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The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn’t show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell. Every human cell contains nearly two meters of DNA, which is carefully packaged to form a dense structure known as chromatin. The building block of chromatin is the nucleosome, a unit composed of a short section of DNA tightly wound up around a spool-like core of proteins called histones. The tight structure of the nucleosome prevents the cell from accessing and ‘reading’ the genes in the packaged DNA, effectively switching off these genes. So the exact placement of nucleosomes helps manage which genes are turned on. Changing the position of the nucleosomes can ‘free’ the DNA and make genes available to the cell. Enzymes called chromatin remodelers move nucleosomes around – for example, they can make the histone core slide on the DNA strand. However, it is still unclear how these enzymes recognize nucleosomes. Previous research indicates that many proteins bind to nucleosomes by using a surface on the histone proteins called the acidic patch. Could chromatin remodelers also work by interacting with this acidic patch? To address this further, Gamarra et al. investigate how a chromatin remodeler enzyme known as SNF2h interacts with a nucleosome. By default, SNF2h is inactive because two of its regions called AutoN and NegC act as brakes. The experiments show that the acidic patch helps to bypass this inactivation and switches on SNF2h. Gamarra et al. propose that, when SNF2h docks on to the nucleosome, the patch provides a landing pad for the AutoN and NegC modules; this interaction activates the enzyme, which can then start remodeling the nucleosome. However, another type of chromatin remodeler also uses the patch to interact with nucleosomes but it does not have the AutoN and NegC regions. This suggests that chromatin remodelers work with the acidic patch in different ways. Overall, the findings deepen our understanding of how DNA is packaged in cells, and how this process may go wrong and cause disease.

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks 1 . As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle 2 – 5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters 6 , 7 , recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division. WDR5 and TBP recruit anaphase-promoting complex to specific transcription start sites in mitosis, initiating a ubiquitin-dependent mechanism that preserves cell identity by linking gene expression and cell division.

Eukaryotic genomes are packaged into chromatin: a highly heterogeneous structure composed of nucleic acids and proteins. This packaging controls access to the underlying DNA sequences and, as a result plays a critical role in nearly all genomic processes. The primary molecular structure of chromatin is the nucleosome: ~147 bp of DNA wrapped around a core of histone proteins. Both the location and status of nucleosomes in the genome are critical for the proper packaging of chromatin. As a result, cells have evolved several sophisticated molecular machines to disrupt or modify nucleosomes to achieve specific packaging states. A critical member of these machines are the SWI2/SNF2 superfamily of ATP-dependent chromatin remodeling enzymes, which are DNA translocases that harness the energy of ATP hydrolysis to physically disrupt nucleosomes. Because of their central role in control nucleosome structure throughout the genome, remodelers play roles in virtually all DNA-dependent process, but the precise mechanisms of how remodelers disrupt nucleosomes and how this disruption is coupled to other molecular events remains very poorly understood. In this thesis we focus on understanding the remodeling mechanisms of two subfamilies of SWI2/SNF2 remodelers that slide nucleosomes: INO80 and ISWI. To understand how these and other SWI2/SNF2 ATP-dependent remodelers might cooperate with nuclear machinery to enable biological processes, we first review our broad understanding of remodeling mechanism as it compares to another molecular motor that disrupts nucleosomes: RNA polymerase. We then speculate on how these two distinct families cooperate to accomplish transcription on chromatin templates. After this, we set out to uncover elements of nucleosome that control remodeler activity and identify a conserved surface of the nucleosome known as the acidic patch that is required to activate both ISWI and INO80 family remodelers. Using a combination of biochemical and biophysical assays, we show that this surface activates remodeling by these two families after they bind the nucleosome. For the ISWI remodeler SNF2h, the acidic patch activates remodeling by serving as a landing pad for the binding of autoinhibitory domains while INO80 uses a separate mechanism. We then solve the near-atomic CryoEM structure of SNF2h bound to the nucleosome. Unexpectedly, we find that SNF2h binding in an activated state asymmetrically distorts the histone core of the nucleosome and that this may be important in regulating the activity of the enzyme. Finally, we test the hypothesis that by measuring remodeling activity of nucleosomes with site-specific restraints in the histone core. We find that specifically restraining histone dynamics in locations across all 4 histone proteins inhibits SNF2h-mediated nucleosome sliding. Taken together, these results suggest that remodelers rely on the structure and dynamics of both the DNA and protein components of the nucleosome to accomplish their activities.

Metazoan development requires robust proliferation of progenitor cells, whose identities are established by tightly controlled transcriptional networks 1. As gene expression is globally inhibited during mitosis, the transcriptional programs defining cell identity must be restarted in each cell cycle 2-5, yet how this is accomplished is poorly understood. Here, we identified a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters 6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites (TSS) during mitosis. This allows APC/C to decorate histones with K11/K48-branched ubiquitin chains that recruit p97/VCP and the proteasome and ensure rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and transcription re-initiation are thus controlled by the same regulator, APC/C, which provides a robust mechanism to maintain cell identity through cell division.

Long-read sequencing assays that detect base modifications are becoming increasingly important research tools for the study of epigenetic regulation, especially with the development of DiMeLo-seq and similar methods that deposit non-native base modifications to mark a range of epigenetic features such as protein-DNA interactions and chromatin accessibility. A main benefit of these methods is their inherent capacity for multimodality, enabling the encoding of multiple genomic signals onto single nucleic acid molecules. However, there are limited tools available for visualization and statistical analysis of this type of multimodal data. Here we introduce dimelo-toolkit, a python package built to enable flexible visualizations and easy integration into custom data processing workflows. We demonstrate the utility of dimelo-toolkit's preset visualizations of multiple base modifications in long-read single-molecule sequencing data with a novel extension of the DiMeLo-seq protocol that can capture three separate aspects of chromatin state on the same single reads: target protein binding, CpG methylation, and chromatin accessibility. We apply this multimodal method to simultaneously map chromatin accessibility, CpG methylation, and LMNB1 and CTCF binding patterns, respectively, in GM12878 cells. Additionally, we use dimelo-toolkit to investigate technical biases that arise when working with this type of multimodal data. This software tool will pave the way for developing well-optimized protocols and help unlock previously inaccessible biological insights.

Genome regulation relies on complex and dynamic interactions between DNA and proteins. Recently, powerful methods have emerged that leverage third-generation sequencing to map protein-DNA interactions genome-wide. For example, Directed Methylation with Long-read sequencing (DiMeLo-seq) enables mapping of protein-DNA interactions along long, single chromatin fibers, including in highly repetitive genomic regions. However, DiMeLo-seq involves lossy centrifugation-based wash steps that limit its applicability to many sample types. To address this, we developed DiMeLo-cito, a single-tube, wash-free protocol that maximizes the yield and quality of genomic DNA obtained for long-read sequencing. This protocol enables the interrogation of genome-wide protein binding with as few as 100,000 cells and without the requirement of a nuclear envelope, enabling confident measurement of protein-DNA interactions during mitosis. Using this protocol, we detected strong binding of CTCF to mitotic chromosomes in diploid human cells, in contrast with earlier studies in karyotypically unstable cancer cell lines, suggesting that CTCF \"bookmarks\" specific sites critical for maintaining genome architecture across cell divisions. By expanding the capabilities of DiMeLo-seq to a broader range of sample types, DiMeLo-cito can provide new insights into genome regulation and organization.

Abstract Elucidating the mechanisms by which ATP-dependent chromatin remodeling enzymes disrupt nucleosome structure is essential to understanding how chromatin states are established and maintained. A key finding informing remodeler mechanism is the observation that the dynamics of protein residues buried within the histone core of the nucleosome are used by specific remodelers to mobilize the nucleosome1. Recently, a study obtaining cryo-electron microscopy (cryo-EM) structures of ISWI-family remodelernucleosome complexes failed to observe stable conformational rearrangements in the histone octamer2. The authors of this study also failed to replicate the earlier finding that site-specifically restraining histone dynamics inhibits nucleosome sliding by ISWI-family remodelers1,2. In contrast, a recent cryo-EM structure detected asymmetric histone dynamics in an ISWI-nucleosome complex3. Here, using two different protocols, we replicate the original finding in Sinha et al.1 that dynamics within the histone core are important for nucleosome sliding by the human ISWI remodeler, SNF2h. These results firmly establish histone dynamics as an essential feature of ISWI-mediated nucleosome sliding and highlight the care required in designing and performing biochemical experiments investigating nucleosome dynamics using disulfide linkages. Competing Interest Statement The authors have declared no competing interest. Footnotes * Arising from: Yan et al. (2019) Nature Structure and Molecular Biology https://doi.org/10.1038/s41594-019-0199-9 * Additional experiments are presented in supplemental figures 1-5 which further bolster the arguments presented in the first two versions of the manuscript. Additional revisions are made to the text to improve clarity and quality of our arguments.

Genomic DNA is highly compacted in the nucleus of eukaryotic cells as a nucleoprotein assembly called chromatin. The basic unit of chromatin is the nucleosome, where ~146 base pair increments of the genome are wrapped and compacted around the core histone proteins. Further genomic organization and compaction occur through higher order assembly of nucleosomes. This organization regulates many nuclear processes, and is controlled in part by histone post-transtranslational modifications and chromatin-binding proteins. Mechanisms that regulate the assembly and compaction of the genome remain unclear. Here we show that in the presence of physiologic concentrations of mono- and divalent salts, histone tail-driven interactions drive liquid-liquid phase separation (LLPS) of nucleosome arrays, resulting in substantial condensation. Phase separation of nucleosomal arrays is inhibited by histone acetylation, whereas histone H1 promotes phase separation, further compaction, and decreased dynamics within droplets, mirroring the relationship between these modulators and the accessibility of the genome in cells. These results indicate that under physiologically relevant conditions, LLPS is an intrinsic behavior of the chromatin polymer, and suggest a model in which the condensed phase reflects a genomic 'ground state' that can produce chromatin organization and compaction in vivo. The dynamic nature of this state could enable known modulators of chromatin structure, such as post-translational modifications and chromatin binding proteins, to act upon it and consequently control nuclear processes such as transcription and DNA repair. Our data suggest an important role for LLPS of chromatin in the organization of the eukaryotic genome.