Nature structural & molecular biology最新文献

Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable ‘confined target search’ mechanism. PTFs such as FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-PTF MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domains, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested that transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.

RNA-guided endonucleases, once thought to be exclusive to prokaryotes, have been recently identified in eukaryotes and are called Fanzors. They are classified into two clades, Fanzor1 and Fanzor2. Here we present the cryo-electron microscopy structure of Acanthamoeba polyphaga mimivirus Fanzor2, revealing its ωRNA architecture, active site and features involved in transposon-adjacent motif recognition. A comparison to Fanzor1 and TnpB structures highlights divergent evolutionary paths, advancing our understanding of RNA-guided endonucleases.

Long noncoding RNAs (lncRNAs) are increasingly appreciated for their important functions in mammalian cells. However, how their functional capacities are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA-binding proteins, but the structural principles that underlie this mode of regulation are unknown. The NORAD lncRNA is a known decoy for Pumilio proteins, which modulate the translation and stability of hundreds of messenger RNAs and, consequently, a regulator of genomic stability and aging. Here we probed the RNA structure and long-range RNA–RNA interactions formed by human NORAD inside cells under different stressful conditions. We discovered a highly modular structure consisting of well-defined domains that contribute independently to NORAD function. Following arsenite stress, most structural domains undergo relaxation and form interactions with other RNAs that are targeted to stress granules. We further revealed a unique structural organization that spatially clusters the multiple Pumilio binding sites along NORAD and consequently contributes to the derepression of Pumilio targets. We then applied these structural principles to design an effective artificial decoy for the let-7 microRNA. Our work demonstrates how the sequence of a lncRNA spatially clusters its function into separated domains and how structural principles can be employed for the rational design of lncRNAs with desired activities.

Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix. It is a heteropolysaccharide of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) reaching several megadaltons in healthy tissues. HA is synthesized and translocated in a coupled reaction by HA synthase (HAS). Here, structural snapshots of HAS provide insights into HA biosynthesis, from substrate recognition to HA elongation and translocation. We monitor the extension of a GlcNAc primer with GlcA, reveal the coordination of the uridine diphosphate product by a conserved gating loop and capture the opening of a translocation channel to coordinate a translocating HA polymer. Furthermore, we identify channel-lining residues that modulate HA product lengths. Integrating structural and biochemical analyses suggests an avenue for polysaccharide engineering based on finely tuned enzymatic activity and HA coordination.

Activation of the chemokine receptor CXCR4 by its chemokine ligand CXCL12 regulates diverse cellular processes. Previously reported crystal structures of CXCR4 revealed the architecture of an inactive, homodimeric receptor. However, many structural aspects of CXCR4 remain poorly understood. Here, we use cryo-electron microscopy to investigate various modes of human CXCR4 regulation. CXCL12 activates CXCR4 by inserting its N terminus deep into the CXCR4 orthosteric pocket. The binding of US Food and Drug Administration-approved antagonist AMD3100 is stabilized by electrostatic interactions with acidic residues in the seven-transmembrane-helix bundle. A potent antibody blocker, REGN7663, binds across the extracellular face of CXCR4 and inserts its complementarity-determining region H3 loop into the orthosteric pocket. Trimeric and tetrameric structures of CXCR4 reveal modes of G-protein-coupled receptor oligomerization. We show that CXCR4 adopts distinct subunit conformations in trimeric and tetrameric assemblies, highlighting how oligomerization could allosterically regulate chemokine receptor function.

DNA double-strand breaks that initiate meiotic recombination are formed by the topoisomerase-relative enzyme Spo11, supported by conserved auxiliary factors. Because high-resolution structural data have not been available, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-electron microscopy structures at up to 3.3-Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104 and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3′-OH and first 5′ overhanging nucleotide, establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.

Human silencers have been shown to regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated, such as whether they can form ‘super-silencers’ and whether they are linked to cancer progression. Here, we show two silencer components of the FGF18 gene can cooperate through compensatory chromatin interactions to form a super-silencer. Double knockout of two silencers exhibited synergistic upregulation of FGF18 expression and changes in cell identity. To perturb the super-silencers, we applied combinational treatment of an enhancer of zeste homolog 2 inhibitor GSK343, and a repressor element 1-silencing transcription factor inhibitor, X5050 (‘GR’). Interestingly, GR led to severe loss of topologically associated domains and loops, which were associated with reduced CTCF and TOP2A mRNA levels. Moreover, GR synergistically upregulated super-silencer-controlled genes related to cell cycle, apoptosis and DNA damage, leading to anticancer effects in vivo. Overall, our data demonstrated a super-silencer example and showed that GR can disrupt super-silencers, potentially leading to cancer ablation.

Translesion DNA synthesis (TLS) is a cellular process that enables the bypass of DNA lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase κ (Polκ) has the distinctive ability to bypass minor groove DNA adducts in vitro. However, Polκ is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base-editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Polκ during lesion bypass. Strikingly, we show that Polκ has two main functions during TLS, which are differentially regulated by Rev1 binding. On the one hand, Polκ is essential to replicate across a minor groove DNA lesion in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, through its cooperative interaction with Rev1 and ubiquitylated PCNA, Polκ appears to stabilize the Rev1–Polζ extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of Polκ’s catalytic activity. Together, our work identifies catalytic and noncatalytic functions of Polκ in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases.