Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

ParB-type DNA Segregation Proteins Are CTP-Dependent Molecular Switches.

During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.

Immunomimetic Designer Cells Protect Mice from MRSA Infection.

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis.

During neurodegenerative disease, the toxic accumulation of aggregates and misfolded proteins is often accompanied with widespread changes in peripheral metabolism, even in cells in which the aggregating protein is not present. The mechanism by which the central nervous system elicits a distal reaction to proteotoxic stress remains unknown. We hypothesized that the endocrine communication of neuronal stress plays a causative role in the changes in mitochondrial homeostasis associated with proteotoxic disease states. We find that an aggregation-prone protein expressed in the neurons of C. elegans binds to mitochondria, eliciting a global induction of a mitochondrial-specific unfolded protein response (UPR(mt)), affecting whole-animal physiology. Importantly, dense core vesicle release and secretion of the neurotransmitter serotonin is required for the signal's propagation. Collectively, these data suggest the commandeering of a nutrient sensing network to allow for cell-to-cell communication between mitochondria in response to protein folding stress in the nervous system.

Completing genome replication outside of S phase.

Mitotic DNA synthesis (MiDAS) is an unusual form of DNA replication that occurs during mitosis. Initially, MiDAS was characterized as a process associated with intrinsically unstable loci known as common fragile sites that occurs after cells experience DNA replication stress (RS). However, it is now believed to be a more widespread "salvage" mechanism that is called upon to complete the duplication of any under-replicated genomic region. Emerging data suggest that MiDAS is a DNA repair process potentially involving two or more pathways working in parallel or sequentially. In this review, we introduce the causes of RS, regions of the human genome known to be especially vulnerable to RS, and the strategies used to complete DNA replication outside of S phase. Additionally, because MiDAS is a prominent feature of aneuploid cancer cells, we will discuss how targeting MiDAS might potentially lead to improvements in cancer therapy.

O-GlcNAcylation of Raptor transduces glucose signals to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient levels. Dysregulation of mTORC1 results in a broad spectrum of diseases. Glucose is the primary energy supply of cells, and therefore, glucose levels must be accurately conveyed to mTORC1 through highly responsive signaling mechanisms to control mTORC1 activity. Here, we report that glucose-induced mTORC1 activation is regulated by O-GlcNAcylation of Raptor, a core component of mTORC1, in HEK293T cells. Mechanistically, O-GlcNAcylation of Raptor at threonine 700 facilitates the interactions between Raptor and Rag GTPases and promotes the translocation of mTOR to the lysosomal surface, consequently activating mTORC1. In addition, we show that AMPK-mediated phosphorylation of Raptor suppresses Raptor O-GlcNAcylation and inhibits Raptor-Rags interactions. Our findings reveal an exquisitely controlled mechanism, which suggests how glucose coordinately regulates cellular anabolism and catabolism.

The multifaceted roles of mitochondria.

Much more than the "powerhouse" of the cell, mitochondria have emerged as critical hubs involved in metabolism, cell death, inflammation, signaling, and stress responses. To open our mitochondria focus issue, we asked several scientists to share the unanswered questions, emerging themes, and topics of investigation that excite them.

Biased allosteric activation of ketone body receptor HCAR2 suppresses inflammation.

Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body ß-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for inflammation-related diseases. HCAR2 mediates distinct pathophysiological events by activating Gi/o protein or ß-arrestin effectors. Here, we characterize compound 9n as a Gi-biased allosteric modulator (BAM) of HCAR2 and exhibit anti-inflammatory efficacy in RAW264.7 macrophages via a specific HCAR2-Gi pathway. Furthermore, four structures of HCAR2-Gi complex bound to orthosteric agonists (niacin or monomethyl fumarate), compound 9n, and niacin together with compound 9n simultaneously reveal a common orthosteric site and a unique allosteric site. Combined with functional studies, we decipher the action framework of biased allosteric modulation of compound 9n on the orthosteric site. Moreover, co-administration of compound 9n with orthosteric agonists could enhance anti-inflammatory effects in the mouse model of colitis. Together, our study provides insight to understand the molecular pharmacology of the BAM and facilitates exploring the therapeutic potential of the BAM with orthosteric drugs.

The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

E3 ligase RNF167 and deubiquitinase STAMBPL1 modulate mTOR and cancer progression.

The mTOR complex 1 (mTORC1) is an essential metabolic hub that coordinates cellular metabolism with the availability of nutrients, including amino acids. Sestrin2 has been identified as a cytosolic leucine sensor that transmits leucine status signals to mTORC1. In this study, we identify an E3 ubiquitin ligase RING finger protein 167 (RNF167) and a deubiquitinase STAMBPL1 that function in concert to control the polyubiquitination level of Sestrin2 in response to leucine availability. Ubiquitination of Sestrin2 promotes its interaction with GATOR2 and inhibits mTORC1 signaling. Bioinformatic analysis reveals decreased RNF167 expression and increased STAMBPL1 expression in gastric and colorectal tumors. Knockout of STAMBPL1 or correction of the heterozygous STAMBPL1 mutation in a human colon cancer cell line suppresses xenograft tumor growth. Lastly, a cell-permeable peptide that blocks the STAMBPL1-Sestrin2 interaction inhibits mTORC1 and provides a potential option for cancer therapy.

RAD51 protects human cells from transcription-replication conflicts.

Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.

Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut.

A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that downregulation of the conserved cholinergic enzyme acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific Egr5 (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+, which spreads in the epithelium through Innexin2-Innexin7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial Ca2+ currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury.

Clathrin-associated AP-1 controls termination of STING signalling.

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Nonlinear mechanics of human mitotic chromosomes.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.

Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Replication Stress Induces ATR/CHK1-Dependent Nonrandom Segregation of Damaged Chromosomes.

Nonrandom DNA segregation (NDS) is a mitotic event in which sister chromatids carrying the oldest DNA strands are inherited exclusively by one of the two daughter cells. Although this phenomenon has been observed across various organisms, the mechanism and physiological relevance of this event remain poorly defined. Here, we demonstrate that DNA replication stress can trigger NDS in human cells. This biased inheritance of old template DNA is associated with the asymmetric DNA damage response (DDR), which derives at least in part from telomeric DNA. Mechanistically, we reveal that the ATR/CHK1 signaling pathway plays an essential role in mediating NDS. We show that this biased segregation process leads to cell-cycle arrest and cell death in damaged daughter cells inheriting newly replicated DNA. These data therefore identify a key role for NDS in the maintenance of genomic integrity within cancer cell populations undergoing replication stress due to oncogene activation.

Two Hippo signaling modules orchestrate liver size and tumorigenesis.

The Hippo pathway is a central regulator of organ size and tumorigenesis and is commonly depicted as a kinase cascade, with an increasing number of regulatory and adaptor proteins linked to its regulation over recent years. Here, we propose that two Hippo signaling modules, MST1/2-SAV1-WWC1-3 (HPO1) and MAP4K1-7-NF2 (HPO2), together regulate the activity of LATS1/2 kinases and YAP/TAZ transcriptional co-activators. In mouse livers, the genetic inactivation of either HPO1 or HPO2 module results in partial activation of YAP/TAZ, bile duct hyperplasia, and hepatocellular carcinoma (HCC). On the contrary, inactivation of both HPO1 and HPO2 modules results in full activation of YAP/TAZ, rapid development of intrahepatic cholangiocarcinoma (iCCA), and early lethality. Interestingly, HPO1 has a predominant role in regulating organ size. HPO1 inactivation causes a homogenous YAP/TAZ activation and cell proliferation across the whole liver, resulting in a proportional and rapid increase in liver size. Thus, this study has reconstructed the order of the Hippo signaling network and suggests that LATS1/2 and YAP/TAZ activities are finetuned by HPO1 and HPO2 modules to cause different cell fates, organ size changes, and tumorigenesis trajectories.

The transcription factor MYB110 regulates plant height, lodging resistance, and grain yield in rice.

The high-yielding Green Revolution varieties of cereal crops are characterized by a semidwarf architecture and lodging resistance. Plant height is tightly regulated by the availability of phosphate (Pi), yet the underlying mechanism remains obscure. Here, we report that rice (Oryza sativa) R2R3-type Myeloblastosis (MYB) transcription factor MYB110 is a Pi-dependent negative regulator of plant height. MYB110 is a direct target of PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) and regulates OsPHR2-mediated inhibition of rice height. Inactivation of MYB110 increased culm diameter and bending resistance, leading to enhanced lodging resistance despite increased plant height. Strikingly, the grain yield of myb110 mutants was elevated under both high- and low-Pi regimes. Two divergent haplotypes based on single nucleotide polymorphisms in the putative promoter of MYB110 corresponded with its transcript levels and plant height in response to Pi availability. Thus, fine-tuning MYB110 expression may be a potent strategy for further increasing the yield of Green Revolution cereal crop varieties.

SAR1B senses leucine levels to regulate mTORC1 signalling.

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.