Whole-Genome Sequencing of a Healthy Aging Cohort.

Studies of long-lived individuals have revealed few genetic mechanisms for protection against age-associated disease. Therefore, we pursued genome sequencing of a related phenotype-healthy aging-to understand the genetics of disease-free aging without medical intervention. In contrast with studies of exceptional longevity, usually focused on centenarians, healthy aging is not associated with known longevity variants, but is associated with reduced genetic susceptibility to Alzheimer and coronary artery disease. Additionally, healthy aging is not associated with a decreased rate of rare pathogenic variants, potentially indicating the presence of disease-resistance factors. In keeping with this possibility, we identify suggestive common and rare variant genetic associations implying that protection against cognitive decline is a genetic component of healthy aging. These findings, based on a relatively small cohort, require independent replication. Overall, our results suggest healthy aging is an overlapping but distinct phenotype from exceptional longevity that may be enriched with disease-protective genetic factors. VIDEO ABSTRACT.

Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3.

Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq.

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost 'capture' method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.

m6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin.

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin.

Deficient LEF1 expression is associated with lithium resistance and hyperexcitability in neurons derived from bipolar disorder patients.

Bipolar disorder (BD) is a psychiatric condition characterized by depressive and manic episodes that affect 2% of the world population. The first-line long-term treatment for mood stabilization is lithium (Li). Induced pluripotent stem cell modeling of BD using hippocampal dentate gyrus-like neurons derived from Li-responsive (LR) and Li-non-responsive (NR) patients previously showed neuronal hyperexcitability. Li treatment reversed hyperexcitability only on the LR neurons. In this study we searched for specific targets of Li resistance in NR neurons and found that the activity of Wnt/ß-catenin signaling pathway was severely affected, with a significant decrease in expression of LEF1. Li targets the Wnt/ß-catenin signaling pathway by inhibiting GSK-3ß and releasing ß-catenin that forms a nuclear complex with TCF/LEF1, activating the Wnt/ß-catenin transcription program. Therefore, we propose that downregulation of LEF1 may account for Li resistance in NR neurons. Our results show that valproic acid (VPA), a drug used to treat NR patients that also acts downstream of GSK-3ß, upregulated LEF1 and Wnt/ß-catenin gene targets, increased transcriptional activity of complex ß-catenin/TCF/LEF1, and reduced excitability in NR neurons. In addition, decreasing LEF1 expression in control neurons using shLEF1 caused hyperexcitability, confirming that the impact of VPA on excitability in NR neurons was connected to changes in LEF1 and in the Wnt/ß-catenin pathway. Our results suggest that LEF1 may be a useful target for the discovery of new drugs for BD treatment.

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS: We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. RESULTS: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. CONCLUSIONS: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

BART: bioinformatics array research tool.

BACKGROUND: Microarray experiments comprise more than half of all series in the Gene Expression Omnibus (GEO). However, downloading and analyzing raw or semi-processed microarray data from GEO is not intuitive and requires manual error-prone analysis and a bioinformatics background. This is due to a lack of standardization in array platform fabrication as well as the lack of a simple interactive tool for clustering, plotting, differential expression testing, and testing for functional enrichment. RESULTS: We introduce the Bioinformatics Array Research Tool (BART), an R Shiny web application that automates the microarray download and analysis process across diverse microarray platforms. It provides an intuitive interface, automatically downloads and parses data from GEO, suggests groupings of samples for differential expression testing, performs batch effect correction, outputs quality control plots, converts probe IDs, generates full lists of differentially expressed genes, and performs functional enrichment analysis. We show that BART enables a more comprehensive analysis of a wider range of microarray datasets on GEO by comparing it to four leading online microarray analysis tools. CONCLUSIONS: BART allows a scientist with no bioinformatics background to extract knowledge from their own microarray data or microarray experiments available from GEO. BART is functional on more microarray experiments and provides more comprehensive analyses than extant microarray analysis tools. BART is hosted on bart.salk.edu , includes a user tutorial, and is available for download from https://bitbucket.org/Luisa_amaral/bart .

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

SG-ADVISER CNV: copy-number variant annotation and interpretation.

PURPOSE: Copy-number variants have been associated with a variety of diseases, especially cancer, autism, schizophrenia, and developmental delay. The majority of clinically relevant events occur de novo, necessitating the interpretation of novel events. In this light, we present the Scripps Genome ADVISER CNV annotation pipeline and Web server, which aims to fill the gap between copy number variant detection and interpretation by performing in-depth annotations and functional predictions for copy number variants. METHODS: The Scripps Genome ADVISER CNV suite includes a Web server interface to a high-performance computing environment for calculations of annotations and a table-based user interface that allows for the execution of numerous annotation-based variant filtration strategies and statistics. RESULTS: The annotation results include details regarding location, impact on the coding portion of genes, allele frequency information (including allele frequencies from the Scripps Wellderly cohort), and overlap information with other reference data sets (including ClinVar, DGV, DECIPHER). A summary variant classification is produced (ADVISER score) based on the American College of Medical Genetics and Genomics scoring guidelines. We demonstrate >90% sensitivity/specificity for detection of pathogenic events. CONCLUSION: Scripps Genome ADVISER CNV is designed to allow users with no prior bioinformatics expertise to manipulate large volumes of copy-number variant data. Scripps Genome ADVISER CNV is available at http://genomics.scripps.edu/ADVISER/.

A genome sequencing program for novel undiagnosed diseases.

PURPOSE: The Scripps Idiopathic Diseases of Man (IDIOM) study aims to discover novel gene-disease relationships and provide molecular genetic diagnosis and treatment guidance for individuals with novel diseases using genome sequencing integrated with clinical assessment and multidisciplinary case review. Here we describe the operational protocol and initial results of the IDIOM study. METHODS: A total of 121 cases underwent first-tier review by the principal investigators to determine whether the primary inclusion criteria were satisfied, 59 (48.8%) underwent second-tier review by our clinician-scientist review panel, and 17 patients (14.0%) and their family members were enrolled. RESULTS: 60% of cases resulted in a plausible molecular diagnosis, and 18% of cases resulted in a confirmed molecular diagnosis. Two of three confirmed cases led to the identification of novel gene-disease relationships. In the third confirmed case a previously described but unrecognized disease was revealed. In all three confirmed cases a new clinical management strategy was initiated based on the genetic findings. CONCLUSION: Genome sequencing provides tangible clinical benefit for individuals with idiopathic genetic disease, not only in the context of molecular genetic diagnosis of known rare conditions but also in cases where prior clinical information regarding a new genetic disorder is lacking.

Aging compromises human islet beta cell function and identity by decreasing transcription factor activity and inducing ER stress.

Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.

Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

Activity-dependent modulation of synapse-regulating genes in astrocytes.

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.

Altered Neuronal Support and Inflammatory Response in Bipolar Disorder Patient-Derived Astrocytes.

Bipolar disorder (BD) is characterized by cyclical mood shifts. Studies indicate that BD patients have a peripheral pro-inflammatory state and alterations in glial populations in the brain. We utilized an in vitro model to study inflammation-related phenotypes of astrocytes derived from induced pluripotent stem cells (iPSCs) generated from BD patients and healthy controls. BD astrocytes showed changes in transcriptome and induced a reduction in neuronal activity when co-cultured with neurons. IL-1ß-stimulated BD astrocytes displayed a unique inflammatory gene expression signature and increased secretion of IL-6. Conditioned medium from stimulated BD astrocytes reduced neuronal activity, and this effect was partially blocked by IL-6 inactivating antibody. Our results suggest that BD astrocytes are functionally less supportive of neuronal excitability and this effect is partially mediated by IL-6. We confirmed higher IL-6 in blood in a distinct cohort of BD patients, highlighting the potential role of astrocyte-mediated inflammatory signaling in BD neuropathology.

Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.

Dynamical Electrical Complexity Is Reduced during Neuronal Differentiation in Autism Spectrum Disorder.

Neuronal activity can be modeled as a nonlinear dynamical system to yield measures of neuronal state and dysfunction. The electrical recordings of stem cell-derived neurons from individuals with autism spectrum disorder (ASD) and controls were analyzed using minimum embedding dimension (MED) analysis to characterize their dynamical complexity. MED analysis revealed a significant reduction in dynamical complexity in ASD neurons during differentiation, which was correlated to bursting and spike interval measures. MED was associated with clinical endpoints, such as nonverbal intelligence, and was correlated with 53 differentially expressed genes, which were overrepresented with ASD risk genes related to neurodevelopment, cell morphology, and cell migration. Spatiotemporal analysis also showed a prenatal temporal enrichment in cortical and deep brain structures. Together, we present dynamical analysis as a paradigm that can be used to distinguish disease-associated cellular electrophysiological and transcriptional signatures, while taking into account patient variability in neuropsychiatric disorders.

The Aging Astrocyte Transcriptome from Multiple Regions of the Mouse Brain.

Aging brains undergo cognitive decline, associated with decreased neuronal synapse number and function and altered metabolism. Astrocytes regulate neuronal synapse formation and function in development and adulthood, but whether these properties change during aging, contributing to neuronal dysfunction, is unknown. We addressed this by generating aged and adult astrocyte transcriptomes from multiple mouse brain regions. These data provide a comprehensive RNA-seq database of adult and aged astrocyte gene expression, available online as a resource. We identify astrocyte genes altered by aging across brain regions and regionally unique aging changes. Aging astrocytes show minimal alteration of homeostatic and neurotransmission-regulating genes. However, aging astrocytes upregulate genes that eliminate synapses and partially resemble reactive astrocytes. We further identified heterogeneous expression of synapse-regulating genes between astrocytes from different cortical regions. We find that alterations to astrocytes in aging create an environment permissive to synapse elimination and neuronal damage, potentially contributing to aging-associated cognitive decline.

The RNA-Binding Protein NONO Coordinates Hepatic Adaptation to Feeding.

The mechanisms by which feeding and fasting drive rhythmic gene expression for physiological adaptation to daily rhythm in nutrient availability are not well understood. Here we show that, upon feeding, the RNA-binding protein NONO accumulates within speckle-like structures in liver cell nuclei. Combining RNA-immunoprecipitation and sequencing (RIP-seq), we find that an increased number of RNAs are bound by NONO after feeding. We further show that NONO binds and regulates the rhythmicity of genes involved in nutrient metabolism post-transcriptionally. Finally, we show that disrupted rhythmicity of NONO target genes has profound metabolic impact. Indeed, NONO-deficient mice exhibit impaired glucose tolerance and lower hepatic glycogen and lipids. Accordingly, these mice shift from glucose storage to fat oxidation, and therefore remain lean throughout adulthood. In conclusion, our study demonstrates that NONO post-transcriptionally coordinates circadian mRNA expression of metabolic genes with the feeding/fasting cycle, thereby playing a critical role in energy homeostasis.