Human promoters are intrinsically directional.

Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in HeLa cells revealed that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that about half of promoters are unidirectional and that unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process but rather the consequence of the presence of both forward- and reverse-directed core promoters.

Identification and activity of the functional complex between hnRNPL and the pseudoexfoliation syndrome-associated lncRNA, LOXL1-AS1.

Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

DNA methylation signatures of aggression and closely related constructs: A meta-analysis of epigenome-wide studies across the lifespan.

DNA methylation profiles of aggressive behavior may capture lifetime cumulative effects of genetic, stochastic, and environmental influences associated with aggression. Here, we report the first large meta-analysis of epigenome-wide association studies (EWAS) of aggressive behavior (N = 15,324 participants). In peripheral blood samples of 14,434 participants from 18 cohorts with mean ages ranging from 7 to 68 years, 13 methylation sites were significantly associated with aggression (alpha = 1.2 × 10-7; Bonferroni correction). In cord blood samples of 2425 children from five cohorts with aggression assessed at mean ages ranging from 4 to 7 years, 83% of these sites showed the same direction of association with childhood aggression (r = 0.74, p = 0.006) but no epigenome-wide significant sites were found. Top-sites (48 at a false discovery rate of 5% in the peripheral blood meta-analysis or in a combined meta-analysis of peripheral blood and cord blood) have been associated with chemical exposures, smoking, cognition, metabolic traits, and genetic variation (mQTLs). Three genes whose expression levels were associated with top-sites were previously linked to schizophrenia and general risk tolerance. At six CpGs, DNA methylation variation in blood mirrors variation in the brain. On average 44% (range = 3-82%) of the aggression-methylation association was explained by current and former smoking and BMI. These findings point at loci that are sensitive to chemical exposures with potential implications for neuronal functions. We hope these results to be a starting point for studies leading to applications as peripheral biomarkers and to reveal causal relationships with aggression and related traits.

Latent transcriptional variations of individual Plasmodium falciparum uncovered by single-cell RNA-seq and fluorescence imaging.

Malaria parasites follow a complex life cycle that consists of multiple stages that span from the human host to the mosquito vector. Among the species causing malaria, Plasmodium falciparum is the most lethal, with clinical symptoms manifesting during the intraerythrocytic developmental cycle (IDC). During the IDC, P. falciparum progresses through a synchronous and continuous cascade of transcriptional programming previously established using population analyses. While individual parasites are known to exhibit transcriptional variations to evade the host immune system or commit to a sexual fate, such rare expression heterogeneity is largely undetectable on a population level. Therefore, we combined single-cell RNA-sequencing (scRNA-seq) on a microfluidic platform and fluorescence imaging to delineate the transcriptional variations among individual parasites during late asexual and sexual stages. The comparison between asexual and sexual parasites uncovered a set of previously undefined sex-specific genes. Asexual parasites were segregated into three distinct clusters based on the differential expression of genes encoding SERAs, rhoptry proteins, and EXP2 plus transporters. Multiple pseudotime analyses revealed that these stage-specific transitions are distinct. RNA fluorescent in situ hybridization of cluster-specific genes validated distinct stage-specific expression and transitions during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.

Effect of Bicarbonate on Net Acid Excretion, Blood Pressure, and Metabolism in Patients With and Without CKD: The Acid Base Compensation in CKD Study.

RATIONALE & OBJECTIVE: Patients with CKD are at elevated risk of metabolic acidosis due to impaired net acid excretion (NAE). Identifying early markers of acidosis may guide prevention in chronic kidney disease (CKD). This study compared NAE in participants with and without CKD, as well as the NAE, blood pressure (BP), and metabolomic response to bicarbonate supplementation. STUDY DESIGN: Randomized order, cross-over study with controlled feeding. SETTING & PARTICIPANTS: Participants consisted of 8 patients with CKD (estimated glomerular filtration rate 30-59mL/min/1.73m2 or 60-70mL/min/1.73m2 with albuminuria) and 6 patients without CKD. All participants had baseline serum bicarbonate concentrations between 20 and 28 mEq/L; they did not have diabetes mellitus and did not use alkali supplements at baseline. INTERVENTION: Participants were fed a fixed-acid-load diet with bicarbonate supplementation (7 days) and with sodium chloride control (7 days) in a randomized order, cross-over fashion. OUTCOMES: Urine NAE, 24-hour ambulatory BP, and 24-hour urine and plasma metabolomic profiles were measured after each period. RESULTS: During the control period, mean NAE was 28.3±10.2 mEq/d overall without differences across groups (P=0.5). Urine pH, ammonium, and citrate were significantly lower in CKD than in non-CKD (P<0.05 for each). Bicarbonate supplementation reduced NAE and urine ammonium in the CKD group, increased urine pH in both groups (but more in patients with CKD than in those without), and increased; urine citrate in the CKD group (P< 0.2 for interaction for each). Metabolomic analysis revealed several urine organic anions were increased with bicarbonate in CKD, including 3-indoleacetate, citrate/isocitrate, and glutarate. BP was not significantly changed. LIMITATIONS: Small sample size and short feeding duration. CONCLUSIONS: Compared to patients without CKD, those with CKD had lower acid excretion in the form of ammonium but also lower base excretion such as citrate and other organic anions, a potential compensation to preserve acid-base homeostasis. In CKD, acid excretion decreased further, but base excretion (eg, citrate) increased in response to alkali. Urine citrate should be evaluated as an early and responsive marker of impaired acid-base homeostasis. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and the Duke O'Brien Center for Kidney Research. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT02427594.

Characterizing genetic and environmental influences on variable DNA methylation using monozygotic and dizygotic twins.

Variation in DNA methylation is being increasingly associated with health and disease outcomes. Although DNA methylation is hypothesized to be a mechanism by which both genetic and non-genetic factors can influence the regulation of gene expression, little is known about the extent to which DNA methylation at specific sites is influenced by heritable as well as environmental factors. We quantified DNA methylation in whole blood at age 18 in a birth cohort of 1,464 individuals comprising 426 monozygotic (MZ) and 306 same-sex dizygotic (DZ) twin pairs. Site-specific levels of DNA methylation were more strongly correlated across the genome between MZ than DZ twins. Structural equation models revealed that although the average contribution of additive genetic influences on DNA methylation across the genome was relatively low, it was notably elevated at the highly variable sites characterized by intermediate levels of DNAm that are most relevant for epigenetic epidemiology. Sites at which variable DNA methylation was most influenced by genetic factors were significantly enriched for DNA methylation quantitative trait loci (mQTL) effects, and overlapped with sites where inter-individual variation correlates across tissues. Finally, we show that DNA methylation at sites robustly associated with environmental exposures such as tobacco smoking and obesity is also influenced by additive genetic effects, highlighting the need to control for genetic background in analyses of exposure-associated DNA methylation differences. Estimates of the contribution of genetic and environmental influences to DNA methylation at all sites profiled in this study are available as a resource for the research community (http://www.epigenomicslab.com/online-data-resources).

Noncanonical autophagy in dermal dendritic cells mediates immunosuppressive effects of UV exposure.

BACKGROUND: Control of the inflammatory response is critical to maintaining homeostasis, and failure to do so contributes to the burden of chronic inflammation associated with several disease states. The mechanisms that underlie immunosuppression, however, remain largely unknown. Although defects in autophagy machinery have been associated with inflammatory pathologic conditions, we now appreciate that autophagic components participate in noncanonical pathways distinct from classical autophagy. We have previously demonstrated that LC3-associated phagocytosis (LAP), a noncanonical autophagic process dependent on Rubicon (rubicon autophagy regulator [RUBCN]), contributes to immunosuppression. OBJECTIVE: We used Rubcn-/- mice to examine the role of the LAP pathway in mediating the UV-induced immunotolerant program in a model of contact hypersensitivity (CHS). METHODS: Flow cytometry and transcriptional analysis were used to measure immune cell infiltration and activation in the skin of Rubcn+/+ and Rubcn-/- mice during the CHS response. RESULTS: Here, we demonstrate that LAP is required for UV-induced immunosuppression and that UV exposure induces a broadly anti-inflammatory transcriptional program dependent on Rubicon. Rubcn-/- mice are resistant to UV-induced immunosuppression and instead display exaggerated inflammation in a model of CHS. Specifically, RUBCN deficiency in CD301b+ dermal dendritic cells results in their increased antigen presentation capacity and subsequent hyperactivation of the CD8+ T-cell response. CONCLUSIONS: LAP functions to limit the immune response and is critical in maintaining the balance between homeostasis and inflammation.

A polygenic score for age-at-first-birth predicts disinhibition.

BACKGROUND: A recent genome-wide association study identified molecular-genetic associations with age-at-first-birth. However, the meaning of these genetic discoveries is unclear. Drawing on evidence linking early pregnancy with disinhibitory behavior, we tested the hypothesis that genetic discoveries for age-at-first-birth predict disinhibition. METHODS: We included participants with genotype data from the two-decade-long Environmental Risk (E-Risk) Study (N = 1,999) and the four-decade-long Dunedin Study (N = 918). We calculated a genome-wide polygenic score for age-at-first-birth and tested whether it was associated with a range of disinhibitory outcomes across the life course, including low childhood self-control; risk for externalizing psychopathology; officially recorded criminal offending; substance dependence; informant reports of disinhibitory problems; and number of lifetime sexual partners. We further tested whether associations were attributable to accelerated pubertal maturation. RESULTS: In both cohorts, the age-at-first-birth polygenic score predicted low childhood self-control, externalizing psychopathology, officially recorded criminal offending, substance dependence, and number of sexual partners. Associations were modest, but robust across replication. Childhood disinhibition partly mediated associations between the polygenic score and reproductive behaviors. In contrast, associations were not attributable to accelerated pubertal timing. CONCLUSIONS: Genomic discoveries for age-at-first-birth are about more than reproductive biology: They provide insight into the disinhibitory traits and behaviors that accompany early parenthood. Age-at-first-birth is a useful proxy phenotype for researchers interested in disinhibition. Further, interventions that improve self-regulation abilities may benefit young parents and their children.

A Polygenic Score for Higher Educational Attainment is Associated with Larger Brains.

People who score higher on intelligence tests tend to have larger brains. Twin studies suggest the same genetic factors influence both brain size and intelligence. This has led to the hypothesis that genetics influence intelligence partly by contributing to the development of larger brains. We tested this hypothesis using four large imaging genetics studies (combined N = 7965) with polygenic scores derived from a genome-wide association study (GWAS) of educational attainment, a correlate of intelligence. We conducted meta-analysis to test associations among participants' genetics, total brain volume (i.e., brain size), and cognitive test performance. Consistent with previous findings, participants with higher polygenic scores achieved higher scores on cognitive tests, as did participants with larger brains. Participants with higher polygenic scores also had larger brains. We found some evidence that brain size partly mediated associations between participants' education polygenic scores and their cognitive test performance. Effect sizes were larger in the population-based samples than in the convenience-based samples. Recruitment and retention of population-representative samples should be a priority for neuroscience research. Findings suggest promise for studies integrating GWAS discoveries with brain imaging to understand neurobiology linking genetics with cognitive performance.

Using DNA From Mothers and Children to Study Parental Investment in Children's Educational Attainment.

This study tested implications of new genetic discoveries for understanding the association between parental investment and children's educational attainment. A novel design matched genetic data from 860 British mothers and their children with home-visit measures of parenting: the E-Risk Study. Three findings emerged. First, both mothers' and children's education-associated genetics, summarized in a genome-wide polygenic score, were associated with parenting-a gene-environment correlation. Second, accounting for genetic influences slightly reduced associations between parenting and children's attainment-indicating some genetic confounding. Third, mothers' genetics were associated with children's attainment over and above children's own genetics, via cognitively stimulating parenting-an environmentally mediated effect. Findings imply that, when interpreting parents' effects on children, environmentalists must consider genetic transmission, but geneticists must also consider environmental transmission.

Arginase1 Deficiency in Monocytes/Macrophages Upregulates Inducible Nitric Oxide Synthase To Promote Cutaneous Contact Hypersensitivity.

The innate immune components that modulate allergic contact hypersensitivity (CHS) responses are poorly defined. Using human skin from contact dermatitis patients and a mouse model of CHS, we find that hapten allergens disrupt the Arginase1 (Arg1) and inducible NO synthase (iNOS) dynamic in monocytes/macrophages (mono/MΦ), which renders those cells ineffectual in suppressing skin inflammation. Mice lacking Arg1 in MΦ develop increased CHS characterized by elevated ear thickening, mono/MΦ-dominated dermal inflammation, and increased iNOS and IL-6 expression compared with control mice. Treatment of Arg1flox/flox; LysMCre+/- mice with a selective NOS inhibitor or knockout of Nos2, encoding iNOS, significantly ameliorates CHS. Our findings suggest a critical role for Arg1 in mono/MΦ in suppressing CHS through dampening Nos2 expression. These results support that increasing Arg1 may be a potential therapeutic avenue in treating allergic contact dermatitis.

Correction of Biochemical Abnormalities and Improved Muscle Function in a Phase I/II Clinical Trial of Clenbuterol in Pompe Disease.

This 52-week, phase I/II double-blind, randomized, placebo-controlled study investigated the novel use of clenbuterol in late-onset Pompe disease (LOPD) stably treated with ERT. Eleven of thirteen participants completed the study. No serious adverse events were related to clenbuterol, and transient minor adverse events included mild elevations of creatine kinase, muscle spasms, and tremors. At week 52, the 6-min walk test distance increased by a mean of 16 m (p = 0.08), or a mean of 3% of predicted performance (p = 0.03), and the maximum inspiratory pressure increased 8% (p = 0.003) for the clenbuterol group. The quick motor function test score improved by a mean of seven points (p = 0.007); and the gait, stairs, gower, chair test improved by a mean of two points (p = 0.004). Clenbuterol decreased glycogen content in the vastus lateralis by 50% at week 52. Transcriptome analysis revealed more normal muscle gene expression for 38 of 44 genes related to Pompe disease following clenbuterol. The placebo group demonstrated no significant changes over the course of the study. This study provides initial evidence for safety and efficacy of adjunctive clenbuterol in patients with LOPD (NCT01942590).

Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability.

RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

Eleven Telomere, Epigenetic Clock, and Biomarker-Composite Quantifications of Biological Aging: Do They Measure the Same Thing?

The geroscience hypothesis posits that therapies to slow biological processes of aging can prevent disease and extend healthy years of life. To test such "geroprotective" therapies in humans, outcome measures are needed that can assess extension of disease-free life span. This need has spurred development of different methods to quantify biological aging. But different methods have not been systematically compared in the same humans. We implemented 7 methods to quantify biological aging using repeated-measures physiological and genomic data in 964 middle-aged humans in the Dunedin Study (New Zealand; persons born 1972-1973). We studied 11 measures in total: telomere-length and erosion, 3 epigenetic-clocks and their ticking rates, and 3 biomarker-composites. Contrary to expectation, we found low agreement between different measures of biological aging. We next compared associations between biological aging measures and outcomes that geroprotective therapies seek to modify: physical functioning, cognitive decline, and subjective signs of aging, including aged facial appearance. The 71-cytosine-phosphate-guanine epigenetic clock and biomarker composites were consistently related to these aging-related outcomes. However, effect sizes were modest. Results suggested that various proposed approaches to quantifying biological aging may not measure the same aspects of the aging process. Further systematic evaluation and refinement of measures of biological aging is needed to furnish outcomes for geroprotector trials.

FMRP targets distinct mRNA sequence elements to regulate protein expression.

Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1(-/-) mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.

Quantification of biological aging in young adults.

Antiaging therapies show promise in model organism research. Translation to humans is needed to address the challenges of an aging global population. Interventions to slow human aging will need to be applied to still-young individuals. However, most human aging research examines older adults, many with chronic disease. As a result, little is known about aging in young humans. We studied aging in 954 young humans, the Dunedin Study birth cohort, tracking multiple biomarkers across three time points spanning their third and fourth decades of life. We developed and validated two methods by which aging can be measured in young adults, one cross-sectional and one longitudinal. Our longitudinal measure allows quantification of the pace of coordinated physiological deterioration across multiple organ systems (e.g., pulmonary, periodontal, cardiovascular, renal, hepatic, and immune function). We applied these methods to assess biological aging in young humans who had not yet developed age-related diseases. Young individuals of the same chronological age varied in their "biological aging" (declining integrity of multiple organ systems). Already, before midlife, individuals who were aging more rapidly were less physically able, showed cognitive decline and brain aging, self-reported worse health, and looked older. Measured biological aging in young adults can be used to identify causes of aging and evaluate rejuvenation therapies.

The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery.

The TCT motif (polypyrimidine initiator) encompasses the transcription start site of nearly all ribosomal protein genes in Drosophila and mammals. The TCT motif is required for transcription of ribosomal protein gene promoters. The TCT element resembles the Inr (initiator), but is not recognized by TFIID and cannot function in lieu of an Inr. However, a single T-to-A substitution converts the TCT element into a functionally active Inr. Thus, the TCT motif is a novel transcriptional element that is distinct from the Inr. These findings reveal a specialized TCT-based transcription system that is directed toward the synthesis of ribosomal proteins.