Chromosome structure is complex, and many aspects of chromosome organization are still not understood. Measuring the stiffness of chromosomes offers valuable insight into their structural properties. In this study, we analyzed the stiffness of chromosomes from metaphase I (MI) and metaphase II (MII) oocytes. Our results revealed a tenfold increase in stiffness (Young’s modulus) of MI chromosomes compared to somatic chromosomes. Furthermore, the stiffness of MII chromosomes was found to be lower than that of MI chromosomes. We examined the role of meiosis-specific cohesin complexes in regulating chromosome stiffness. Surprisingly, the stiffness of chromosomes from three meiosis-specific cohesin mutants did not significantly differ from that of wild-type chromosomes, indicating that these cohesins may not be primary determinants of chromosome stiffness. Additionally, our findings revealed an age-related increase of chromosome stiffness for MI oocytes. Since aging is associated with elevated levels of DNA damage, we investigated the impact of etoposide-induced DNA damage on chromosome stiffness and found that it led to a reduction in stiffness in MI oocytes. Overall, our study underscores the dynamic and cyclical nature of chromosome stiffness, modulated by both the cell cycle and age-related factors.

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin and bound by the sequence specific binding protein CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromere's stiffness and the role of the CCAN. We degraded CENP-C and CENP-N using auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching approximately 3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.

Molecular crowding influences DNA mechanics and DNA-protein interactions and is ubiquitous in living cells. Quantifying the effects of molecular crowding on DNA supercoiling is essential to relating in vitro experiments to in vivo DNA supercoiling. We use single-molecule magnetic tweezers to study DNA supercoiling in the presence of dehydrating or crowding cosolutes. To study DNA supercoiling, we apply a stretching force of 0.8 pN to the DNA and then rotate one end of the DNA to induce supercoiling. In a 200 mM NaCl buffer without cosolutes, negatively supercoiled DNA absorbs some of the tortional stress by forming locally melted DNA regions. The base pairs in these locally melted regions are believed to adopt a configuration where nucleotide base pairing is disrupted. We find that the presence of a dehydrating cosolute such as glycerol further destabilizes base pairs in negatively supercoiled DNA. The presence of polyethylene glycol, commonly used as a crowding agent, suppresses local strand separation and results in plectoneme formation even when DNA is negatively supercoiled. The results presented in this letter suggest further directions for studies of DNA supercoiling and supercoiled DNA-protein interactions in molecular conditions that approximate in vivo molecular composition.

Spindles are essential for accurate chromosome segregation in all eukaryotic cells. This study presents a novel approach for isolating fresh mammalian spindles from mouse oocytes, establishing it as a valuable in vitro model system for a wide range of possible studies. Our method enables the investigation of the physical properties and migration force of meiotic spindles in oocytes. We found that the spindle length decreases upon isolation from the oocyte. Combining this observation with direct measurements of spindle mechanics, we examined the forces governing spindle migration during oocyte asymmetric division. Our findings suggest that spindle migration is regulated by a pulling force and a net tensile force of approximately 680 pN is applied to the spindle in vivo during the migration process. This method, unveiling insights into spindle dynamics, holds promise as a robust model for future investigations into spindle formation and chromosome separation. We also found that the same approach could not isolate spindles from somatic cells, indicative of mammalian oocytes having a unique spindle organization amenable to isolation.

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.

The highly and slightly condensed forms of chromatin, heterochromatin and euchromatin, respectively, segregate in the cell nucleus. Heterochromatin is more abundant in the nucleus periphery. Here we study the mechanism of heterochromatin segregation by modeling interphase chromosomes as diblock ring copolymers confined in a rigid spherical shell using molecular dynamics simulations. In our model, heterochromatin and euchromatin are distinguished by their bending stiffnesses only, while an interaction potential between the spherical shell and chromatin is used to model lamin-associated proteins. Our simulations indicate that in the absence of attractive interactions between the nuclear shell and the chromatin, most heterochromatin segregates towards the nuclear interior due to the depletion of less flexible heterochromatin segments from the nuclear periphery. This inverted chromatin distribution,which is opposite to the conventional case with heterochromatin dominating at the periphery, is in accord with experimental observations in rod cells. This "inversion" is also found to be independent of the heterochromatin concentration and chromosome number. The chromatin distribution at the periphery found in vivo can be recovered by further increasing the bending stiffness of heterochromatin segments or by turning on attractive interactions between the nuclear shell and heterochromatin. Our results indicate that the bending stiffness of chromatin could be a contributor to chromosome organization along with differential effects of HP1α-driven phase segregation and of loop extruders and interactions with the nuclear envelope and topological constraints.

ABSTRACTStructural Maintenance of Chromosomes (SMC) complexes play essential roles in genome organization across all domains of life. To determine how the activities of these large ( 50 nm) complexes are controlled by ATP binding and hydrolysis, we developed a molecular dynamics model that accounts for conformational motions of the SMC and DNA. The model combines DNA loop capture with an ATP-induced “power stroke” to translocate the SMC complex along DNA. This process is sensitive to DNA tension: at low tension (0.1 pN), the model makes loop-capture steps of average 60 nm and up to 200 nm along DNA (larger than the complex itself), while at higher tension, a distinct inchworm-like translocation mode appears. By tethering DNA to an experimentally-observed additional binding site (“safety belt”), the model SMC complex can perform loop extrusion (LE). The dependence of LE on DNA tension is distinct for fixed DNA tension vs. when fixed DNA end points: LE reversal occurs above 0.5 pN for fixed tension, while LE stalling without reversal occurs at about 2 pN for fixed end points. Our model matches recent experimental results for condensin and cohesin, and makes testable predictions for how specific structural variations affect SMC function.

Intro Force measurements of the nucleus, the strongest organelle, have propelled the field of mechanobiology to understand the basic mechanical components of the nucleus and how these components properly support nuclear morphology and function. Micromanipulation force measurement provides separation of the relative roles of nuclear mechanical components chromatin and lamin A. Methods To provide access to this technique, we have developed a universal micromanipulation apparatus for inverted microscopes. We outline how to engineer and utilize this apparatus through dual micromanipulators, fashion and calibrate micropipettes, and flow systems to isolate a nucleus and provide force vs. extensions measurements. This force measurement approach provides the unique ability to measure the separate contributions of chromatin at short extensions and lamin A strain stiffening at long extensions. We then investigated the apparatus' controllable and programmable micromanipulators through compression, isolation, and extension in conjunction with fluorescence to develop new assays for nuclear mechanobiology. Results Using this methodology, we provide the first rebuilding of the micromanipulation setup outside of its lab of origin and recapitulate many key findings including spring constant of the nucleus and strain stiffening across many cell types. Furthermore, we have developed new micromanipulation-based techniques to compress nuclei inducing nuclear deformation and/or rupture, track nuclear shape post-isolation, and fluorescence imaging during micromanipulation force measurements. Conclusion We provide the workflow to build and use a micromanipulation apparatus with any inverted microscope to perform nucleus isolation, force measurements, and various other biophysical techniques.

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1α I165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.

Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.

While there is a prevalent genome organization in eukaryotic cells, with heterochromatin concentrated at the nuclear periphery, anomalous cases do occur. Deviations of chromatin distribution are frequent, for example, upon aging, under malignant diseases, or even naturally in rod cells of nocturnal mammals. Using molecular dynamic simulations, we study the segregation of heterochromatin in the cell nucleus by modeling interphase chromosomes as diblock ring copolymers confined in a rigid spherical shell. In our model, heterochromatin and euchromatin are distinguished by their bending stiffnesses, while an interaction potential between the spherical shell and chromatin is used as a proxy for lamin-associated proteins. Our simulations indicate that in the absence of attractive interactions between the nuclear shell and the chromatin, the majority of heterochromatin segregates towards the nuclear interior due to depletion of less flexible heterochromatin segments from the nuclear periphery. This inverted chromatin distribution is in accord with experimental observations in rod cells. This “inversion” is also found to be independent of the heterochromatin concentration and chromosome number, and is further enhanced by additional attractive interactions between heterochromatin segments. as well as by allowing bond-crossing to emulate topoisomerase activity. The usual chromatin distribution, with heterochromatin at the periphery, can be recovered by further increasing the bending stiffness of heterochromatin segments or by turning on attractive interactions between the nuclear shell and heterochromatin. Overall, our results indicate that bending stiffness of chromatin could be a contributor to chromosome organization along with differential effects of HP1 α -driven phase segregation and of loop extruders, and interactions with the nuclear envelope and topological constraints.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Loop extrusion by structural maintenance of chromosomes (SMC) complexes has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, whereas cohesin extrudes loops symmetrically in interphase. Our data show that loop extrusion is a general mechanism underlying DNA organization, with dynamic and structural properties that are biochemically regulated during the cell cycle. Introduction Chromatin undergoes a dramatic reorganization during the cell cycle (Hirano and Mitchison, 1991; Rowley and Corces, 2018; Nagano et al., 2017). In interphase, chromatin is organized into compartments and topological-associating domains (TADs) that are cell-type specific (Bonev and Cavalli, 2016; Dekker and Mirny, 2016; Rao et al., 2014). TADs are composed of chromatin loops that have been hypothesized to regulate gene expression by spatially restricting contacts between genes and regulatory elements (Smith et al., 2016; Lupiáñez et al., 2015; Ren et al., 2017; Schoenfelder and Fraser, 2019). In metaphase, chromosomes undergo large-scale compaction, leading to the loss of specific boundaries and the shutdown of transcription, which is achieved by arranging chromatin into an array of condensed loops (Marsden and Laemmli, 1979; Earnshaw and Laemmli, 1983; Naumova et al., 2013; Goloborodko et al., 2016; Uhlmann, 2016; Kinoshita and Hirano, 2017). These different degrees of organization require the coordinated activity of protein complexes such as structural maintenance of chromosomes (SMCs) proteins (Nasmyth, 2001; Yatskevich et al., 2019; Fudenberg et al., 2016; Nuebler et al., 2018; Hirano et al., 1997; Bouwman and de Laat, 2015), but how these complexes organize chromatin dynamically during the cell cycle is still unknown. SMCs are thought to organize DNA by actively extruding DNA loops (Rao et al., 2014; Fudenberg et al., 2016; Alipour and Marko, 2012; Sanborn et al., 2015). Recent experimental studies have shown that yeast condensin extrudes DNA loops in a one-sided manner in vitro (Ganji et al., 2018). Although consistent with the loop-extrusion hypothesis, it is inconsistent with the requirement for two-sided loop extrusion predicted by theory (Banigan and Mirny, 2018; Banigan et al., 2019). One reason for this discrepancy could be that the properties of loop extrusion in cellular contexts differ from those in vitro and may be regulated during the cell cycle (Abramo et al., 2019; Losada et al., 1998). Notably, condensin complexes do not structure the genome during interphase (Abdennur, 2018), which raises intriguing questions about the molecular players that regulate DNA architecture in interphase. Recent in vitro work demonstrated that cohesin can extrude DNA loops symmetrically (Davidson et al., 2019; Kim et al., 2019), though this activity has not been directly visualized in cellular contexts (Rao et al., 2017; Schwarzer et al., 2017; Hansen et al., 2017). To bridge the gap between in vitro biochemical assays and physiological conditions, we used histone H3/H4-depleted Xenopus laevis egg extracts to reconstitute loop formation on single DNA molecules. These extracts can be cycled between metaphase and interphase and recapitulate many sub-cellular biological processes, such as the formation of mitotic chromatids and interphase nuclei (Hirano and Mitchison, 1991; Murray, 1991). Results To visualize DNA loop formation in Xenopus laevis egg extracts, we attached lambda-phage DNA to a cover slide using biotin-streptavidin linkers (Ganji et al., 2016) in custom-built microfluidic chambers (Figure 1A). Addition of either metaphase-arrested or interphase Xenopus egg extracts into the chamber triggered the formation of small DNA enrichments, consistent with nucleosomal deposition (Yan et al., 2007; Gruszka et al., 2019), that rapidly reduced any slack in the DNA molecules (Figure 1—figure supplement 1A and Figure 1—videos 1–2). To increase the amount of available slack to allow for loop extrusion, we abolished nucleosomal assembly along the strand by depleting ~90–95% of soluble H3-H4 heterodimers in the extract (Zierhut et al., 2014; Figure 1—figure supplement 1B). This led to the formation of compacted DNA clusters that grew in size over time in both metaphase and interphase (Figure 1B and Videos 1–2; Figure 1—videos 3–6). To investigate whether these clusters exhibited a topology consistent with DNA loops, we hydrodynamically stretched the DNA strand by applying a flow in the perpendicular direction to the strand. This procedure revealed DNA clusters with a characteristic loop topology in both inter- and metaphase extracts (Figure 1C, Figure 1—figure supplement 1C and Figure 2—video 1; Figure 1—videos 7–9). In mock-depleted extracts, loops also formed but at a much lower frequency (Figure 1—figure supplement 1D and Figure 1—video 10) and seemed to compete with nucleosomes for available DNA slack. These results show that DNA loop extrusion can be reconstituted in Xenopus egg extracts in metaphase and interphase. Figure 1 with 11 supplements see all Download asset Open asset Single DNA molecule assay for direct visualization of DNA looping in Xenopus egg extracts. (A) (i) Side and top view schematics of a single strand of λ-phage DNA attached to a functionalized cover slip via biotin-streptavidin linkers. (ii) Xenopus egg extract is flowed into the microfluidic chamber. (iii) Side and top view schematics visualizing how soluble active loop-extruding factors extrude loops in H3-H4-depleted extract. (B) Dynamics of the formation of DNA loops induced by H3-H4-depleted extract in metaphase (upper) and interphase (lower). Snapshot of a single molecule of λ-phage DNA visualized using Sytox Orange preceding treatment with H3-H4-depleted extract (left). Kymograph of DNA signal over time displaying a looping event upon addition of H3-H4-depleted extract (middle). Snapshot of steady-state DNA looping event after ~60 s (right). (C) Hydrodynamic flows reveal loop topology within DNA cluster. (i) Schematic of the loop topology revealed upon flow. (ii) Topology of extract-induced DNA loops in metaphase (upper) and interphase (lower) visualized using Sytox Orange revealed upon flow in the direction of the arrow. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Example of DNA loop formation in H3-H4-depleted egg extract arrested in metaphase visualized using Sytox Orange. The movie duration is 87 s and the scale bar is 5 μm. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Example of DNA loop formation in H3-H4-depleted egg extract in interphase visualized using Sytox Orange. The movie duration is 80 s and the scale bar is 2 μm. To characterize the dynamic properties of loop formation in Xenopus egg extracts, we quantified the DNA distribution inside the loop and to the left and right of the loop as a function of time (Figure 2). We computed the loop extrusion rate from the DNA amount that entered the loop over time, and could determine whether this DNA came from one or both of the non-looped regions. Briefly, we summed the fluorescence intensity of the DNA along the perpendicular direction to the DNA strand, and tracked the loop position defined by the local maximum of the DNA intensity. We then fitted a Gaussian function to the loop region and defined the loop boundaries as ±2 standard deviations away from the maximum value of the fit (Figure 2A). We obtained the amount of DNA inside the loop as the difference between the integrated intensity in the loop region minus the offset intensity from the Gaussian fit. Finally, the amount of DNA to the left and right of the loop corresponded to the integrated intensity of the DNA strands outside the loop region (see Supplementary Methods). This assay allows us to observe loop extrusion in extract, to quantify the partitioning of DNA between the looped and non-looped regions, and to examine the symmetry of the underlying DNA extrusion process. Figure 2 with 7 supplements see all Download asset Open asset Symmetry of DNA loop extrusion is cell cycle-dependent with similar extrusion rates and stalling forces. (A) Method to track DNA-loop dynamics through space and time. Upper: Schematic of the top view of a DNA-looping event segmented into three regions: region I (orange), region II (green), and the loop region (blue). Middle: Snapshot of DNA-looping event where DNA is labelled using Sytox Orange. Bottom: The integrated fluorescence intensity of the DNA generated by summing the intensity values along the perpendicular axis of the strand. The dashed red line represents a Gaussian fit to the data. Signal values above the fit’s offset define the looped region given in blue; signal values below this threshold correspond to the non-looped regions I and II, given in orange and in green. To convert the signal into DNA length, the integrated intensity of each region is divided by the total summed intensity of the DNA strand and multiplied by the total length of λ-phage DNA (48.5 kb). (B) Dynamics of DNA looping in H3-H4-depleted extract in metaphase and interphase. (Bi,iv left) DNA amount as a function of time computed for the looped region (blue) and non-looped regions I and II (green and orange). The dots represent experimental data and the solid lines represent exponential fits to the data. (Bi,iv right) The redistribution of DNA during the looping events shown in the left panel quantified as the change in DNA content in each region. (Bii,v) Change in the amount of DNA in the looped region and non-loop regions I and II (as in Bi,iv right) for the entire population of meta- and interphase looping events plotted as a function of the initial relative DNA extension of the corresponding molecules. Error bars correspond to standard deviations of data clustered by proximity. Points represent raw data. (Biii, vi) Analysis of loop extrusion symmetry shows predominantly non-symmetric extrusion (symmetry score ~1) in metaphase (Biii) and symmetric extrusion (symmetry score ~0) in interphase (Bvi). (C) Initial growth rates of DNA loop extrusion in metaphase (red) and interphase (blue) as a function of initial relative DNA extension. These rates were obtained from the slopes of the exponential fits to the loop data at time t = 0 for the subset of loop extrusion events that allowed for a fitting that converged within a tolerance (10−8relative change of the cost function), that corresponded to N = 21 out of 30 in interphase, and N = 24 out of 34 for metaphase. Error bars were obtained from error propagation of the uncertainties of the exponential fit parameters. (D) Box plots of the stall forces for DNA loop extrusion in metaphase and interphase obtained from the final relative extension of the DNA strand at the end of loop formation. When applied to metaphase-arrested, H3-H4-depleted extract (n=7 extract days), this assay showed that DNA loops are initially extruded at 2.36 ± 0.35 kb/s (mean ± SEM) (Figure 2Bi, C; Figure 2—figure supplement 1A). However, loop growth rapidly slowed down as more DNA was pulled into the looped region (Figure 2—figure supplement 2A), suggesting that extrusion rates depend on DNA tension. To examine the relationship between loop extrusion and DNA tension, we used the worm-like chain model (Marko and Siggia, 1995) that relates the relative extension of the DNA outside the loop to the corresponding force on the DNA strand (see supplementary methods, Figure 2—figure supplement 2 and Figure 2—figure supplement 26). The relative extension outside the loop is a dynamic quantity defined as: RE(t)=L/(CLλ-DNAloop(t)), where L represents the end-to-end binding distance of the DNA molecule on the coverslip, CLλ is the contour length of lambda-phage DNA, and DNAloop is the amount of DNA in the looped region. These results show that the relationship between extrusion rates and DNA tension is generally conserved for all looping events (Figure 2—figure supplement 2F). Finally, loop extrusion stopped when the relative extension of DNA outside of the loop reached on average ~65%, corresponding to a stall force of 0.16 pN ± 0.01 pN (mean ± SEM), (Figure 2D). In rare cases, we observed individual looping events stalling at DNA extensions of up to ~85%, corresponding to forces up to ~1 pN (Figure 2—figure supplement 2C–5C). To characterize the extrusion symmetry in metaphase, we quantified the total decrease in DNA from the left and right regions of the loop between the onset of loop formation and the final steady-state size of the loop (Figure 2Bii). We used these quantities to define a symmetry score as the relative difference between the decrease of these two regions and the total amount of DNA extruded (supplementary methods). The majority of metaphase looping events had a symmetry score close to 1, which corresponds to one-sided (non-symmetric) loop extrusion (Figure 2Biii). However, a small population of ~20% of all metaphase looping events were two-sided (as defined by a symmetry score of less than 0.5, Figure 2—figure supplement 3A). To complement these symmetry score results, we tracked loop movement along the DNA strand (Figure 2—figure supplement 4). Consistent with one-sided loop extrusion, loops that were displaced during loop formation, moved with equal probability towards the boundaries or the center of the strand. This behavior is suggestive of non-symmetric DNA extruding factors landing in a random orientation on the DNA molecule. Taken together, our analysis demonstrates that DNA loop extrusion in metaphase is predominantly one-sided, with extrusion speeds and stall forces similar to those measured in vitro (Ganji et al., 2018; Strick et al., 2004; Kong et al., 2019). Next, we used interphase H3-H4-depleted extract (n = 8 extract days) to investigate whether the dynamics of loop extrusion share similar properties throughout the cell cycle (Figure 2 Civ-vi). Loop extrusion in interphase displayed a similar distribution of extrusion rates, with a mean of 1.94 ± 0.26 kb/s, and average stall forces of 0.18 pN ±0.03 pN, with maximal forces of up to 0.82 pN (Figure 2C and D, Figure 2—figure supplement 2F). However, the distribution of symmetry scores of these looping events peaked towards zero, indicating that these loops are symmetrically extruded. Similar to metaphase looping, we observed that a sub-population of ~20% of all loops had the opposite symmetry (symmetry score larger than 0.5, Figure 2—figure supplement 3B). As predicted for symmetric extrusion, loops that started off-center on the strand displayed a strong bias to move towards the DNA boundary of the shorter DNA portion (Figure 2—figure supplement 4). Thus, we conclude that the mechanisms of DNA loop extrusion are different in interphase and metaphase. The different dynamic properties of DNA loop formation that we observe in interphase and metaphase suggest that different molecular activities may be responsible for loop formation during the cell cycle (Dekker and Mirny, 2016; Losada et al., 1998). The cell cycle-dependent activities of condensin and cohesin could account for the transition between symmetric and non-symmetric loop extrusion (Banigan et al., 2019; Abramo et al., 2019). To assess the role of cohesin and condensin during loop extrusion in interphase and metaphase, we selectively depleted these protein complexes in Xenopus egg extract. We used custom-made antibodies against XSMC1 and XRad21 for cohesin, and XCAP-C and XCAP-E (SMC2 and SMC4) for simultaneous depletion of condensin I and II (Figure 3—figure supplement 1). We then tested for loop extrusion activity in each depleted condition. We found that, in metaphase (n = 3 extract days), the occurrence of DNA loop extrusion was significantly reduced (p<0.01) upon depletion of condensin I and II but was unaffected by cohesin depletion (Figure 3A). In contrast, there was a significant decrease (p<0.01) in loop extrusion following cohesin depletion in interphase (n = 3 extract days), but was unaffected by condensin depletion (Figure 3A). We confirmed these depletions with immunostainings that showed colocalization of cohesin and condensin with the DNA loops observed in interphase and metaphase, respectively (Figure 3B). Additionally, we tested for ATPase activity of the loop extrusion factors by enzymatically depleting ATP using apyrase—which for technical reasons was limited to interphase in extract (see supplementary methods). When applied to interphase extract, apyrase-mediated ATP depletion resulted in a near-complete elimination of DNA looping activity , suggesting that cohesin actively extrudes loops in an ATP-dependent manner. Altogether, our results show that cohesin actively extrudes DNA loops symmetrically during interphase, whereas condensin extrudes DNA loops non-symmetrically in metaphase. This demonstrates that the molecular mechanisms of DNA loop extrusion are differentially regulated during the cell cycle. Figure 3 with 1 supplement see all Download asset Open asset Condensin extrudes DNA loops in metaphase and cohesin extrudes loops in interphase. (A) DNA loop extrusion probability—the frequency at which looping occurs on a DNA strand with sufficient slack—in metaphase and interphase under different depletion conditions. In metaphase, co-depleting condensin I, condensin II, and H3-H4 (using anti-XCAP-C/E and anti-H4K12Ac) significantly (** represents p<0.01, Binomial test) reduced loop extrusion probability, whereas the same depletion condition in interphase had no effect on loop extrusion probability compared to the control H3-H4-depleted extract. However, co-depleting cohesin and H3-H4 (using anti-XRAD21/XSMC1 and anti-H4K12Ac) had no effect in metaphase, though significantly (p<0.01) decreased loop extrusion probability in interphase compared to H3-H4-depleted extract. (B) Snapshots of antibody stainings of representative loops in metaphase and interphase. (Top) In metaphase, Alexa488-labeled anti-XRad21 bound to cohesin does not localize to the DNA loop, whereas in interphase (right panels), the anti-XRad21 co-localizes to the loop. (Bottom) Alexa488-labeled anti-XCAP-C bound to condensin localizes to the DNA loop in metaphase, but does not localize to the loop in interphase. Discussion Our findings provide the first direct evidence that loop extrusion is a general mechanism of DNA organization in a cellular context, and, furthermore, that it is differentially regulated during the cell cycle. This regulation is achieved by the distinct activities of cohesin (Rao et al., 2017; Schwarzer et al., 2017; Losada et al., 1998) and condensin (Kinoshita and Hirano, 2017; Hirano et al., 1997; Shintomi et al., 2017) during interphase and metaphase, and may control different levels of DNA organization during the cell cycle: from chromatin that is mostly decondensed and spatially organized into TAD structures during interphase to highly compacted chromosomes in metaphase (Abramo et al., 2019). Symmetric loop extrusion by cohesin in interphase may ensure the formation of specific TAD boundaries by bringing together distal CTCF sites (Sanborn et al., 2015; Tang et al., 2015). In metaphase, reorganization of loosely packed interphase chromatin into condensed chromosomes leads to the loss of TAD boundaries and the shutdown of transcription (Nagano et al., 2017; Naumova et al., 2013), which may be achieved by condensin activity (Goloborodko et al., 2016). However, many questions remain regarding how the cell cycle regulates condensin and cohesin activities. Previous studies have shown that condensin binds to chromatin in metaphase, but is largely undetected on chromatin in interphase (Hirano and Mitchison, 1994); whereas cohesin is bound to chromatin in interphase, but not as strongly in metaphase (Losada et al., 1998; Sumara et al., 2000). The CDK1-mediated phosphorylation of condensin HEAT subunits in metaphase may be the biochemical signal that triggers the association of condensin to chromatin (Hirano et al., 1997). In contrast, most cohesin is released from chromatin by a mechanism that involves the phosphorylation of cohesin’s SA subunit (Losada and Hirano, 2001; Hauf et al., 2005). Thus, the different affinities of condensin and cohesin for chromatin during the cell cycle could be a natural explanation for the different DNA loop extrusion activities that we see in our experiments. Our demonstration of predominantly non-symmetric DNA loop extrusion during metaphase is consistent with recent in vitro data, but it is at odds with the theoretical requirements to fully compact chromosomes in metaphase (Banigan and Mirny, 2018; Banigan et al., 2019). However, these studies suggest that a small fraction of two-sided loop extruders—including extrusion events that reel in DNA at different rates from left and right—can facilitate higher levels of chromosome compaction. Our metaphase data suggest that loops with symmetry scores below 0.8 could be considered ‘slow’ two-sided extrusion events, as DNA is reeled into the loop from both sides, but at different rates. These events account for about 50% of the total population of metaphase looping events, which, according to theoretical predictions, could be sufficient to achieve 100-fold linear chromosome compaction (Banigan and Mirny, 2018). Thus, the mixed populations of loop extrusion symmetries we observe could play a crucial role for proper chromosome organization in metaphase. What is the origin of the small population of symmetric loop extrusion in metaphase? One possibility is that condensin I and condensin II compact DNA using different symmetries (Kong et al., 2019). In Xenopus egg extract, the relative abundances of condensin I and condensin II is roughly 5:1 (Ono et al., 2003), which would be consistent with the fraction of nearly symmetric loop extrusion events that we observe in metaphase (~20%). One limitation of our work, however, is that our antibodies simultaneously depleted those two complexes. As a consequence, we cannot rule out that the small population of symmetric loop extrusion may arise from residual cohesin activity in metaphase. In the future it will be interesting to investigate the origin of the different looping symmetries by using specific antibodies for condensin I and II. In addition, we observe a small sub-population (~20%) of non-symmetric loop extrusion events in interphase, suggesting a differential regulation of extrusion symmetries in both cell cycle phases. Despite the differences in loop extrusion symmetries between interphase and metaphase, extrusion rates and stall forces seem to be conserved during the cell cycle. The mean extrusion rates we observe, however, are three to four times higher than those observed in previous in vitro studies (Ganji et al., 2018; Kim et al., 2019) for cohesin and condensin respectively. One possibility for this discrepancy could be that, in extract, several extruding factors participate in the extrusion of the same DNA loop in a cooperative manner. However, the average stall forces we estimate are about five to seven times lower than previous estimates in vitro (Ganji et al., 2018; Kim et al., 2019). We speculate that in cytoplasmic context of the H3-H4-depleted egg extract, many other DNA-binding factors—such as linker histone (Xiao et al., 2012)—may compete with the loop extrusion machinery for DNA slack. The large spread in the distribution of stall forces, with individual examples reaching values that compare to those reported in vitro, may suggest that secondary factors could cause the loop extrusion machinery to stop prematurely, and, consequently, we may underestimate the magnitude of the looping stall forces. We wonder, however, how condensin and cohesin share such similar extrusion rates, even though condensin predominantly extrudes non-symmetrically while cohesin extrudes loops symmetrically. The similar loop growth velocities would suggest that condensin reels in DNA from one side at twice the rate that cohesin reels in DNA from each of its two sides. This assumes, however, that cohesin functions by simultaneously extruding DNA from two sides. Alternatively, cohesin may be a one-sided motor that alternates its extrusion direction (Banigan et al., 2019)—though we did not observe this kind of switching within the temporal resolution of our measurements. We speculate that symmetric cohesin loop extrusion could originate from the dimerization of two identical non-symmetric motors, though recent in vitro work shows that this idea is controversial (Davidson et al., 2019; Kim et al., 2019). Interestingly, our results comparing extrusion velocities and corresponding symmetries in interphase show that, on average, symmetric loop extrusion rates are higher (roughly twice) compared to the non-symmetric events (Figure 2—figure supplement 5A). This difference in extrusion rates would be consistent with symmetric and non-symmetric loop extrusion mediated by a dimer and a monomer respectively. Our assay will allow for the dissection of the biochemical underpinnings of these processes, and more generally make it possible to reconstitute complex processes such as the formation of boundary elements and the interplay between transcription, replication, and loop extrusion in cellular contexts. Materials and methods Xenopus laevis egg extract preparation, immunodepletions, and ATP depletion Request a detailed protocol Cytostatic factor (CSF)-arrested Xenopus laevis (RRID:XEP_XIa_100) egg extract was prepared as described previously (Murray, 1991). In brief, unfertilized oocytes were dejellied and crushed by centrifugation, generating an extract that was arrested in meiosis II. We added protease inhibitors (LPC: leupeptin, pepstatin, chymostatin) and cytochalasin D (CyD) to a final concentration of 10 μg/ml each to the extract. In order to generate interphase extracts, CaCl2 was added to a final concentration of 0.4 mM. To immunodeplete soluble H3-H4 heterodimers from the extract (Zierhut et al., 2014), we coupled 130 μg of a mouse monoclonal anti-H4K12Ac to 12.5 μl rProtein A Sepharose (GE Healthcare) slurry in antibody coupling buffer (10 mM K-HEPES pH = 8, 150 mM NaCl), rotating overnight at 4°C. After several washes with a wash buffer (10 mM HEPES pH = 7.7, 100 mM KCl, 150 mM Sucrose, 1 mM MgCl2), we combined 50 μl fresh CSF extract with the beads and incubated the bead-extract mixture for 1.5 hr on ice, occasionally flicking the tubes in order to prevent the beads settling to the bottom. After recovering the extract from the beads, we immediately proceeded with the experiment. We generated mock-depleted extracts with the same protocol using 130 μg random mouse IgG antibodies (IgG from Mouse (polyclonal)-unconjugated, Jackson Immuno Research) in 50 μl of fresh CSF extract. To co-deplete H3-H4 and both condensin I and condensin II, we coupled 130 μg anti-H4K12Ac and 10 μg rabbit polyclonal antibodies of both anti-XCAP-C and anti-XCAP-E to 15 μl rProtein A Sepharose slurry and performed the same H3-H4 depletion method. To co-deplete H3-H4 and cohesin, we coupled 130 µg anti-H4K12Ac and 10 μg rabbit polyclonal anti-XRad21 and 10 μg anti-XSMC1 to 15 μl rProtein A Sepharose and performed the same H3-H4 depletion method. ATP was depleted by adding 0.03 U/μl apyrase (A6410; Sigma-Aldrich) to the extract reaction in the presence of 5 mM CaCl2, followed by a 15 min incubation at room temperature. The ATP-depleted extract was then introduced into the DNA channels as described below. Western blots Request a detailed protocol We prepared 1:25 dilutions of immunodepleted extract in 1X sample loading buffer (50 mM Tris-HCl, pH = 6.8, 2% SDS, 10% glycerol, 0.006% bromophenol blue, 100 mM DTT), ran a gel electrophoresis on a gradient gel, transferred to a nitrocellulose membrane with a semi-dry transfer approach, and performed primary antibody incubation with polyclonal rabbit antibodies anti-H3 (1:10000, ab1791, RRID:AB_302613), anti-XSMC1 (1:2500, MPI-CBG antibody facility), anti-XCAP-C (1:2000, MPI-CBG antibody facility) and monoclonal mouse antibodies to detect tubulin using anti-DM1a (1:10000, MPI-CBG antibody facility). We detected primary antibodies using LI-COR IRDye secondary antibodies and imaged the western blots using an Odyssey Infrared Imaging System. We analyzed the blots using FIJI. Antibody production and labeling Request a detailed protocol We raised rabbit polyclonal antibodies for immunodepletion against peptides SDIVATPGPRFHTV and DLTKYPDANPNPND corresponding to antibodies that targeted cohesin’s XRAD21 and XSMC1 subunits. We also raised rabbit polyclonal antibodies against peptides AAKGLAEMQSVG and SKTKERRNRMEVDK corresponding to antibodies that targeted XCAP-C and XCAP-E for both condensin I and II for immunodepletion (Hirano et al., 1997). We added a cysteine residue on the peptide’s N-termi

Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.

ABSTRACTBraided DNAs are significant structural intermediates in cellular processes, yet little has been experimentally demonstrated about their higher-order structure and twisting torques. We use magnetic tweezers to measure braid extensions at forces ranging from 0.3 to 8 piconewtons, and then apply a thermodynamic Maxwell relation to calculate the torque. Experimentally inferred torques in unbuckled braids take on values up to 76 pN·nm, which depends on force, and inter-tether distance. As predicted using a statistical mechanical model, the twist modulus of the braids increases with catenation prior to buckling or formation of plectoneme, and is comparable to that of single DNA.

The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.

SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a ‘one-sided’ manner. We therefore simulate one-sided loop extrusion to investigate whether ‘one-sided’ complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by ‘two-sided’ loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization.