Mechanistic roles for many lncRNAs are poorly understood, in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs ...and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin, and that localization responds to cues involved in the transcription process.
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•High-resolution genomic binding sites for the human lncRNAs NEAT1 and MALAT1•NEAT1 and MALAT1 exhibit distinct colocalized binding at active chromatin•Transcription impacts NEAT1 localization patterns•CHART-MS identifies proteins associated with endogenous RNAs in vivo
NEAT1 and MALAT1 are two of the most abundant mammalian lncRNAs. West et al. map the genomic binding of NEAT1 and MALAT1 and identify interacting proteins via RNA pull-down followed by mass spectrometry, showing these RNAs play a synergistic role in nuclear organization linking active genes and nuclear subdomains.
Memory CD4+ T helper type 2 (Th2) cells drive allergic asthma, yet the mechanisms whereby tissue-resident memory Th2 (Th2 Trm) cells and circulating memory Th2 cells collaborate in vivo remain ...unclear. Using a house dust mite (HDM) model of allergic asthma and parabiosis, we demonstrate that Th2 Trm cells and circulating memory Th2 cells perform nonredundant functions. Upon HDM rechallenge, circulating memory Th2 cells trafficked into the lung parenchyma and ignited perivascular inflammation to promote eosinophil and CD4+ T cell recruitment. In contrast, Th2 Trm cells proliferated near airways and induced mucus metaplasia, airway hyperresponsiveness, and airway eosinophil activation. Transcriptional analysis revealed that Th2 Trm cells and circulating memory Th2 cells share a core Th2 gene signature but also exhibit distinct transcriptional profiles. Th2 Trm cells express a tissue-adaptation signature, including genes involved in regulating and interacting with extracellular matrix. Our findings demonstrate that Th2 Trm cells and circulating memory Th2 cells are functionally and transcriptionally distinct subsets with unique roles in promoting allergic airway disease.
X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene ...expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.
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► Deleting Xist causes blood cancer ► Overdosage of the X chromosome causes cancer ► Xist is required for hematopoietic stem cell survival and function ► Deleting Xist causes marrow fibrosis, leukemia, and histiocytic sarcoma
Deletion of Xist in the blood cells of female mice causes an aggressive and highly penetrant hematopoietic cancer.
Communication within the glial cell ecosystem is essential for neuronal and brain health
. The influence of glial cells on the accumulation and clearance of β-amyloid (Aβ) and neurofibrillary tau in ...the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions
. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of Aβ deposits, microglia increase their expression of IL-3Rα-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of Aβ and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.
Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using ...several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.
Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant ...clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.
Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which ...contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20–140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.
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•PRC1-repressed loci form isolated self-interacting domains of compacted chromatin•Decompaction of developmentally regulated loci follows PRC1 loss upon differentiation•Chromatin compaction requires canonical PRC1, but not variant PRC1 or H2AK119Ub•PRC1-associated domains are distinct from topologically associated domains
Kundu et al. determined the 3D chromatin architecture of developmentally regulated genes repressed by PRC1 in pluripotent ESCs. PRC1 target genes are compacted into discrete domains whose formation requires the Polyhomeotic protein. These domains are lost when PRC1 binding is lost. These structures could convey epigenetic memory of gene repression.
Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood.
We ...performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF.
We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7;
<0.0001).
These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.
Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of ...valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium's response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.
The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic ...links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.
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