
  4. Pim J Huis in 't Veld
    Reviewer; Max Planck Institute of Molecular Physiology, Germany

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "The TFIIH Complex is Required to Establish and Maintain Mitotic Chromosome Structure" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Silke Hauf as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Rebecca Heald (Reviewer #2); Pim J Huis in 't Veld (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this summary to help you prepare a revised submission.

All reviewers felt that you are reporting a novel, interesting, and likely impactful observation. The quality of the data was generally considered high, but several experiments require additional quantifications or controls.

Essential revisions:

1) Throughout the paper, images of "representative" chromosome morphologies are shown, but some form of quantification is required – either scoring condensation states subjectively, or – if possible – an objective (automated) quantification. Perhaps solidity measurements would help. It should also be stated how many times an experiment was replicated and how many structures were evaluated.

Quantifications would be helpful to strengthen the conclusions on some results that are currently difficult to interpret. For example, RNase H treatment and THZ1 treatment (Figure 1 – supplement 1) are interpreted as not showing an effect, but the chromatids look thinner in RNase H treatment, and somewhat uncondensed in the THZ1 treatment. In Figure 2A the +TPL at 25 min looks qualitatively different, as if it is taking longer for the effect to manifest. Is this the case?

2) It would be informative to see to what degree condensin and XPB co-localize on the chromosome axis at higher magnification, if this is possible.

3) All experiments, where no effect on condensation is observed (such as DRB and α-amanitin in Figure 1B or ERCC1 depletion in Figure 1G/H) would profit from showing that the drug or treatment did have an effect – to the extent that this is possible.

4) The authors could be yet more careful with the conclusions drawn. For example, the authors claim that TFIIH promotes both loading and activity of condensin from comparing the kinetics of chromosome morphology changes and condensin occupancy on chromatin. Other models would be equally consistent, e.g. TFIIH depletion could affect the binding or activity of some other molecule, which then causes condensin to fall off, like histone H1 which was shown to have an antagonistic relationship with condensin binding (see 10.7554/eLife.68918).

In the overall conclusions, the authors propose that TFIIH changes chromatin structure in a way that is conducive for condensin binding. This interpretation is similarly problematic since many proteins are competing for binding sites on chromatin and effects could be indirect and alternative models would not require changes in chromatin structure.

Additional suggestions (non-essential):

These are topics that came up in the discussion. If you can address any of them, that'd be great, but they are not required to meet the standard for publication.

1) Given the potential difficulties, this is not a requirement for publication, but the paper would obviously greatly profit from examining the role of TFIIH in condensation in other situations. For example, it would be interesting to know if the triptolide effect is specific to sperm chromosomes, or if mitotic chromosomes isolated from tissue culture cells, or formed from nuclei isolated from later developmental stages of embryogenesis, are similarly affected. Does triptolide block condensation in a somatic Xenopus cell line, or in a human cell line? Does TFIIH localize to mitotic chromosomes in somatic cells?

2) Given that the authors propose that TFIIH alters chromatin for condensin action, the paper would also be strengthened by examining nucleosome occupancy on sperm chromatin with and without TFIIH inhibition.

3) The interaction between condensin and TFIIH is potentially interesting. However, it seems that only a very small fraction of condensin is co-immunoprecipitated with XPB. Can the interaction be detected when immunoprecipitating condensin? Please also provide the full information from the mass spectrometry experiment. Was CAP-E the only condensin subunit detected? Do you have any evidence that the interaction is direct?

4) For the H3/H4 reduction + TPL, it would be very interesting to see not only the endpoint, but also the time course. Are chromosomes also resistant to decondensation in the first 5-10 minutes after TPL addition when histone levels are reduced?

5) It's striking that the TPL effects were abolished with mild (20-30%) H3/H4 depletions. Is it possible to determine the H3/H4 reduction that renders chromosome formation TPL insensitive in a more systematic way. What happens when H3/H4 are, for example, 5%, 10%, 20%, 40% or 80% reduced?

6) If extracts with histones are added to the histone-depleted chromosomes that condensed in the presence of TPL, would that result in decondensation? Does that depend on the inclusion of TPL in the add-back extract?

7) The authors could support the CDK7-independent conclusions of the paper by a) excluding abnormal CDK7 effects in the presence of TPL by inhibiting CDK7 simultaneously with XPB and b) excluding effects of TPL on overall CDK1 activity by immunoblotting for the phosphorylation of the activation loop of CDK1.

8) Are XPB levels reduced from chromatin when condensin I and/or condensin II is depleted from chromatin? Is it feasible to conduct this experiment?

9) The following paper might be interesting to discuss: https://www.nature.com/articles/s41467-019-09270-2

https://doi.org/10.7554/eLife.75475.sa1

Author response

Essential revisions:

1) Throughout the paper, images of "representative" chromosome morphologies are shown, but some form of quantification is required – either scoring condensation states subjectively, or – if possible – an objective (automated) quantification. Perhaps solidity measurements would help. It should also be stated how many times an experiment was replicated and how many structures were evaluated.

To quantify the chromosome morphologies in each experiment, we used the “condensation parameter” metric. This metric represents the percentage of pixels below a defined cutoff of fluorescence intensity for each image, and we feel it accurately reports the change in the shape of the fluorescence intensity distribution of chromatin that occurs during condensation. This approach was originally reported by the Desai and Oegema labs and used to quantify mitotic chromosome condensation in worm embryos and examine the effects of condensin depletion (Maddox et al., PNAS, 2006). We have provided this data both in the main figures, and in extensive scatter plots in: Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 3, Figure 2, Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 4, Figure 4—figure supplement 1, Figure 5, and Figure 5—figure supplement 2.

To put these metrics into perspective, we depleted condensins from extracts and determined the condensation parameters under this condition. As shown in Figure 1B and C, condensin depletion and TPL treatment have similar drastic effects on condensation (83% of pixels for DMSO at 180 min, 4% for condensin depleted, and 5% for TPL treated). We feel this further demonstrates the validity of the quantification method, and the similarities of the loss of condensation induced by triptolide treatment, TFIIH depletion, and condensin depletion.

We have also included information on the number of times experiments were replicated and how many structures were evaluated in the methods and figure legends of the manuscript.

Quantifications would be helpful to strengthen the conclusions on some results that are currently difficult to interpret. For example, RNase H treatment and THZ1 treatment (Figure 1 – supplement 1) are interpreted as not showing an effect, but the chromatids look thinner in RNase H treatment, and somewhat uncondensed in the THZ1 treatment. In Figure 2A the +TPL at 25 min looks qualitatively different, as if it is taking longer for the effect to manifest. Is this the case?

We believe that the condensation parameters now included in the manuscript should help address these concerns.

Regarding the RNase H treatment morphologies, we agree that the chromatids are a bit thinner than in wild type. However, this effect was also observed in the heat-inactivated RNase H condition as well (Figure 1—figure supplement 1C), and thus it is likely this is caused by one of the buffer components that the enzyme is provided in (e.g. KCl and/or EDTA), and not the activity of the enzyme.

We repeated the THZ1 treatment experiments multiple times and saw no obvious effect of the drug on chromosome morphology, and this is supported by the condensation parameter quantifications (Figure 1—figure supplement 3D). We have replaced the original images, which were from experiments in which the extracts were spotted directly onto a coverslip and fixed (“squashes”), with images from experiments in which we spun the chromosome-containing extracts through a glycerol cushion onto coverslips (Figure 1—figure supplement 1F), which provides a sharper view of individual chromosome morphology.

Regarding the TPL additions at 25 mins in Figure 2A, the new quantifications of the condensation parameters demonstrates that there is a drastic and rapid effect of TPL addition at all indicated timepoints, and that there is a slightly higher condensation parameter 5 mins after addition of TPL than at longer times (e.g. 7% at 30’ vs 4% at 185’ for the TPL @ 25’ condition; Figure 2—figure supplement 1). However, the controls demonstrate that the condensation parameter starts at around 40% at 25’, and plateaus around 80% at 180’. Thus, although there is a small and gradual decrease in condensation from 5 mins to 155 mins after TPL addition, the magnitude of the immediate effect of TPL addition is much larger.

2) It would be informative to see to what degree condensin and XPB co-localize on the chromosome axis at higher magnification, if this is possible.

We performed co-staining of XPB and CAP-E on chromatids, and this data is now included in Figure 3—figure supplement 1D.

3) All experiments, where no effect on condensation is observed (such as DRB and α-amanitin in Figure 1B or ERCC1 depletion in Figure 1G/H) would profit from showing that the drug or treatment did have an effect – to the extent that this is possible.

Data supporting the activity of the small molecules (THZ1, DRB, α-amanitin, and triptolide) is now provided in Figure 1—figure supplement 2. Although the first zygotic transcript is not produced until the 8-cell stage of Xenopus embryonic development (Jukam et al., 2017), multiple reports have demonstrated that active transcription occurs at the centromere repeats in egg extracts prior to zygotic activation (Grenfell et al., JCB,2016; Blower, Cell Reports, 2016; Perea-Resa et al., Mol. Cell, 2020). Indeed, we observed co-localization of both RNA Pol II S2ph (elongating) and RNA Pol II S5ph (paused) at centromeres. As expected, the elongation inhibitors α-amanitin and DRB blocked centromeric accumulation of S2ph, but not S5ph. Furthermore, THZ1 treatment, which inhibits the kinase responsible for S5ph that is upstream of the kinases required for S2ph, blocked centromeric accumulation of both forms of RNA Pol II. Likewise, triptolide, which inhibits the release of RNA Pol II from the pre-initiation complex, blocked centromeric accumulation of both forms of RNA Pol II. Thus, our new data show clear efficacy of each small molecule, and further support our claims of a specific role for the TFIIH complex in condensation.

We were unable to perform nucleotide excision repair (NER) assays to further evaluate the effects of ERCC1 depletion due to technical limitations. However, we have now quantified our ERCC1 depletion efficiency (Figure 1—figure supplement 2), and this level of depletion was sufficient to block NER in egg extracts using the same source of antibodies (Klein Douwel et al., EMBO, 2017; Mol. Cell, 2014).

4) The authors could be yet more careful with the conclusions drawn. For example, the authors claim that TFIIH promotes both loading and activity of condensin from comparing the kinetics of chromosome morphology changes and condensin occupancy on chromatin. Other models would be equally consistent, e.g. TFIIH depletion could affect the binding or activity of some other molecule, which then causes condensin to fall off, like histone H1 which was shown to have an antagonistic relationship with condensin binding (see 10.7554/eLife.68918).

In the overall conclusions, the authors propose that TFIIH changes chromatin structure in a way that is conducive for condensin binding. This interpretation is similarly problematic since many proteins are competing for binding sites on chromatin and effects could be indirect and alternative models would not require changes in chromatin structure.

We agree with the reviewers and have extensively incorporated these suggestions throughout the revised manuscript. We discuss alternative models of TFIIH action and make clear that TFIIH could be acting directly or indirectly in these processes. Further, we have changed “chromatin structure” to “chromatin environment” to include both nucleosomes and chromatin binding proteins and enzymes.

Additional suggestions (non-essential):

1) Given the potential difficulties, this is not a requirement for publication, but the paper would obviously greatly profit from examining the role of TFIIH in condensation in other situations. For example, it would be interesting to know if the triptolide effect is specific to sperm chromosomes, or if mitotic chromosomes isolated from tissue culture cells, or formed from nuclei isolated from later developmental stages of embryogenesis, are similarly affected. Does triptolide block condensation in a somatic Xenopus cell line, or in a human cell line? Does TFIIH localize to mitotic chromosomes in somatic cells?

Although we were unable to perform such experiments due to COVID and technical limitations, we agree that these are exciting experiments. Since submission of our manuscript, we discovered multiple earlier papers from the Zurita lab that reported defects in genome integrity, chromatin remodeling, and chromosome condensation in TFIIH mutants in Drosophila mitotic embryos and neuroblasts, and have incorporated these previous results in the Discussion. We feel this helps support a conserved role of the TFIIH in condensation during embryogenesis.

2) Given that the authors propose that TFIIH alters chromatin for condensin action, the paper would also be strengthened by examining nucleosome occupancy on sperm chromatin with and without TFIIH inhibition.

Unfortunately, we were unable to perform such experiments due to COVID and technical limitations, but hope to perform these exciting experiments in the future.

3) The interaction between condensin and TFIIH is potentially interesting. However, it seems that only a very small fraction of condensin is co-immunoprecipitated with XPB. Can the interaction be detected when immunoprecipitating condensin? Please also provide the full information from the mass spectrometry experiment. Was CAP-E the only condensin subunit detected? Do you have any evidence that the interaction is direct?

We performed an immunoprecipitation of condensin and we were unable to detect an interaction with XPB (Figure 3—figure supplement 1C). We have incorporated this data and our new data on XPB and condensin co-localization (Figure 3—figure supplement 1D) and the lack of an effect of condensin depletion on TFIIH chromatin levels (Figure 3—figure supplement 1A), in the Results section. We believe these findings demonstrate that any interaction of TFIIH and condensins is weak and not necessarily direct. This is in line with our H3/H4 partial depletion data (Figure 5) that shows that chromosomal condensin levels can be substantially restored in the absence of chromatin-bound TFIIH, and further supports our model that TFIIH acts upstream of condensin to maintain a chromatin environment that is conducive to condensin loading and function.

4) For the H3/H4 reduction + TPL, it would be very interesting to see not only the endpoint, but also the time course. Are chromosomes also resistant to decondensation in the first 5-10 minutes after TPL addition when histone levels are reduced?

We have included new data for this experiment detailing chromosome condensation 10 minutes after TPL or DMSO addition (Figure 5A and in Figure 5—figure supplement 2A). We found that chromosomes were resistant to decondensation in the H3/H4 reduction + TPL condition 10 minutes after drug addition, which we feel supports a primary effect of H3/H4 reduction in rescuing condensation.

5) It's striking that the TPL effects were abolished with mild (20-30%) H3/H4 depletions. Is it possible to determine the H3/H4 reduction that renders chromosome formation TPL insensitive in a more systematic way. What happens when H3/H4 are, for example, 5%, 10%, 20%, 40% or 80% reduced?

Unfortunately, we were unable to perform such experiments due to COVID and limited quantities of depletion-competent antibodies. We hope to perform these exciting experiments in the future.

6) If extracts with histones are added to the histone-depleted chromosomes that condensed in the presence of TPL, would that result in decondensation? Does that depend on the inclusion of TPL in the add-back extract?

Unfortunately, we were unable to perform such experiments due to COVID and limited quantities of depletion-competent antibodies. We hope to perform these exciting experiments in the future.

7) The authors could support the CDK7-independent conclusions of the paper by a) excluding abnormal CDK7 effects in the presence of TPL by inhibiting CDK7 simultaneously with XPB and b) excluding effects of TPL on overall CDK1 activity by immunoblotting for the phosphorylation of the activation loop of CDK1.

We have provided new data that exclude abnormal CDK7 effects in response to TPL treatment in Figure 1—figure supplement 1F. We have also provided new data that demonstrate no effect of TPL on CDK1 activation loop phosphorylation in extracts in Figure 1—figure supplement 1G.

8) Are XPB levels reduced from chromatin when condensin I and/or condensin II is depleted from chromatin? Is it feasible to conduct this experiment?

We have provided new data that demonstrate that depletion of condensins I and II have no effect on XPB levels on chromatin (Figure 3—figure supplement 1A).

9) The following paper might be interesting to discuss: https://www.nature.com/articles/s41467-019-09270-2

We have included this in the Discussion.

https://doi.org/10.7554/eLife.75475.sa2

Article and author information

Author details

  1. Julian Haase

    Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5891-4241

  2. Richard Chen

    Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, United States
    Present address
    Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review and editing
    Contributed equally with
    Wesley M Parker
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5447-8646

  3. Wesley M Parker

    Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review and editing
    Contributed equally with
    Richard Chen
    Competing interests
    No competing interests declared

  4. Mary Kate Bonner

    Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, United States
    Contribution
    Data curation, Formal analysis, Methodology, Resources, Validation, Writing – review and editing
    Competing interests
    No competing interests declared

  5. Lisa M Jenkins

    Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1245-1338

  6. Alexander E Kelly

    Laboratory of Biochemistry & Molecular Biology, National Cancer Institute, NIH, Bethesda, United States
    Contribution
    Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing – review and editing
    For correspondence
    alexander.kelly@nih.gov
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3395-6012

Funding

National Cancer Institute (Intramural Research Program)

  • Julian Haase
  • Richard Chen
  • Wesley M Parker
  • Mary Kate Bonner
  • Lisa M Jenkins
  • Alexander E Kelly

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Takashi Akera, Chongyi Chen, and Michael Lichten for critical reading of the manuscript, and members of the LBMB for comments and support, and A Arnaoutov, Y Azuma, M Dasso, H Kimura, P Knipscheer, and A Nussenzweig for kindly providing reagents. We thank Jacob Paiano for assistance with mouse sperm purification, and Mia Rosenfeld for initial work with inhibitors.

Ethics

This study was performed in strict accordance with the care standards provided by the 8th edition of the Guide for the Care and Use of Laboratory Animals. African clawed frogs, Xenopus laevis, which were maintained and handled according to approved institutional animal care and use committee (NCI IACUC) protocol (LBMB-001-1) of the National Cancer Institute, which is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited research facility.

Senior Editor

  1. Jessica K Tyler, Weill Cornell Medicine, United States

Reviewing Editor

  1. Silke Hauf, Virginia Tech, United States

Reviewers

  1. Rebecca Heald, UC Berkeley, United States
  2. Pim J Huis in 't Veld, Max Planck Institute of Molecular Physiology, Germany

Publication history

  1. Preprint posted: November 7, 2021 (view preprint)
  2. Received: November 11, 2021
  3. Accepted: March 15, 2022
  4. Accepted Manuscript published: March 16, 2022 (version 1)
  5. Version of Record published: March 25, 2022 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 1,458
    Page views
  • 229
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Julian Haase
  2. Richard Chen
  3. Wesley M Parker
  4. Mary Kate Bonner
  5. Lisa M Jenkins
  6. Alexander E Kelly
(2022)
The TFIIH complex is required to establish and maintain mitotic chromosome structure
eLife 11:e75475.
https://doi.org/10.7554/eLife.75475

Categories and tags

Research organisms

Further reading

    1. Cell Biology

    Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture

    Sandipan Dasgupta, Daniella Y Dayagi ... Jeffrey E Gerst
    Research Article Updated

    Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the ‘transferome’) is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically,<1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

    1. Cell Biology
    2. Neuroscience

    Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain

    Elizabeth L Wagner, Jun-Sub Im ... Jung-Bum Shin
    Research Article

    Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as ‘gaps’ in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s

    Wenxin Zhang, Taki Nishimura ... Sharon A Tooze
    Research Article

    Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.