Chromatin remodeling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair, and transcription. To understand how these complexes find their target sites on chromatin, we use genetically encoded photo-cross-linker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC complex, on nucleosomes in living yeast. We find that H3 K14 acetylation induces the interaction of the Sth1 bromodomain with the H3 tail and mediates the interaction of RSC with neighboring nucleosomes rather than recruiting it to chromatin. RSC preferentially resides on H2B SUMOylated nucleosomes in vivo and shows a moderately enhanced affinity due to this modification in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but changes its mode of nucleosome binding. Our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications likely in combination with histone variants and transcription factors.
The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes.
Chromatin remodeler (CR) complexes play crucial roles in regulating chromosomal architectural and act to modify nucleosomal DNA contacts by repositioning nucleosomes. The mechanistic details of the CR family of proteins are of importance because each remodeler complex contributes to unique chromatin structural maintenance. Here, we study the RSC complex of yeast to better characterize its dynamics in the living nucleus. Utilizing an expanded genetic code, we incorporate the photo‐crosslinking amino acid, p‐benzoyl‐L‐phenylalanine (pBPA), site‐specifically into histone proteins in order to monitor non‐histone protein interactions at the nucleosomal surface, in vivo . We map the interface between the RSC motor protein, Sth1, and the nucleosome core particle, revealing that crosslinking efficiency to the H3 N‐terminal tail is dependent upon the acetylation of H3 K14. This modification is also a known binding site of the RSC subunit, Rsc4, suggesting that the remodeler favors binding symmetrical H3 K14ac marks on the same nucleosome. Additionally, we reveal that RSC binding is dependent on SUMOylation of histone H2B. Pairing crosslinking with temporal control of the cell cycle we observe that RSC has differential binding modes as the chromatin architectures alter during compaction and passage into mitosis. All together, this data provides greater mechanistic insight to the functional dynamics of the RSC remodeler under true physiological conditions. Support or Funding Information National Institute Of General Medical Sciences of the National Institutes of Health under [Award Number R15GM124600] German Research Foundation (DFG) [Grants NE1589/5‐1 and 6‐1]
Chromatin remodelling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair and transcription. A key question is how these complexes find their target sites on chromatin. Here, we use genetically encoded photo-crosslinker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC (remodels the structure of chromatin) complex, on the nucleosome in living yeast. We find that the interaction of the Sth1 bromodomain with the H3 tail depends on K14 acetylation by Gcn5. This modification does not recruit RSC to chromatin but mediates its interaction with neighbouring nucleosomes. We observe a preference of RSC for H2B SUMOylated nucleosomes in vivo and show that this modification moderately enhances RSC binding to nucleosomes in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but its mode of nucleosome binding differs between interphase and mitosis. In sum, our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications most likely in combination with other components such as histone variants and transcription factors.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes. https://doi.org/10.7554/eLife.10396.001 eLife digest DNA in humans, yeast and other eukaryotic organisms is packaged in structures called chromosomes. When a cell divides these chromosomes are copied and then the matching pairs are separated so that each daughter cell has a full set of its genome. To enable these events to take place, the DNA must become more tightly packed so that the chromosomes become rigid units with projections called arms. Any failure in this chromosome “condensation” leads to the loss of chromosomes during cell division. Within a chromosome, sections of DNA are wrapped around groups of proteins to make a series of linked units called nucleosomes, which resemble beads on a string. These units and other scaffold proteins together make a structure called chromatin and establish the overall shape of the chromosome. However, it is not exactly clear how the nucleosomes and scaffold proteins are rearranged during condensation. Kruitwagen et al. used microscopy to study chromosome condensation in budding yeast. The experiments reveal that condensation involves two separate processes. First, modifications to the nucleosomes result in these units becoming more tightly packed in a process called short-range compaction. Second, a group of proteins called condensin is responsible for rearranging the compacted chromatin to enforce higher-order structure on the arms of the condensed chromosome (long-range contraction). Further experiments suggest that an enzyme called Hst2 may help to co-ordinate these processes to ensure that chromosomes adopt the right shape before the cell divides. For example, Hst2 ensures that longer chromosomes condense more than shorter ones. A future challenge will be to find out whether chromosome condensation works in a similar way in humans and other large eukaryotes, which form much larger chromosomes with more complicated structures than yeast. https://doi.org/10.7554/eLife.10396.002 Introduction The DNA molecule at the core of any eukaryotic chromosome is a hundred to million times longer than the average diameter of the cell that hosts it. Thus, cells need to fold their genetic material in order to fit it in the interphase nucleus; they need to pack it further during mitosis, in order to move sister-chromatids safely and symmetrically apart. Furthermore, chromatin folding must be dynamic to allow transcription and replication during interphase, and such that exceptionally large chromosomes can hyper-condense during anaphase in order to fit the size of the spindle and prevent chromosome missegregation (Neurohr et al., 2011; Titos et al., 2014). Moreover, mitotic condensation also facilitates the decatenation of sister chromatids during their separation (Charbin et al., 2014), and might help to ‘cleanse’ chromosomes from transcription, replication and cohesion factors (Yanagida, 2009). This is thought to ‘reset’ the transcriptional state of genes, and prevent displaced factors from interfering with chromosome segregation. However, despite their importance for chromosome segregation, the events ensuring the mitotic condensation of chromosomes are still only partially understood. Early studies made evident that nucleosomes play a critical role in DNA packaging. In favor of the idea that they play specific roles in chromatin condensation, histone H3 is phosphorylated by aurora B throughout mitosis on a serine at position 10 in most, if not all, eukaryotes. Furthermore, aurora B inactivation leads to chromosome condensation and segregation defects in budding yeast (Lavoie et al., 2004), fission yeast (Petrova et al., 2013; Tada et al., 2011), HeLa cells (Tada et al., 2011) and roundworms (Hagstrom et al., 2002). However, the precise role of H3 S10 phosphorylation has remained unclear (Ajiro and Nishimoto, 1985). Recent data demonstrated that H3 S10 phosphorylation promotes the recruitment of the sirtuin-related deacetylase Hst2, which in turn deacetylates, at least, lysine 16 of histone H4 (Wilkins et al., 2014). This unmasks a basic patch, allowing H4 to interact with the acidic patch on H2A, most probably on an adjacent nucleosome (Robinson et al., 2008; Gordon et al., 2005). Thus, this cascade of events initiated by H3 phosphorylation is thought to tighten the interaction between neighboring nucleosomes. However, all studies carried out so far have failed to reveal strong phenotypes for H3 serine 10 to alanine mutations in a plethora of model organisms (de la Barre et al., 2001; Afonso et al., 2014; Ditchfield et al., 2003). Furthermore, mutation of this residue in budding yeast did not affect axial contraction of chromosomes and the condensation of the rDNA during regular mitoses (Neurohr et al., 2011; Lavoie et al., 2004; Lavoie et al., 2002). Indeed, the only phenotype identified upon replacement of H3 S10 with alanine in yeast so far is limited to the reduced ability to hyper-condense artificially long chromosomes in order to fit them in the spindle (Neurohr et al., 2011). Thus, it remains unclear whether H3 phosphorylation and H4 deacetylation play any general role in mitotic chromosome condensation. The discovery that mitotic extracts of frog eggs lacking any one of the subunits of a protein complex called condensin largely failed to condense chromosomes (Hirano and Mitchison, 1994) opened new perspectives for understanding chromosome condensation (Piazza et al., 2013; Thadani et al., 2012). Condensin is a ring-shaped pentameric protein complex. The core of the ring is formed by two structural maintenance of chromosome (SMC) subunits, Smc2 and Smc4. Three non-SMC proteins (Brn1, Ycg1 and Ycs4 in budding yeast) close the ring. The mechanism of condensin loading on chromatin is not understood, but seems to depend on the activity of the kinase aurora B (Lavoie et al., 2004; Tada et al., 2011). Furthermore, the non-SMC subunits were recently shown to directly bind DNA (Piazza et al., 2014), potentially followed by topological entrapment of chromatin inside the condensin ring (Cuylen et al., 2011). How condensin performs its functions in chromosome condensation is unclear, but it has been proposed that condensin’s role might be structural, by inducing loops within the same DNA strand (Cuylen et al., 2011; Cuylen and Haering, 2011) or might be enzymatic by promoting positive DNA supercoiling (Baxter and Aragón, 2012), both assisting in a decrease in length of mitotic chromatids. Mitigating the central role of condensin in chromosome condensation, however, were observations in model organisms as diverse as fission yeast, fly, chicken and mammalian cells that indicate that chromosomes can still, at least partially, condense in the absence of condensin (Petrova et al., 2013; Coelho et al., 2003; Vagnarelli et al., 2006; Gerlich et al., 2006). Thus, although condensin was established as a key player in chromosome condensation, it cannot be the sole factor shaping mitotic chromosomes. In order to gather insights into whether and how H2A-H4 interaction contributes to the organization of mitotic chromosomes, we sought for a method to assay the condensation state of chromatin in vivo. Here, we use a fluorescence-based assay to investigate short-range chromatin compaction and use it to study the relationships between condensin and histone modifications during chromosome condensation in mitotic cells. Results A microscopy-based assay to measure chromatin fiber compaction In order to develop a chromatin condensation assay, we reasoned that increased nucleosome-nucleosome interaction might render chromatin less accessible to DNA-binding proteins. To test this idea directly, we asked whether chromatin condensation restricted access for heterologous reporter proteins to their binding sites when those are introduced at a chosen chromosomal locus. Therefore, we used a yeast strain in which a set of Tet operator (TetO) repeats are inserted at the TRP1 locus on chromosome IV, 15 kb from CEN4, and constitutively expressing the TetR-mCherry fusion protein, which efficiently binds the TetO repeat. As a consequence, these cells exhibit a red dot in their nucleus throughout the cell cycle (Figure 1A). To test whether the intensity of TetR-mCherry fluorescence possibly varied over the cell cycle, we measured the fluorescence intensity of this dot in G1 cells (unbudded), when the chromatid is decondensed, but not replicated yet, and in late anaphase mother cells, when the chromatid is separated from its sister and has reached full condensation (Figures 1B and 3C; (Neurohr et al., 2011; Sullivan et al., 2004; D'Amours et al., 2004) and see below). After subtracting background fluorescence, we noticed a highly significant (p<0.0001), 2–2.5-fold decrease in mCherry fluorescence intensity at the TetO repeats on the anaphase compared with the G1-phase chromosomes (Figure 1A, B). Figure 1 Download asset Open asset Fluorescence intensity of TetO/TetR-mCherry as a read-out for chromatin compaction. (A) Representative images of a cell in G1 and anaphase, containing a TetO array at the TRP1 locus and expressing TetR-mCherry (red). Fluorescence intensity of a focus is measured by determining the total fluorescence and subtracting the background, giving the corrected fluorescence intensity. Scale bar is 2 µm. (B) TetR-mCherry intensities for the indicated wild type (WT) and mutant strains in G1 and anaphase mother cells. One way Analysis Of Variance ( ANOVA) was performed to test significance. (C) Fluorescence intensity for a wild type strain containing LYS4:LacO and expressing LacI-GFP. Student’s t-test was performed to determine significance. (D) Anaphase TetR-mCherry intensities for the indicated strains, synchronized in G1 by alpha-factor treatment and released at the indicated temperatures. Intensities for G1 were determined 5 min after release from alpha-factor induced arrest. All data are means and standard deviation for n>30 cells. **** p<0.0001 and n.s. not significant. https://doi.org/10.7554/eLife.10396.003 We next asked whether the variations of fluorescence intensity at the TetO repeats reflected changes in H2A/H4 interaction. Supporting this view, mutating key residues in the H3 phosphorylation and H4 deacetylation pathway established by (Wilkins et al., 2014) affected these variations (Figure 1B,C). Strikingly, mutations that abrogate the mitotic interaction between H2A and H4, such as H3 S10A, hst2∆ and the H4 ∆9–16 mutations, all abolished the reduction in brightness of the TetR-mCherry focus normally observed in anaphase cells (Figure 1B). In reverse, mutations that promote constitutive H2A/H4 interaction, such as H3 S10D and H4 K16R, caused the TetR-mCherry focus to constitutively show, that is, even in G1 cells, the low fluorescence intensity normally specific of anaphase cells. The effect of the H3 S10D mutation was indeed mediated by the recruitment of Hst2, since the hst2Δ mutation suppressed it; the H3 S10D hst2∆ double mutant cells showed constitutive high brightness, similar to hst2∆ single mutant cells. Interestingly, however, introducing the H4 K16R mutation in the hst2∆ mutant cells did not restore the intensity drop normally observed during anaphase, suggesting that H4 K16 is not the sole residue that Hst2 deacetylates to promote nucleosome-nucleosome interaction (Figure 1B). To test whether the observed fluctuations in fluorescence intensity were specific for the TRP1 locus or TetO/TetR-mCherry, we also measured the fluorescence intensity at the LYS4 locus, in the middle of the right arm of chromosome IV, where we integrated LacO repeats in cells expressing LacI fused to Green Fluorescent Protein (GFP). Although the effect was slightly less pronounced, we observed a similar, and significant, decrease in reporter brightness in anaphase compared with G1 cells at this locus (Figure 1C). As for the TRP1 locus, mutants in the H3 S10 pathway also affected fluctuations in intensity at the CEN distal locus: hst2∆, H3 S10A and H3S10D hst2∆ showed a continuously higher fluorescent intensity on the LacO repeats near the LYS4 gene and the H3 S10D mutation also resulted in a continuously lower fluorescent signal. These data indicated that changes in chromatin organization during mitosis indeed affected either the recruitment or the fluorescence intensity of TetR-mCherry and LacI-GFP on two distant chromatin loci, one close to the centromere and the second in the middle of the second longest yeast chromosome arm. Since chromatin condensation is regulated by the kinase aurora B (Ipl1 in budding yeast), we last asked whether Ipl1 activity is required for the intensity decrease of TetR-mCherry at the TRP1 locus in mitotic cells. We arrested wild type yeast cells and cells containing the temperature sensitive ipl1-321 allele in G1 with alpha-factor, released them at the restrictive temperature of 35ºC and determined TetO/TetR-mCherry fluorescence intensity in the same G1 or following anaphase (Figure 1D). Whereas wild type cells showed no significant difference in G1 and anaphase TetO/TetR-mCherry fluorescence intensity, the ipl1-321 strain showed a significantly brighter dot when undergoing anaphase at the restrictive temperature. Compaction in G1 of ipl1-321 cells at the restrictive temperature was not affected, presumably due to the fact that this protein has no activity in G1, even in wild type cells (Buvelot et al., 2003). Thus, we conclude that the enhanced H2A-H4 interaction triggered by aurora B-dependent recruitment of the deacetylase Hst2 onto chromatin indeed affects the intensity of the TetR-mCherry signal on the chromosome. Fluorophore concentration quenching causes fluctuations in brightness We next wanted to better understand the molecular processes and structural changes of chromatin that were underlying the fluorescence variation at the TetO array over the cell cycle. Assuming enhanced nucleosome-nucleosome interaction promotes chromatin compaction, three models may explain the observed decrease of fluorescence in mitosis. First, chromatin compaction might reduce access of DNA-binding proteins, such as TetR-mCherry, to their binding site on DNA and cause their removal, as postulated by the chromosome cleansing hypothesis. Second, chromatin compaction might increase the local packing of TetR-mCherry, leading to quenching of the fluorophore (Lakowicz, 2013); these two first models are depicted in Figure 2A. Third, the changed local environment of mitotic chromatin might reduce the intrinsic fluorescence of mCherry and GFP. Figure 2 Download asset Open asset Fluorophore quenching causes changes of TetO/TetR intensity over the cell cycle. (A) Two models to explain anaphase-specific decrease in fluorescence brightness (cleansing and quenching, see text for explanations). Shown are the consequences of each model in G1 and anaphase, in the case of cells carrying TRP1:TetOs and either expressing only TetR-mCherry or TetR-mCherry and TetR-GFP. (B) G1 and anaphase TetO/TetR-mCherry intensities in cells carrying TetO and expressing only TetR-mCherry (left) or TetR-mCherry and TetR-GFP (right). Data are means and standard deviations, unpaired Student’s t tests were performed to test significance, **** p<0.0001 and n.s. not significant. (C) FRET values for indicated strains. Plotted are mean values and standard deviation. Unpaired Student’s t tests were performed to test significance, ** p<0.01 and n.s. not significant. https://doi.org/10.7554/eLife.10396.004 In order to better distinguish between these models, we rationalized that coexpressing TetR-GFP with TetR-mCherry would not protect mCherry from a cleansing effect (model 1) or a change in local environment (model 3), but should strongly reduce any quenching, due to intercalation of a second fluorophore with a different excitation spectrum. Furthermore, in this context, quenching might be replaced by Förster Resonance Energy Transfer (FRET) between the TetR-GFP and TetR-mCherry molecules. Remarkably, unlike the cells expressing only TetR-mCherry, cells expressing both versions of TetR failed to show significant variation of the fluorescence signal for either mCherry or GFP at the TetO array between anaphase and G1 (Figure 2B). Thus, cleansing and a general change in the local environment of the fluorophores are unlikely to explain the fluorescence drop observed at the TetO array during anaphase in the cells expressing solely TetR-mCherry. Supporting the idea that the intensity drop was due to a quenching effect, FRET was indeed observed upon exciting in the GFP and recording emission in the red channel in cells expressing both TetR-mCherry and TetR-GFP, but not in cells expressing TetR-mCherry alone (Figure 2C). Moreover, FRET was significantly increased during anaphase compared with G1-phase (Figure 2B,C), indicating that the fluorophores are indeed brought in closer proximity during anaphase compared with interphase. We conclude that increased H2A/H4 interaction results in a tighter packing of fluorophores and their quenching, establishing that H2A/H4 interaction leads to compaction of mitotic chromatin in vivo. Furthermore, cell cycle dependent changes of TetR-mCherry or TetR-GFP signals on TetO arrays is a reliable measure of short-range compaction of the underlying chromatin. Chromatin compaction precedes the axial shortening of chromosomes Next, we investigated the dynamics of chromatin compaction during the cell cycle. To this end, we visualized both the TetO/TetR-mCherry (at TRP1) and LacO/LacI-GFP (at LYS4) loci simultaneously.This presence of two labeled loci on the same chromosome allowed measuring the physical distanceseparating them and hence the long-range contraction of the chromosome arm along its longitudinal axis during anaphase. Using this strain, we first recorded time-lapse movies (Figure 3A) in which we measured the intensity of the LacI-GFP fluorescence at the LYS4 locus in cells progressing through mitosis (Figure 3B). Upon averaging the signal of at least 15 (t = -18 minutes) to maximum 31 (t = 0 minutes) such traces, we observed that the intensity of the signal was indeed lowest during the first 12 minutes of anaphase, while starting to increase as soon as the cells started to exit mitosis (Figure 3B, blue line indicates the formation of the first bud in the population). We also noticed that fluorescence intensity at the LacO locus was highly variable throughout every single movie, leading to high standard deviations. This variation was lowest during anaphase and started to increase as soon as chromatin was decondensing, consistent with the idea that chromatin is more constrained when it is most compacted and fluorophore quenching is highest. The source of this fluorescence variation is not known, but might reflect breathing movements of the underlying chromatin or complex photochemistry effects. In either case, this intrinsic cell-to-cell variability precludes drawing conclusions at the single cell level and emphasizes the fact that the quenching assay introduced here is statistical in nature. Figure 3 Download asset Open asset Dynamics of chromatin compaction and chromosome arm contraction. (A) Example of a cell going from metaphase to the next G1 phase with TRP1 and LYS4 loci marked with TetR-mCherry and LacI-GFP, respectively. (B) Background normalized, mean GFP-intensity values of mitotic cells, aligned at mid-anaphase (red dashed line: GFP dot split). Blue line indicates formation of the first bud. Standard deviations are shown. (C) Upper panel: normalized (to G1) intensity of TetR-mCherry and LacI-GFP foci in mother cells in the indicated cell cycle stages. Lower panel: mother TRP1:TetO - LYS4:LacO distances in indicated cell cycle stages. Shown are mean and standard deviation for n>30 cells. (D) Nuclear diameter of G1 and late anaphase cells in wild type and hst2∆ cells containing Nup170-GFP. Box shows median value, whiskers all data points n>50 cells. Scale bars are 2 µm. https://doi.org/10.7554/eLife.10396.005 Next, we wanted to determine whether the dynamics of chromatin compaction could be related to the contraction of the chromosome arm measured using the TRP1-LYS4 distance, as described previously by us and others (Neurohr et al., 2011; Petrova et al., 2013; Guacci et al., 1994; Vas et al., 2007). To avoid variations due to photobleaching, we used snapshot images of cells at precise and representative time points in mitosis (Figure 3C): metaphase (large buds but neither the TRP1 nor the LYS4 loci were separated), early anaphase (sister TRP1 loci – in red – are separated, but the two LYS4 loci are not), mid-anaphase (both loci have undergone separation but the LYS4 locus still lags behind), late anaphase (all loci are separated and moved to the opposite poles of the cell) and G1 (unbudded cell). For each of these stages (>25 cells each), we measured both the intensity of the fluorescence on the two arrays and the distance between them. In this study, we focused specifically on the loci segregated to the mother cell, as we showed before that mother and bud are not directly comparable (Neurohr et al., 2011). Analysis of this data set indicated that the two marked loci underwent compaction and decompaction with slightly different kinetics (Figure 3C, upper panel). During early anaphase, both the TetO and LacO array seemed to be compacted to some extent already. As cells progressed to mid anaphase, the CEN4 proximal TetO arrays seemed slightly more compacted than the distal LacO arrays. In late anaphase, the TetO array was already starting to unpack, whereas the LacO remained compacted. Both dots had recovered their full intensity in the G1 cells, demonstrating that the intensity decrease in anaphase was not solely the consequence of sister-chromatid separation, which is expected to reduce fluorescence intensity by half, down to its G1 level until the next S-phase. In the same cells, measuring the TetO-LacO distance (Figure 3C, lower panel) indicated that the chromosome first stretched out upon anaphase onset, to subsequently contract, reaching their shortest length in late anaphase, as reported (Guacci et al., 1994; Harrison et al., 2009). The TetO-LacO distance re-extended then to its steady state average in G1 cells. The changes in distance between the two loci could be due to changes in nuclear diameter, which would constrain the maximal distance that the loci can move apart by random motion. However, we did not observe any significant changes in nuclear diameter (as determined by GFP-tagging the nucleoporin Nup170) when comparing late anaphase and the G1 phase in wild type and hst2Δ cells (Figure 3D). Thus, the shortening of the TRP1-LYS4 distance in late anaphase cells truly reflects the effect of chromatin condensation by axial contraction of the chromosomes. Furthermore, our results established that short-range chromatin compaction was not strictly concomitant with long-range axial contraction of the chromosome, but rather preceded it. Condensin is not involved in local chromatin compaction These observations suggested that chromatin compaction and axial contraction of mitotic chromosomes might be distinct processes. Thus, we asked whether condensin, which is essential for axial chromosome contraction, contributed to short-range compaction of chromatin. We analyzed the brightness of the TetR-mCherry focus in yeast cells carrying the smc2-8 allele, a temperature sensitive mutation in the condensin subunit Smc2 (Figure 4A). Remarkably, condensin inactivation for 90 min at the restrictive temperature had no effect on the changes in mCherry brightness between the anaphase and G1-phase of the cell cycle, whereas it indeed abrogated shortening of the TetO-LacO distance during anaphase (see below). We therefore conclude that condensin, unlike histone 3 phosphorylation and histone 4 deacetylation, does not promote nucleosome-nucleosome interaction. To test this idea further, we directly probed H2A/H4 interaction by using genetically encoded Ultraviolet (UV) inducible crosslinking (Wilkins et al., 2014). We arrested cells in G1 with alpha-factor and released them in the presence of nocadozole under wild type and smc2-8 conditions at 37ºC. Fluorescence-Activated Cell Sorting (FACS) analysis showed that the release and arrest was equally efficient in both cells (Figure 4—figure supplement 1). In wild type cells, as reported before, H4/H2A crosslinking is observed in mitosis and correlated strongly with H4 K16 deacetylation (Figure 4B). In fitting with the microscopy data (Figure 4A), the crosslinking between H4 and H2A showed no difference in kinetics in the condensin inactivated and in the wild type cells (Figure 4B). Thus, condensin function is not required for proper, short-range compaction of mitotic chromatin. Figure 4 with 1 supplement see all Download asset Open asset Condensin does not impact chromatin compaction. (A) TetR-mCherry intensities in the mother cell for the indicated strains and cell cycle stages. To inactivate Smc2, cells were shifted to 37ºC for 90 min. One way ANOVA was performed to test significance, **** p<0.0001 and n>40. (B) Yeast cells producing H2A Y58BPA were synchronized with alpha-factor at permissive temperature and then released into medium containing nocodazole at restrictive temperature. Samples were taken at indicated times, irradiated with UV and histones extracted with acid from isolated nuclei. Western blot against H4 detects the H2A-H4 crosslink (upper row), bulk H4 (lower row) and blotting against H4 K16Ac shows cell cycle progression. https://doi.org/10.7554/eLife.10396.006 The chromatin compaction pathway does not contribute to axial contraction of chromosomes In order to investigate in more detail how the phosphorylation of H3 S10 and the subsequent activation of Hst2 contributed to chromosome condensation, we next characterized if these events promoted axial contraction of chromosome IV, using the LacO-TetO distance as a readout (see Figure 3D). Confirming the role of aurora B/Ipl1 in chromosome condensation, the ipl1-321 mutant cells failed to undergo chromosome contraction when shifted to the restrictive temperature prior to mitosis, compared with wild type cells at these temperatures (Figure 5A). As expected from previous studies (Neurohr et al., 2011; Lavoie et al., 2002), the function of Ipl1 in the contraction of regular chromosomes was unlikely to require H3 S10 phosphorylation and H2/H4 interaction, since the mutations H3 S10A and H4 Δ9–16 did not impair anaphase contraction (Figure 5B,C ). Thus, Ipl1 promotes the axial contraction of chromosomes independently of phosphorylating H3 S10 and of promoting H4/H2A interaction, but possibly by promoting condensin function (see discussion). Figure 5 Download asset Open asset Chromatin compaction does not influence axial chromosome contraction. (A) TRP1-LYS4 distances for the indicated strains, synchronized in G1 by alpha-factor treatment and released at the indicated temperatures. (B) TRP1-LYS4 distances were determined in the mother cell for the indicated strains and cell cycle stages. Box shows median value, whiskers all data points n>30 cells. One way ANOVA was performed to test significances, ** p<0.01, **** p<0.0001, n.s. not significant. (C) Example cells containing the indicated mutations and their impact on chromosome length as determined by the TRP1 (red) to LYS4 (green) distance. (D) TRP1-LYS4 distances were determined for the indicated strains in anaphase. Box shows median value, whiskers all data points n>45. One way ANOVA was performed to test significances, *** p<0.001, * p<0.05, n.s. not significant. https://doi.org/10.7554/eLife.10396.008 In contrast, the hst2∆ mutation did abrogate the proper contraction of the chromosome during mitosis, implying that Hst2 acts in both axial chromosome contraction and short-range chromatin compaction (Figures 5C and 1B). Even more remarkably, the H3 S10D phospho-mimicking allele caused chromosome IV to remain in a constitutive state of axial contraction throughout the cell cycle (Figure 5B,C). This most probably reflected constitutive rec
Exploiting an expanded genetic code in yeast, we can site‐specifically encode unnatural amino acids into the native chromatin landscape of the living cell. Utilizing the UV‐activated photo‐crosslinking amino acid, p‐benzoylphenylalanine (pBPA), we can covalently trap protein‐protein interactions when contacts between proteins are made within binding distances of ~ 0.4 nm. We have mapped the RSC remodeler complex protein, Sth1, to the nucleosome and shown that its binding is regulated by posttranslational modifications, including acetylation and SUMOylation. RSC has been linked to promoter sites and we want to further our binding map with an understanding of its occupancy across the genome. To map the loci of binding sites we use a double IP chromatin immunoprecipitation (ChIP) technique that utilizes the power of in vivo protein‐protein crosslinking from histones. Histones are well associated with DNA and efficient targets for ChIP analysis; therefore, we propose that if the histone protein is first crosslinked to a nucleosomal protein target, that the histone can be initially precipitated in tandem with its crosslinked protein and associated DNA fragments. The target protein can then be further precipitated to purify DNA, via its interaction with the histone. Our first goal was to establish histone occupancy of pBPA‐containing histones. Using ChIP, we were able to isolate DNA from both wild type and mutant histones where we can achieve a quantitate analysis of the difference with qPCR. Building on this we will move to a double IP system to determine Sth1 occupancy. This work aims to establish a novel approach in ChIP targeting of proteins that are difficult to analyze due to more transient interactions at the nucleosomal core. Support or Funding Information NIH R15 grant
Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis.
Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.
