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Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1–3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4–7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

The properties of the cell types that are most vulnerable in the Huntington’s disease (HD) cortex, the nature of somatic CAG expansion ofmHTTin these cells, and their importance in CNS circuitry have not been delineated. Here we have employed serial fluorescence activated nuclear sorting (sFANS), deep molecular profiling, and single nucleus RNA sequencing (snRNAseq) to demonstrate that layer 5a pyramidal neurons are selectively vulnerable in primary motor cortex and other cortical areas. Extensive somaticmHTT-CAG expansion occurs in vulnerable layer 5a pyramidal cells, and in Betz cells, layer 6a, layer 6b neurons that are not lost in HD. Retrograde tracing experiments in the macaque brain identify the vulnerable layer 5a neurons as corticostriatal pyramidal cells. Our data establish thatmHTT-CAG expansion is not sufficient for cell loss in the cerebral cortex of HD, and suggest that cortico-striatal disconnection in early-stage HD patients may play an important role in neurodegeneration.

SUMMARYTissue-specific somatic expansion of the mutant Huntingtin (mHTT) CAG tract and regional degeneration of the brain are key features of Huntington’s disease (HD). However, the relationships between somatic CAG expansion, death of specific cell types, and molecular events associated with these processes have not been established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. Expansion of themHTTCAG tract occurs in striatal MSNs and cholinergic interneurons, in cerebellar Purkinje cells, and at the mutantATXN3locus in MSN nuclei from SCA3 donors. Somatic CAG tract instability in MSNs is associated with higher levels of MSH2 and MSH3. Our data indicate that somatic CAG tract expansion is not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansion and toxicity in the human striatum.

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15 + HLADR hi and CD48 high S100A2 pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular stress can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily post-mitotic and aids regeneration in mammals. In the present study we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of its target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.Significance StatementIn humans and other mammals, many organs regenerate through the proliferation of cells that replace those that have succumbed to aging or injury. However, proliferation is largely absent in certain critical organs, including the heart, the central nervous system, and sensory organs such as the inner ear and retina. The Hippo-Yap biochemical signaling pathway, a cascade of proteins that—when active—inhibits cell division, constitutes one impediment to proliferation. We earlier identified a small molecule that interrupts Hippo-Yap signaling and thus relieves this block for some non-proliferating cells in vitro. In the present investigation, we have chemically modified the original substance to yield a more potent analog that is effective for several hours in mammalian tissues in vivo and initiates the proliferation of heart-muscle cells after cryolesioning. After further refinements, compounds of this family might prove useful in regenerative therapies.

Fanconi anemia (FA) pathway is required for the repair of DNA interstrand crosslinks (ICL). ICLs are caused by genotoxins, such as chemotherapeutic agents or reactive aldehydes. Inappropriately repaired ICLs contribute to hematopoietic stem cell (HSC) failure and tumorigenesis. While endogenous acetaldehyde and formaldehyde are known to induce HSC failure and leukemia in humans with FA, the effects of other toxic metabolites in FA pathogenesis have not been systematically investigated. Using a metabolism-focused CRISPR screen, we found that ALDH9A1 deficiency causes synthetic lethality in FA pathway-deficient cells. Combined deficiency of ALDH9A1 and FANCD2 causes genomic instability, apoptosis, and decreased hematopoietic colony formation. Fanca−/−Aldh9a1−/− mice exhibited an increased incidence of ovarian tumors. A suppressor CRISPR screen revealed that the loss of ATP13A3, a polyamine transporter, resulted in improved survival of FANCD2−/−ALDH9A1−/− cells. These findings implicate high intracellular polyamines and the resulting 3-aminopropanal or acrolein in the pathogenesis of FA. In addition, we find that ALDH9A1 variants may be modifying disease onset in FA patients.Statement of SignificanceALDH9A1 deficiency is a previously unrecognized source of endogenous DNA damage. If not repaired by the Fanconi anemia pathway, such damage leads to increased genomic instability and tumorigenesis. Limiting substrates that accumulate when ALDH9A1 is absent can reduce aldehyde production and rescue synthetic lethality in the combined deficiency of ALDH9A1/FANCD2.

Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals1–4. However, these interactions have not been studied systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality.

When cells encounter stressful situations, they activate the integrated stress response (ISR), which limits total protein synthesis and redirects translation to proteins that help the cells to cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in squamous cell carcinoma (SCC) stem cells. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during stress, the ISR also functions in promoting cellular recovery once the stress has subsided. This finding exposes a vulnerability to SCC stem cells that could be exploited therapeutically.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

Double-strand break (DSB) repair relies on DNA damage response (DDR) factors including BRCA1, BRCA2, and RAD51, which promote homology-directed repair (HDR); 53BP1, which affects single-stranded DNA formation; and proteins that mediate end-joining. Here we show that the CRL4/DDB1/WDR70 complex (CRL4

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRNVglut3) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRNVglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRNVglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRNVglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders.

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP’s potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington’s Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP’s transcriptional activity can partially rescue the HD phenotype.

Neurons rely on translation of synaptic mRNAs in order to generate activity-dependent changes in plasticity. Here, we develop a strategy combining compartment-specific crosslinking immunoprecipitation (CLIP) and translating ribosome affinity purification (TRAP) in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. We identify CA1 dendritic transcripts with differentially localized mRNA isoforms generated by alternative polyadenylation and alternative splicing, including many that have altered protein-coding capacity. Among dendritic mRNAs, FMRP targets were found to be overrepresented. Cell-type-specific FMRP-CLIP and TRAP in microdissected CA1 neuropil revealed 383 dendritic FMRP targets and suggests that FMRP differentially regulates functionally distinct modules in CA1 dendrites and cell bodies. FMRP regulates ~15-20% of mRNAs encoding synaptic functions and 10% of chromatin modulators, in the dendrite and cell body, respectively. In the absence of FMRP, dendritic FMRP targets had increased ribosome association, consistent with a function for FMRP in synaptic translational repression. Conversely, downregulation of FMRP targets involved in chromatin regulation in cell bodies suggests a role for FMRP in stabilizing mRNAs containing stalled ribosomes in this compartment. Together, the data support a model in which FMRP regulates the translation and expression of synaptic and nuclear proteins within different compartments of a single neuronal cell type.The brain has over 100 billion neurons that together form vast networks to relay electrical signals. A neuron receives electrical signals from other neurons via branch-like structures known as dendrites. The signals then travel into the cell body of the neuron. If their sum reaches a threshold, they fire a new signal through a single outgoing projection known as the axon, which is connected to the dendrites of other neurons. A single neuron has thousands of dendrites that each receive inputs from different axons, and it is thought that the strengthening and weakening of these dendritic connections enables us to learn and store memories. Dendrites are filled with molecules known as messenger ribonucleic acids (mRNAs) that act as templates to make proteins. Axonal signals reaching the dendrites can trigger these mRNAs to make new proteins that strengthen or weaken the connections between the two neurons, which is believed to be necessary for generating long-term memories. A protein called FMRP is found in both the cell body and dendrites and is able to bind to and regulate the ability of mRNAs to make proteins. A loss of the gene encoding FMRP is the most common cause of inherited intellectual disability and autism in humans, but it remains unclear precisely what role this protein plays in learning and memory. Hale et al. used genetic and bioinformatics approaches to specifically study mRNAs in the dendrites and the cell body of a specific type of neuron involved in memory in mice. The experiments revealed that FMRP played different roles in the dendrites and cell body. In the dendrites, FMRP interacted with mRNAs encoding proteins that can change how the neuron responds to a signal from a neighboring neuron and may alter how strong the connections between the neurons are. On the other hand, FMRP in the cell body modulated the activities of mRNAs encoding proteins that in turn regulate the activities of genes. These findings change the way we think about how memory may work by suggesting that groups of mRNAs encoding proteins with certain activities are found in distinct parts of a single neuron. These observations offer new ways to approach intellectual disabilities and autism spectrum disorder.