Author response: The TFIIH complex is required to establish and maintain mitotic chromosome structure
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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 Decision letter Reviews on Sciety eLife's review process 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 Download 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 (Figure 3B; of The condensin that remained on chromosomes was present in a diffuse pattern with no axis (Figure 3A). the of condensin I in extracts is to than that of condensin II, and condensin I is the major driver of chromosome condensation in egg extracts (Ono et al., 2003; Shintomi and Hirano, 2011), we that triptolide prevents the proper loading and enrichment of both condensin I and condensin triptolide had no significant effect on topo II levels (Figure 3A and B; of control), although topo II was no concentrated on a central A similar effect on topo II localization is upon condensin depletion from egg extracts (Shintomi et al., 2017). a of the complex in both transcription (Zhou et al., and chromosome condensation (Shintomi et al., 2015), was present at similar levels in the presence or absence of triptolide (Figure 3A and B; of CAP-C levels also significantly of control) when fully condensed chromosomes were treated with triptolide at t = for min (Figure 2—figure supplement These results demonstrate that the TFIIH complex is required for the enrichment of condensin on chromosomes during condensation establishment and also suggest that the TFIIH complex condensins and not topo II, TFIIH inhibition condensin but not topo II levels on chromatin (Figure and 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 were with and II), I II), anti-CAP-E I and II), or II of individual chromatids are shown in (B) of fluorescence intensity of indicated proteins from in to n = structures for each condition. Error bars represent SD, and indicate a significant indicates no Two biological replicates were performed, quantified structures are from a single experiment. In we that XPB localization to chromatin was maintained the depletion of condensins (Figure supplement 1A). both TFIIH and condensin levels on chromatin in response to triptolide (Figure 3B), we that TFIIH promote of condensin complexes to chromosomes through We that the condensin II subunit with XPB from high-speed of egg extracts (Figure supplement However, XPB was not with anti-CAP-E we observed with other condensin I and II subunits including (Figure supplement These results suggest that any between TFIIH and condensins are and not Furthermore, TFIIH only partially with condensins on single chromatids, and the TFIIH complex did not demonstrate the axial staining pattern observed for condensins, which a role for TFIIH (Figure supplement 1D). these results show that the TFIIH complex acts of condensin enrichment and associates with the subunit of condensin II, but indicate that the role of TFIIH in condensin enrichment is not through TFIIH inhibition chromosome condensation before condensin and TFIIH are from chromosomes To further how TFIIH promotes condensation and the enrichment of condensins on chromatin, we the of condensin enrichment with and TFIIH we added triptolide 25 min after addition of sperm nuclei to egg extracts and chromatin morphology and condensin levels on chromatin over (Figure As expected, the addition of triptolide to rapid 2A and and Figure supplement 1A), and an of chromatin discernable chromatids was of drug addition. DMSO-treated extracts in a with condensation over (Figure and Figure supplement 1A). In contrast, condensin as by CAP-E increased for the first min after triptolide addition, similar to the first 20 min before to 35% of control over the min (Figure and C, and Figure supplement 2A). condensin II and XPB remained at control levels for min after triptolide addition before to (Figure supplement 2B and These results indicate that TFIIH an role in promoting condensation, and is also required for the enrichment of condensins on chromosomes that occurs throughout the condensation process. Figure with 2 supplements see all Download asset Open asset Triptolide condensation prior to its effects on condensin (A) Schematic of to test the of triptolide-induced defects in condensation and condensin After a DMSO or triptolide (TPL) was and samples were taken at indicated times and for (B) Representative immunofluorescence images of DMSO or triptolide treated extracts at indicated timepoints. were with and anti-CAP-E I and that condensation is in in the first after triptolide addition, as in Figure Mean condensation parameters for each condition are indicated in lower left corner of each image. (C) of fluorescence intensity of CAP-E from in to the 180 min DMSO-treated n = structures for each condition. Error bars represent SD, and indicate a significant Two biological replicates were performed, quantified structures are from a single experiment. formation of nucleosome-free regions the for TFIIH in chromosome condensation lines of suggest that are a to condensin loading and function on chromosomes. In Xenopus egg extracts, condensins a strong for to DNA over et al., 2014; Shintomi et al., 2017; Choppakatla et al., 2021). The of nucleosome-free regions has been proposed to be important for chromosome condensation in et al., 2016), and DNA has been shown to the ATPase activity of condensin (Piazza et al., The TFIIH complex, which acts as a DNA translocase and changes to chromatin, promote condensin and condensation by altering the chromatin environment to allow the of To test this we nucleosome-free regions on chromosomes by using antibodies to partially histones and from egg extracts et al., 2014) and examined the effects of triptolide on chromosome condensation. Xenopus sperm histones and on chromatin (Shintomi et al., 2015), we used mouse which have previously been shown to condense in a condensin-dependent manner in
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Institutions: National Institutes of Health, National Cancer Institute
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