Johannes Waage

microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.

microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

Fish Oil–Derived Fatty Acids in Pregnancy and Wheeze and Asthma in Offspring

BACKGROUND Reduced intake of n-3 long-chain polyunsaturated fatty acids (LCPUFAs) may be a contributing factor to the increasing prevalence of wheezing disorders. We assessed the effect of supplementation with n-3 LCPUFAs in pregnant women on the risk of persistent wheeze and asthma in their offspring. METHODS We randomly assigned 736 pregnant women at 24 weeks of gestation to receive 2.4 g of n-3 LCPUFA (fish oil) or placebo (olive oil) per day. Their children formed the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) cohort and were followed prospectively with extensive clinical phenotyping. Neither the investigators nor the participants were aware of group assignments during follow-up for the first 3 years of the childrens lives, after which there was a 2-year follow-up period during which only the investigators were unaware of group assignments. The primary end point was persistent wheeze or asthma, and the secondary end points included lower respiratory tract infections, asthma exacerbations, eczema, and allergic sensitization. RESULTS A total of 695 children were included in the trial, and 95.5% completed the 3-year, double-blind follow-up period. The risk of persistent wheeze or asthma in the treatment group was 16.9%, versus 23.7% in the control group (hazard ratio, 0.69; 95% confidence interval [CI], 0.49 to 0.97; P=0.035), corresponding to a relative reduction of 30.7%. Prespecified subgroup analyses suggested that the effect was strongest in the children of women whose blood levels of eicosapentaenoic acid and docosahexaenoic acid were in the lowest third of the trial population at randomization: 17.5% versus 34.1% (hazard ratio, 0.46; 95% CI, 0.25 to 0.83; P=0.011). Analyses of secondary end points showed that supplementation with n-3 LCPUFA was associated with a reduced risk of infections of the lower respiratory tract (31.7% vs. 39.1%; hazard ratio, 0.75; 95% CI, 0.58 to 0.98; P=0.033), but there was no statistically significant association between supplementation and asthma exacerbations, eczema, or allergic sensitization. CONCLUSIONS Supplementation with n-3 LCPUFA in the third trimester of pregnancy reduced the absolute risk of persistent wheeze or asthma and infections of the lower respiratory tract in offspring by approximately 7 percentage points, or one third. (Funded by the Lundbeck Foundation and others; ClinicalTrials.gov number, NCT00798226 .).

Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns.

BackgroundNonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2.ResultsWe developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation.ConclusionsOur data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.

C/EBPα Is Required for Long-Term Self-Renewal and Lineage Priming of Hematopoietic Stem Cells and for the Maintenance of Epigenetic Configurations in Multipotent Progenitors

Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.

spliceR: an R package for classification of alternative splicing and prediction of coding potential from RNA-seq data

BackgroundRNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing and prediction of coding potential.ResultsspliceR uses the full-length transcript output from RNA-seq assemblers to detect single or multiple exon skipping, alternative donor and acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. For each of these events spliceR also annotates the genomic coordinates of the differentially spliced elements, facilitating downstream sequence analysis. For each transcript isoform fraction values are calculated to identify transcript switching between conditions. Lastly, spliceR predicts the coding potential, as well as the potential nonsense mediated decay (NMD) sensitivity of each transcript.ConclusionsspliceR is an easy-to-use tool that extends the usability of RNA-seq and assembly technologies by allowing greater depth of annotation of RNA-seq data. spliceR is implemented as an R package and is freely available from the Bioconductor repository (http://www.bioconductor.org/packages/2.13/bioc/html/spliceR.html).

UPF2 Is a Critical Regulator of Liver Development, Function and Regeneration

Background Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full regulatory potential of the NMD pathway in mammals will require the functional assessment of NMD in different tissues. Methodology/Principal Findings Here we use mouse genetics to address the role of UPF2, a core NMD component, in the development, function and regeneration of the liver. We find that loss of NMD during fetal liver development is incompatible with postnatal life due to failure of terminal differentiation. Moreover, deletion of Upf2 in the adult liver results in hepatosteatosis and disruption of liver homeostasis. Finally, NMD was found to be absolutely required for liver regeneration. Conclusion/Significance Collectively, our data demonstrate the critical role of the NMD pathway in liver development, function and regeneration and highlights the importance of NMD for mammalian biology.

Temporal mapping of CEBPA and CEBPB binding during liver regeneration reveals dynamic occupancy and specific regulatory codes for homeostatic and cell cycle gene batteries

Dynamic shifts in transcription factor binding are central to the regulation of biological processes by allowing rapid changes in gene transcription. However, very few genome-wide studies have examined how transcription factor occupancy is coordinated temporally in vivo in higher animals. Here, we quantified the genome-wide binding patterns of two key hepatocyte transcription factors, CEBPA and CEBPB (also known as C/EBPalpha and C/EBPbeta), at multiple time points during the highly dynamic process of liver regeneration elicited by partial hepatectomy in mouse. Combining these profiles with RNA polymerase II binding data, we find three temporal classes of transcription factor binding to be associated with distinct sets of regulated genes involved in the acute phase response, metabolic/homeostatic functions, or cell cycle progression. Moreover, we demonstrate a previously unrecognized early phase of homeostatic gene expression prior to S-phase entry. By analyzing the three classes of CEBP bound regions, we uncovered mutually exclusive sets of sequence motifs, suggesting temporal codes of CEBP recruitment by differential cobinding with other factors. These findings were validated by sequential ChIP experiments involving a panel of central transcription factors and/or by comparison to external ChIP-seq data. Our quantitative investigation not only provides in vivo evidence for the involvement of many new factors in liver regeneration but also points to similarities in the circuitries regulating self-renewal of differentiated cells. Taken together, our work emphasizes the power of global temporal analyses of transcription factor occupancy to elucidate mechanisms regulating dynamic biological processes in complex higher organisms.

Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies

BackgroundThere is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons into operational taxonomic units (OTUs). Strategies for detecting differential relative abundance of OTUs between sample conditions include classical statistical approaches as well as a plethora of newer methods, many borrowing from the related field of RNA-seq analysis. This effort is complicated by unique data characteristics, including sparsity, sequencing depth variation, and nonconformity of read counts to theoretical distributions, which is often exacerbated by exploratory and/or unbalanced study designs. Here, we assess the robustness of available methods for (1) inference in differential relative abundance analysis and (2) beta-diversity-based sample separation, using a rigorous benchmarking framework based on large clinical 16S microbiome datasets from different sources.ResultsRunning more than 380,000 full differential relative abundance tests on real datasets with permuted case/control assignments and in silico-spiked OTUs, we identify large differences in method performance on a range of parameters, including false positive rates, sensitivity to sparsity and case/control balances, and spike-in retrieval rate. In large datasets, methods with the highest false positive rates also tend to have the best detection power. For beta-diversity-based sample separation, we show that library size normalization has very little effect and that the distance metric is the most important factor in terms of separation power.ConclusionsOur results, generalizable to datasets from different sequencing platforms, demonstrate how the choice of method considerably affects analysis outcome. Here, we give recommendations for tools that exhibit low false positive rates, have good retrieval power across effect sizes and case/control proportions, and have low sparsity bias. Result output from some commonly used methods should be interpreted with caution. We provide an easily extensible framework for benchmarking of new methods and future microbiome datasets.

Genetics of allergy and allergic sensitization: common variants, rare mutations.

Our understanding of the specific genetic lesions in allergy has improved in recent years due to identification of common risk variants from genome-wide association studies (GWAS) and studies of rare, monogenic diseases. Large-scale GWAS have identified novel susceptibility loci and provided information about shared genetics between allergy, related phenotypes and autoimmunity. Studies of monogenic diseases have elucidated critical cellular pathways and protein functions responsible for allergy. These complementary approaches imply genetic mechanisms involved in Th2 immunity, T-cell differentiation, TGFβ signaling, regulatory T-cell function and skin/mucosal function as well as yet unknown mechanisms associated with newly identified genes. Future studies, in combination with data on gene expression and epigenetics, are expected to increase our understanding of the pathogenesis of allergy.

Prenatal vitamin D supplementation reduces risk of asthma/recurrent wheeze in early childhood: A combined analysis of two randomized controlled trials

Background We recently published two independent randomized controlled trials of vitamin D supplementation during pregnancy, both indicating a >20% reduced risk of asthma/recurrent wheeze in the offspring by 3 years of age. However, neither reached statistical significance. Objective To perform a combined analysis of the two trials and investigate whether maternal 25-hydroxy-vitamin D (25(OH)D) level at trial entry modified the intervention effect. Methods VDAART (N = 806) and COPSAC2010. (N = 581) randomized pregnant women to daily high-dose vitamin D3 (4,000 IU/d and 2,400 IU/d, respectively) or placebo. All women also received a prenatal vitamin containing 400 IU/d vitamin D3. The primary outcome was asthma/recurrent wheeze from 0-3yrs. Secondary end-points were specific IgE, total IgE, eczema and lower respiratory tract infections (LRTI). We conducted random effects combined analyses of the treatment effect, individual patient data (IPD) meta-analyses, and analyses stratified by 25(OH)D level at study entry. Results The analysis showed a 25% reduced risk of asthma/recurrent wheeze at 0-3yrs: adjusted odds ratio (aOR) = 0.74 (95% CI, 0.57–0.96), p = 0.02. The effect was strongest among women with 25(OH)D level ≥30ng/ml at study entry: aOR = 0.54 (0.33–0.88), p = 0.01, whereas no significant effect was observed among women with 25(OH)D level <30ng/ml at study entry: aOR = 0.84 (0.62–1.15), p = 0.25. The IPD meta-analyses showed similar results. There was no effect on the secondary end-points. Conclusions This combined analysis shows that vitamin D supplementation during pregnancy results in a significant reduced risk of asthma/recurrent wheeze in the offspring, especially among women with 25(OH)D level ≥ 30 ng/ml at randomization, where the risk was almost halved. Future studies should examine the possibility of raising 25(OH)D levels to at least 30 ng/ml early in pregnancy or using higher doses than used in our studies. Trial registration COPSAC2010: ClinicalTrials.gov NCT00856947; VDAART: ClinicalTrials.gov NCT00920621