Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. ...Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa.
Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development ...largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.
To uncover the microRNA (miRNA) interactome of the osteoarthritis (OA) pathophysiological process in the cartilage.
We performed RNA sequencing in 130 samples (n=35 and n=30 pairs for messenger RNA ...(mRNA) and miRNA, respectively) on macroscopically preserved and lesioned OA cartilage from the same patient and performed differential expression (DE) analysis of miRNA and mRNAs. To build an OA-specific miRNA interactome, a prioritisation scheme was applied based on inverse Pearson's correlations and inverse DE of miRNAs and mRNAs. Subsequently, these were filtered by those present in predicted (TargetScan/microT-CDS) and/or experimentally validated (miRTarBase/TarBase) public databases. Pathway enrichment analysis was applied to elucidate OA-related pathways likely mediated by miRNA regulatory mechanisms.
We found 142 miRNAs and 2387 mRNAs to be differentially expressed between lesioned and preserved OA articular cartilage. After applying prioritisation towards likely miRNA-mRNA targets, a regulatory network of 62 miRNAs targeting 238 mRNAs was created. Subsequent pathway enrichment analysis of these mRNAs (or genes) elucidated that genes within the 'nervous system development' are likely mediated by miRNA regulatory mechanisms (familywise error=8.4×10
). Herein
encodes neurotrophin-3, which controls survival and differentiation of neurons and which is closely related to the nerve growth factor.
By an integrated approach of miRNA and mRNA sequencing data of OA cartilage, an OA miRNA interactome and related pathways were elucidated. Our functional data demonstrated interacting levels at which miRNA affects expression of genes in the cartilage and exemplified the complexity of functionally validating a network of genes that may be targeted by multiple miRNAs.
Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, ...we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal development. Analysis of multiple isogenic organ sets shows that organ-specific DNA methylation patterns are highly dynamic between week 9 (W9) and W22 of gestation. We investigate the impact of reprogramming on organ-specific DNA methylation by generating human induced pluripotent stem cell (hiPSC) lines from six isogenic organs. All isogenic hiPSCs acquire DNA methylation patterns comparable to existing hPSCs. However, hiPSCs derived from fetal brain retain brain-specific DNA methylation marks that seem sufficient to confer higher propensity to differentiate to neural derivatives. This systematic analysis of human fetal organs during development and associated isogenic hiPSC lines provides insights in the role of DNA methylation in lineage commitment and epigenetic reprogramming in humans.While DNA methylation and gene expression data are widely available for animal models, comprehensive data from human development is rarer. Here, the authors generated transcriptional and DNA methylation data from 21 organs during human development and 6 isogenic induced pluripotent stem cell lines.
Abstract
Background
Failing of intrinsic chondrocyte repair after mechanical stress is known as one of the most important initiators of osteoarthritis. Nonetheless, insight into these early ...mechano-pathophysiological processes in age-related human articular cartilage is still lacking. Such insights are needed to advance clinical development. To highlight important molecular processes of osteoarthritis mechano-pathology, the transcriptome-wide changes following injurious mechanical stress on human aged osteochondral explants were characterized.
Methods
Following mechanical stress at a strain of 65% (65%MS) on human osteochondral explants (
n
65%MS
= 14 versus
n
control
= 14), RNA sequencing was performed. Differential expression analysis between control and 65%MS was performed to determine mechanical stress-specific changes. Enrichment for pathways and protein-protein interactions was analyzed with Enrichr and STRING.
Results
We identified 156 genes significantly differentially expressed between control and 65%MS human osteochondral explants. Of note,
IGFBP5
(FC = 6.01; FDR = 7.81 × 10
−3
) and
MMP13
(FC = 5.19; FDR = 4.84 × 10
−2
) were the highest upregulated genes, while
IGFBP6
(FC = 0.19; FDR = 3.07 × 10
−4
) was the most downregulated gene. Protein-protein interactions were significantly higher than expected by chance (
P
= 1.44 × 10
−15
with connections between 116 out of 156 genes). Pathway analysis showed, among others, enrichment for cellular senescence, insulin-like growth factor (IGF) I and II binding, and focal adhesion.
Conclusions
Our results faithfully represent transcriptomic wide consequences of mechanical stress in human aged articular cartilage with
MMP13
, IGF binding proteins, and cellular senescence as the most notable results. Acquired knowledge on the as such identified initial, osteoarthritis-related, detrimental responses of chondrocytes may eventually contribute to the development of effective disease-modifying osteoarthritis treatments.
The manipulation of pregnancy diets in animals can lead to changes in DNA methylation with phenotypic consequences in the offspring. Human studies have concentrated on the effects of nutrition during ...early gestation. Lacking in humans is an epigenome-wide association study of DNA methylation in relation to perturbations in nutrition across all gestation periods.
We used the quasi-experimental setting of the Dutch famine of 1944-45 to evaluate the impact of famine exposure during specific 10-week gestation periods, or during any time in gestation, on genome-wide DNA methylation levels at age ∼ 59 years. In addition, we evaluated the impact of exposure during a shorter pre- and post-conception period. DNA methylation was assessed using the Illumina 450k array in whole blood among 422 individuals with prenatal famine exposure and 463 time- or sibling-controls without prenatal famine exposure.
Famine exposure during gestation weeks 1-10, but not weeks 11-20, 21-30 or 31-delivery, was associated with an increase in DNA methylation of CpG dinucleotides cg20823026 (FAM150B), cg10354880 (SLC38A2) and cg27370573 (PPAP2C) and a decrease of cg11496778 (OSBPL5/MRGPRG) (P < 5.9 × 10(-7), PFDR < 0.031). There was an increase in methylation of TACC1 and ZNF385A after exposure during any time in gestation (P < 2.0 × 10(-7), PFDR = 0.034) and a decrease of cg23989336 (TMEM105) after exposure around conception. These changes represent a shift of 0.3-0.6 standard deviations and are linked to genes involved in growth, development and metabolism.
Early gestation, and not mid or late gestation, is identified as a critical time-period for adult DNA methylation changes in whole blood after prenatal exposure to famine.
Summary
The accumulation of epigenetic changes was proposed to contribute to the age‐related increase in the risk of most common diseases. In this study on 230 monozygotic twin pairs (MZ pairs), aged ...18–89 years, we investigated the occurrence of epigenetic changes over the adult lifespan. Using mass spectrometry, we investigated variation in global (LINE1) DNA methylation and in DNA methylation at INS, KCNQ1OT1, IGF2, GNASAS, ABCA1, LEP, and CRH, candidate loci for common diseases. Except for KCNQ1OT1, interindividual variation in locus‐specific DNA methylation was larger in old individuals than in young individuals, ranging from 1.2‐fold larger at ABCA1 (P = 0.010) to 1.6‐fold larger at INS (P = 3.7 × 10−07). Similarly, there was more within‐MZ‐pair discordance in old as compared with young MZ pairs, except for GNASAS, ranging from an 8% increase in discordance each decade at CRH (P = 8.9 × 10−06) to a 16% increase each decade at LEP (P = 2.0 × 10−08). Still, old MZ pairs with strikingly similar DNA methylation were also observed at these loci. After 10‐year follow‐up in elderly twins, the variation in DNA methylation showed a similar pattern of change as observed cross‐sectionally. The age‐related increase in methylation variation was generally attributable to unique environmental factors, except for CRH, for which familial factors may play a more important role. In conclusion, sustained epigenetic differences arise from early adulthood to old age and contribute to an increasing discordance of MZ twins during aging.
Objective
To identify robustly differentially expressed long noncoding RNAs (lncRNAs) with osteoarthritis (OA) pathophysiology in cartilage and to explore potential target messenger RNA (mRNA) by ...establishing coexpression networks, followed by functional validation.
Methods
RNA sequencing was performed on macroscopically lesioned and preserved OA cartilage from patients who underwent joint replacement surgery due to OA (n = 98). Differential expression analysis was performed on lncRNAs that were annotated in GENCODE and Ensembl databases. To identify potential interactions, correlations were calculated between the identified differentially expressed lncRNAs and the previously reported differentially expressed protein‐coding genes in the same samples. Modulation of chondrocyte lncRNA expression was achieved using locked nucleic acid GapmeRs.
Results
By applying our in‐house pipeline, we identified 5,053 lncRNAs that were robustly expressed, of which 191 were significantly differentially expressed (according to false discovery rate) between lesioned and preserved OA cartilage. Upon integrating mRNA sequencing data, we showed that intergenic and antisense differentially expressed lncRNAs demonstrate high, positive correlations with their respective flanking sense genes. To functionally validate this observation, we selected P3H2‐AS1, which was down‐regulated in primary chondrocytes, resulting in the down‐regulation of P3H2 gene expression levels. As such, we can confirm that P3H2‐AS1 regulates its sense gene P3H2.
Conclusion
By applying an improved detection strategy, robustly differentially expressed lncRNAs in OA cartilage were detected. Integration of these lncRNAs with differential mRNA expression levels in the same samples provided insight into their regulatory networks. Our data indicates that intergenic and antisense lncRNAs play an important role in regulating the pathophysiology of OA.
Most disease-associated genetic variants are noncoding, making it challenging to design experiments to understand their functional consequences. Identification of expression quantitative trait loci ...(eQTLs) has been a powerful approach to infer the downstream effects of disease-associated variants, but most of these variants remain unexplained. The analysis of DNA methylation, a key component of the epigenome, offers highly complementary data on the regulatory potential of genomic regions. Here we show that disease-associated variants have widespread effects on DNA methylation in trans that likely reflect differential occupancy of trans binding sites by cis-regulated transcription factors. Using multiple omics data sets from 3,841 Dutch individuals, we identified 1,907 established trait-associated SNPs that affect the methylation levels of 10,141 different CpG sites in trans (false discovery rate (FDR) < 0.05). These included SNPs that affect both the expression of a nearby transcription factor (such as NFKB1, CTCF and NKX2-3) and methylation of its respective binding site across the genome. Trans methylation QTLs effectively expose the downstream effects of disease-associated variants.
Objective
To identify key determinants of the interactive pathophysiologic processes in subchondral bone and cartilage in osteoarthritis (OA).
Methods
We performed RNA sequencing on macroscopically ...preserved and lesional OA subchondral bone from patients in the Research Arthritis and Articular Cartilage study who underwent joint replacement surgery due to OA (n = 24 sample pairs: 6 hips and 18 knees). Unsupervised hierarchical clustering and differential expression analyses were conducted. Results were combined with data on previously identified differentially expressed genes in cartilage (partly overlapping samples) as well as data on recently identified OA risk genes.
Results
We identified 1,569 genes that were significantly differentially expressed between lesional and preserved subchondral bone, including CNTNAP2 (fold change FC 2.4, false discovery rate FDR 3.36 × 10−5) and STMN2 (FC 9.6, FDR 2.36 × 10−3). Among these 1,569 genes, 305 were also differentially expressed, and with the same direction of effect, in cartilage, including the recently recognized OA susceptibility genes IL11 and CHADL. Upon differential expression analysis with stratification for joint site, we identified 509 genes that were exclusively differentially expressed in subchondral bone of the knee, including KLF11 and WNT4. These genes that were differentially expressed exclusively in the knee were enriched for involvement in epigenetic processes, characterized by, e.g., HIST1H3J and HIST1H3H.
Conclusion
IL11 and CHADL were among the most consistently differentially expressed genes OA pathophysiology–related genes in both bone and cartilage. As these genes were recently also identified as robust OA risk genes, they classify as attractive therapeutic targets acting on 2 OA‐relevant tissues.
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