Functional dissection of complex and molecular trait variants at single nucleotide resolution.

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics. The majority of these variants have individually weak effects and lie in non-coding gene-regulatory elements where we lack a complete understanding of how single nucleotide alterations modulate transcriptional processes to affect human phenotypes. To address this, we measured the activity of 221,412 trait-associated variants that had been statistically fine-mapped using a Massively Parallel Reporter Assay (MPRA) in 5 diverse cell-types. We show that MPRA is able to discriminate between likely causal variants and controls, identifying 12,025 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% can plausibly be explained by the disruption of a known transcription factor (TF) binding motif. We dissect the mechanisms of 136 variants using saturation mutagenesis and assign impacted TFs for 91% of variants without a clear canonical mechanism. Finally, we provide evidence that epistasis is prevalent for variants in close proximity and identify multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants underlying complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

Corrigendum for health-related quality of life and hospitalizations in chronic thromboembolic pulmonary hypertension versus idiopathic pulmonary arterial hypertension: And analysis from the Pulmonary Hypertension Association Registry.

[This corrects the article DOI: 10.1177/20458940211053196.].

Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR-FlowFISH.

Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.

N6-Methyladenosine Inhibits Local Ribonucleolytic Cleavage to Stabilize mRNAs in Arabidopsis.

N6-methyladenosine (m6A) is a dynamic, reversible, covalently modified ribonucleotide that occurs predominantly toward 3' ends of eukaryotic mRNAs and is essential for their proper function and regulation. In Arabidopsis thaliana, many RNAs contain at least one m6A site, yet the transcriptome-wide function of m6A remains mostly unknown. Here, we show that many m6A-modified mRNAs in Arabidopsis have reduced abundance in the absence of this mark. The decrease in abundance is due to transcript destabilization caused by cleavage occurring 4 or 5 nt directly upstream of unmodified m6A sites. Importantly, we also find that, upon agriculturally relevant salt treatment, m6A is dynamically deposited on and stabilizes transcripts encoding proteins required for salt and osmotic stress response. Overall, our findings reveal that m6A generally acts as a stabilizing mark through inhibition of site-specific cleavage in plant transcriptomes, and this mechanism is required for proper regulation of the salt-stress-responsive transcriptome.

Publisher Correction: Dysregulation of the epigenetic landscape of normal aging in Alzheimer's disease.

In the version of this article initially published online, the fifth author's name was given as Alexander Amlie-Wolf. The correct name is Alexandre Amlie-Wolf. The error has been corrected in the print, PDF and HTML versions of this article.

Dysregulation of the epigenetic landscape of normal aging in Alzheimer's disease.

Aging is the strongest risk factor for Alzheimer's disease (AD), although the underlying mechanisms remain unclear. The chromatin state, in particular through the mark H4K16ac, has been implicated in aging and thus may play a pivotal role in age-associated neurodegeneration. Here we compare the genome-wide enrichment of H4K16ac in the lateral temporal lobe of AD individuals against both younger and elderly cognitively normal controls. We found that while normal aging leads to H4K16ac enrichment, AD entails dramatic losses of H4K16ac in the proximity of genes linked to aging and AD. Our analysis highlights the presence of three classes of AD-related changes with distinctive functional roles. Furthermore, we discovered an association between the genomic locations of significant H4K16ac changes with genetic variants identified in prior AD genome-wide association studies and with expression quantitative trait loci. Our results establish the basis for an epigenetic link between aging and AD.

Global analysis of ribosome-associated noncoding RNAs unveils new modes of translational regulation.

Eukaryotic transcriptomes contain a major non-protein-coding component that includes precursors of small RNAs as well as long noncoding RNA (lncRNAs). Here, we utilized the mapping of ribosome footprints on RNAs to explore translational regulation of coding and noncoding RNAs in roots of Arabidopsis thaliana shifted from replete to deficient phosphorous (Pi) nutrition. Homodirectional changes in steady-state mRNA abundance and translation were observed for all but 265 annotated protein-coding genes. Of the translationally regulated mRNAs, 30% had one or more upstream ORF (uORF) that influenced the number of ribosomes on the principal protein-coding region. Nearly one-half of the 2,382 lncRNAs detected had ribosome footprints, including 56 with significantly altered translation under Pi-limited nutrition. The prediction of translated small ORFs (sORFs) by quantitation of translation termination and peptidic analysis identified lncRNAs that produce peptides, including several deeply evolutionarily conserved and significantly Pi-regulated lncRNAs. Furthermore, we discovered that natural antisense transcripts (NATs) frequently have actively translated sORFs, including five with low-Pi up-regulation that correlated with enhanced translation of the sense protein-coding mRNA. The data also confirmed translation of miRNA target mimics and lncRNAs that produce trans-acting or phased small-interfering RNA (tasiRNA/phasiRNAs). Mutational analyses of the positionally conserved sORF of TAS3a linked its translation with tasiRNA biogenesis. Altogether, this systematic analysis of ribosome-associated mRNAs and lncRNAs demonstrates that nutrient availability and translational regulation controls protein and small peptide-encoding mRNAs as well as a diverse cadre of regulatory RNAs.

A Global View of RNA-Protein Interactions Identifies Post-transcriptional Regulators of Root Hair Cell Fate.

The Arabidopsis thaliana root epidermis is comprised of two cell types, hair and nonhair cells, which differentiate from the same precursor. Although the transcriptional programs regulating these events are well studied, post-transcriptional factors functioning in this cell fate decision are mostly unknown. Here, we globally identify RNA-protein interactions and RNA secondary structure in hair and nonhair cell nuclei. This analysis reveals distinct structural and protein binding patterns across both transcriptomes, allowing identification of differential RNA binding protein (RBP) recognition sites. Using these sequences, we identify two RBPs that regulate hair cell development. Specifically, we find that SERRATE functions in a microRNA-dependent manner to inhibit hair cell fate, while also terminating growth of root hairs mostly independent of microRNA biogenesis. In addition, we show that GLYCINE-RICH PROTEIN 8 promotes hair cell fate while alleviating phosphate starvation stress. In total, this global analysis reveals post-transcriptional regulators of plant root epidermal cell fate.

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence.

Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including glial fibrillary acidic protein and genes involved in the processing and presentation of antigens by major histocompatibility complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders.

Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.

Genome-Wide Approaches for RNA Structure Probing.

RNA molecules of all types fold into complex secondary and tertiary structures that are important for their function and regulation. Structural and catalytic RNAs such as ribosomal RNA (rRNA) and transfer RNA (tRNA) are central players in protein synthesis, and only function through their proper folding into intricate three-dimensional structures. Studies of messenger RNA (mRNA) regulation have also revealed that structural elements embedded within these RNA species are important for the proper regulation of their total level in the transcriptome. More recently, the discovery of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) has shed light on the importance of RNA structure to genome, transcriptome, and proteome regulation. Due to the relatively small number, high conservation, and importance of structural and catalytic RNAs to all life, much early work in RNA structure analysis mapped out a detailed view of these molecules. Computational and physical methods were used in concert with enzymatic and chemical structure probing to create high-resolution models of these fundamental biological molecules. However, the recent expansion in our knowledge of the importance of RNA structure to coding and regulatory RNAs has left the field in need of faster and scalable methods for high-throughput structural analysis. To address this, nuclease and chemical RNA structure probing methodologies have been adapted for genome-wide analysis. These methods have been deployed to globally characterize thousands of RNA structures in a single experiment. Here, we review these experimental methodologies for high-throughput RNA structure determination and discuss the insights gained from each approach.

The long noncoding RNA landscape in hypoxic and inflammatory renal epithelial injury.

Long noncoding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To identify potential lncRNAs relevant to acute and chronic renal epithelial injury, we performed unbiased whole transcriptome profiling of human proximal tubular epithelial cells (PTECs) in hypoxic and inflammatory conditions. RNA sequencing revealed that the protein-coding and noncoding transcriptomic landscape differed between hypoxia-stimulated and cytokine-stimulated human PTECs. Hypoxia- and inflammation-modulated lncRNAs were prioritized for focused followup according to their degree of induction by these stress stimuli, their expression in human kidney tissue, and whether exposure of human PTECs to plasma of critically ill sepsis patients with acute kidney injury modulated their expression. For three lncRNAs (MIR210HG, linc-ATP13A4-8, and linc-KIAA1737-2) that fulfilled our criteria, we validated their expression patterns, examined their loci for conservation and synteny, and defined their associated epigenetic marks. The lncRNA landscape characterized here provides insights into novel transcriptomic variations in the renal epithelial cell response to hypoxic and inflammatory stress.

Functional analysis and transcriptomic profiling of iPSC-derived macrophages and their application in modeling Mendelian disease.

RATIONALE: An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. OBJECTIVE: To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. METHODS AND RESULTS: We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus. CONCLUSIONS: Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.

Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus.

Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.

Fluoxetine- and norfluoxetine-mediated complex drug-drug interactions: in vitro to in vivo correlation of effects on CYP2D6, CYP2C19, and CYP3A4.

Fluoxetine and its circulating metabolite norfluoxetine comprise a complex multiple-inhibitor system that causes reversible or time-dependent inhibition of the cytochrome P450 (CYP) family members CYP2D6, CYP3A4, and CYP2C19 in vitro. Although significant inhibition of all three enzymes in vivo was predicted, the areas under the concentration-time curve (AUCs) for midazolam and lovastatin were unaffected by 2-week dosing of fluoxetine, whereas the AUCs of dextromethorphan and omeprazole were increased by 27- and 7.1-fold, respectively. This observed discrepancy between in vitro risk assessment and in vivo drug-drug interaction (DDI) profile was rationalized by time-varying dynamic pharmacokinetic models that incorporated circulating concentrations of fluoxetine and norfluoxetine enantiomers, mutual inhibitor-inhibitor interactions, and CYP3A4 induction. The dynamic models predicted all DDIs with less than twofold error. This study demonstrates that complex DDIs that involve multiple mechanisms, pathways, and inhibitors with their metabolites can be predicted and rationalized via characterization of all the inhibitory species in vitro.