Explore the vast range of titles available.

‘Pioneer’ transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming 1 , 2 . Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells 3 . Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription. Cryo-electron microscopy structures of the DNA-binding domains of the pioneer transcription factor SOX2 and its close homologue SOX11 elucidate the role of these factors in initiating chromatin opening and nucleosome remodelling.

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks 1 , 2 . The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription 3 – 6 . Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA–DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed. The topoisomerase Top2 and the chromatin-binding protein Hmo1 maintain under-wound and over-wound DNA at different regions within a gene and thereby modulate the topology of genes.

DNA supercoiling (SC), the over- and under-winding of DNA, is generated by transcription as described in the twin-domain model. Conversely, SC also impacts transcription through torsional stress. SC therefore regulates transcription dynamically and independently of transcription factor binding, particularly in the context of chromosomal topological domains and the activity of topoisomerases in bacteria. In this work, we develop numerical simulations of SC-coupled transcription of a single gene within a topological domain, based on a model incorporating stochastic transcription and activities of topoisomerase I and gyrase. We explore the effect of several parameters not systematically assessed in previous works (role of topoisomerase activities, topological domain size, gene expression strength) and compare the simulation results to a diverse set of experimental observations ranging from in vitro transcription assays to transcriptomics datasets from various species. This model recapitulates the non-monotonic dependence of transcription in vitro with the superhelical density of the plasmid template. Simulations of in vivo transcription in a closed domain exhibit a qualitatively different role for the two topoisomerases, as well as qualitatively different regulatory behaviors depending on the promoter strength. Specifically, topoisomerase I is required for strongly expressed genes that may be hindered by stalled RNA Polymerase, whereas gyrase activity favors the expression of all genes by enhancing transcription initiation and modulating the burstiness of transcription. The simulations exhibit a new mechanism for transcription bursting mediated by negative SC accumulating at the promoter region and modulating the initiation rate, resulting in levels of burstiness compatible with values reported in cells. Finally, we analyze several transcriptomics datasets from a range of evolutionarily distant species and show that topoisomerase inhibition is systematically associated with the repression of highly expressed genes. Simulations show this behavior to occur within a limited parameter range and thus indicate a biologically relevant regime for the simulations. Overall, this work provides a more quantitative description of how SC contributes to differential gene regulation and transcriptional bursting in bacteria.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo , but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3′-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity. The bacterial CRISPR/Cas system acquires short phage sequences known as spacers that integrate between CRISPR repeats and constitute a record of phage infection; this study shows that the Cas1–Cas2 complex is the minimal machinery required for spacer acquisition and the complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases with Cas 1 as the catalytic subunit and Cas2 acting to increase integration activity. Bacterial recognition of viral invaders The once fanciful idea that bacteria might have immunological memory became accepted fact with the discovery that the CRISPR–Cas gene loci evolve rapidly to acquire short phage sequences, or spacers, which then integrate between CRISPR repeats and constitute a record of phage infection. These spacers are transcribed into small CRISPR RNAs (crRNAs) that are used to target the DNA of invading viruses. Two papers published in this issue of Nature describe molecular details about how bacteria create a DNA memory of the invading virus. Jennifer Doudna and colleagues show that the purified Escherichia coli Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA in a manner similar to retroviral integrases and DNA transposases. Cas1 is the catalytic subunit, while Cas2 increases integration activity; together they form the minimal machinery required for spacer acquisition. Luciano Marraffini and colleagues show that in the type II CRISPR–Cas system of Streptococcus pyogenes , the Cas9 nuclease that inactivates invading viral DNA using the crRNA as a guide is also required for the incorporation of new spacer sequences, by a yet to be determined mechanism.

DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIβ. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function. Here, the authors develop an assay to measure DNA supercoiling, revealing DNA supercoiling domains across the human genome that are modulated by topoisomerase and showing their colocalization with nuclear compartments and impact on gene expression.

DNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity. DNA supercoiling can result in underwinding with negative supercoiling or overwinding with positive supercoiling of the DNA double helix. Here the authors reveal insights into the dynamic relationship between DNA supercoiling-induced sequence-dependent disruptions to base pairing, DNA looping, and the shape of the DNA molecule.

RNA polymerase (RNAP) is a processive motor that modulates DNA supercoiling and reshapes DNA structures. The feedback loop between the DNA topology and transcription remains elusive. Here, we investigate the impact of potential G-quadruplex forming sequences (PQS) on transcription in response to DNA supercoiling. We find that supercoiled DNA increases transcription frequency 10-fold higher than relaxed DNA, which lead to an abrupt formation of G-quadruplex (G4) and R-loop structures. Moreover, the stable R-loop relieves topological strain, facilitated by G4 formation. The cooperative formation of G4/R-loop effectively alters the DNA topology around the promoter and suppresses transcriptional activity by impeding RNAP loading. These findings highlight negative supercoiling as a built-in spring that triggers a transcriptional burst followed by a rapid suppression upon G4/R-loop formation. This study sheds light on the intricate interplay between DNA topology and structural change in transcriptional regulation, with implications for understanding gene expression dynamics. Here the authors by applying a single-molecule detection platform enabling FRET measurement on a long plasmid DNA construct, provide detailed insights on how negatively supercoiled DNA impacts co-transcriptional R-loop and G4 formation.

To understand how gyrase interacts with DNA and selects a site of action, we created an ad hoc shape-based recognition methodology to ascertain the DNA sequence from cryoEM density maps as a string of purines and pyrimidines, which matched to the DNA minicircle sequence in our two previous cryoEM structures of negatively supercoiled DNA bound to E. coli gyrase. For one structure, the Gate- or G-segment sequence contains base-pair steps that are among the most flexible in the minicircle, facilitating the bend. The sequence flanking this G-segment is highly inflexible, preventing wrapping the β-pinwheel of gyrase. In the other structure, a flexible DNA minicircle sequence wraps a β-pinwheel of gyrase and the G-segment contains base-pair steps of average deformability. This work highlights how DNA sequence and deformability impact gyrase. It also demonstrates the utility of both identifying DNA sequences from cryoEM structures and assessing base-pair step deformability. Here, the authors develop a shape-based approach to identify DNA sequence from cryoEM density maps. Integrating sequence identification with quantitative analysis of base-pair step deformability, their work reveals that gyrase engages DNA regions with specific mechanical properties.

Type II topoisomerases modulate DNA supercoiling and resolve chromosome entanglements. Type IIB topoisomerases, exemplified by DNA topoisomerase VI (Top6), are used by plants and archaea to support endoreduplication and cell proliferation, respectively; homologs of Top6 further serve to initiate meiotic recombination in eukaryotes and constitute the nuclease portion of MksBEFG/Wadjet/Gabija bacterial defense systems. To understand how such factors act upon DNA, we determine structures of Top6 bound to supercoiled minicircles in cleaved and uncleaved states using single-particle electron cryo-microscopy. The structures show that Top6 binds a curved 74 bp region of the supercoiled minicircle DNA and that it cuts at a distinct deformability motif, explaining its preference for supercoiled substrates and highlighting the role of DNA plasticity in cleavage site selection. Dynamic protein-DNA interactions and an unanticipated tension sensor help recognize bent DNA and couple ATPase disposition to cleavage state activation. Our observations explain how DNA recognition and cleavage by type II topoisomerases are regulated by interdependent structural changes in DNA and the enzyme. DNA recognition and cleavage control in type II topoisomerases are poorly understood processes. Here, the authors determine cleaved and uncleaved structures of supercoiled DNA-bound topoisomerase VI that reveal how the enzyme activates its cleavage state and prefers to act at deformable substrates.

The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity. CRISPR-Cas12a is a programmable endonuclease used for genetic engineering. Here, the authors use real-time single-molecule measurements to show that Cas12a unwinds the target DNA site in dynamic and reversible steps to test for matches with its guide RNA molecule.