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Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR -related cell surface receptor called leucine-rich repeat-containing protein 15 ( LRRC15 ). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans . Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21 st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide. The modification of peptides and proteins for application in drug discovery and chemical biology is currently a rapidly growing field of research. Here, the authors report a photocatalytic diselenide contraction method for the dimerization and site-specific functionalisation of peptides and protein.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 10⁶-fold. Crystal structures of 13 peptide–bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and β-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway. Here, the authors characterise the zinc finger protein ZNF827 as a single stranded DNA binding protein that accumulates at stalled replication forks to activate the ATR-CHK1 pathway and engage homologous-recombination mediated DNA repair.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam 2 Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4 + T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam 2 Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses. Current vaccines against SARS-CoV-2 reduce mortality but are less effective in preventing infection. Here the authors show that intranasal vaccination with a subunit vaccine including an TLR2-stimulating adjuvant induces strong neutralising antibody and T-cell responses against SARS-CoV-2 in the lungs that protect against infection.

Acetylation of histones triggers association with bromodomain-containing proteins that regulate diverse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 \"reads\" acetyl marks on nuclear factors to promote their stable association with chromatin.

β-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG) , which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood, and toxicity limits their use. We identify the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression through a CRISPR/Cas9 screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 causes global demethylation and HBG activation that is reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activates their genes in HUDEP2 or primary CD34 + cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD co-repressor complex, recapitulates the effects of promoter demethylation. Our findings demonstrate that localized CpGmethylation at the HBG promoters facilitates gene silencing and identify a potential therapeutic approach for β-hemoglobinopathies via epigenomic editing. DNA methylation is a critical component for repression of fetal haemoglobin in adult blood cells. Removing DNA methylation from the fetal haemoglobin promoter effectively upregulates the gene, opening avenues for the treatment of blood disorders.

The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.

Although certain human genetic variants are conspicuously loss of function, decoding the impact of many variants is challenging. Previously, we described a patient with leukemia predisposition syndrome (GATA2 deficiency) with a germline GATA2 variant that inserts 9 amino acids between the 2 zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2 deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced granulocyte-macrophage colony-stimulating factor (GM-CSF) and elevated IL-6 signaling. As insufficient GM-CSF signaling caused pulmonary alveolar proteinosis and excessive IL-6 signaling promoted bone marrow failure and GATA2 deficiency patient phenotypes, these results provide insight into mechanisms underlying GATA2-linked pathologies.

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation—even in the absence of nucleotide—triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4–6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding. Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. Here, the authors use single-molecule assays to examine the mechanism of action of CHD4 remodeller, and provide evidence that CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation.