An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.

A genome-scale resource for in vivo tag-based protein function exploration in C. elegans.

Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.

Analysis of cell fate from single-cell gene expression profiles in C. elegans.

The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.

An elt-3/elt-5/elt-6 GATA transcription circuit guides aging in C. elegans.

To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage.

A shared genetic architecture between adhesive capsulitis and Dupuytren disease.

BACKGROUND: The etiology of adhesive capsulitis involves inflammation, thickening, and fibrosis of the shoulder capsule. The underlying genetic factors are poorly understood. The purpose of this study was to identify genetic variants associated with adhesive capsulitis using the UK Biobank (UKB) cohort and compare them with variants associated with Dupuytren disease investigating a common etiology between the 2 fibrotic disorders. METHODS: A genome-wide association study (GWAS) was performed using data from UKB with 10,773 cases of adhesive capsulitis, and a second GWAS was performed with 8891 cases of Dupuytren disease. Next, a comparison of association statistics was performed between adhesive capsulitis and Dupuytren disease using the data from both GWAS. Finally, single-nucleotide polymorphisms (SNPs) previously reported from candidate gene studies for adhesive capsulitis and Dupuytren disease were tested for association with adhesive capsulitis and Dupuytren disease using the summary statistics from their respective GWAS. RESULTS: The UKB GWAS for adhesive capsulitis identified 6 loci that reached genome-wide statistical significance: a cluster of 11 closely linked SNPs on chromosome 1; a single SNP on chromosome 2; a single SNP on chromosome 14; 2 closely linked SNPs on chromosome 21; 33 closely linked SNPs on chromosome 22; and 3 closely linked SNPs on the X chromosome. These SNPs were associated with 8 different genes including TSPAN2/NGF, SATB2, MRPL52/MMP14, ERG, WNT7B, and FGF13. A GWAS for Dupuytren disease was performed and a comparison to the adhesive capsulitis GWAS showed 13 loci significantly associated with both phenotypes. A validation attempt of 6 previously reported SNPs associated with adhesive capsulitis using UKB summary statistics was unable to confirm any of the previously reported SNPs (all P > .19). All 23 previously reported SNPs associated with Dupuytren disease were confirmed using the UKB summary statistics (P < 2.1 × 10-3) CONCLUSION: This GWAS investigating adhesive capsulitis has identified 6 novel loci involving 8 different genes to be associated with adhesive capsulitis. A GWAS investigating Dupuytren disease was performed and compared to the adhesive capsulitis GWAS, and 13 common loci were identified between the 2 disorders with genes involved in pathologic fibrosis. We were unable to validate the SNPs in candidate genes previously reported to be associated with adhesive capsulitis although we were able to confirm all previously reported SNPs associated with Dupuytren disease. The strong genetic overlap between the adhesive capsulitis and Dupuytren disease loci suggests a similar etiology between the 2 diseases.

Maximum reproductive lifespan correlates with CD33rSIGLEC gene number: Implications for NADPH oxidase-derived reactive oxygen species in aging.

Humans and orcas are among the very rare species that have a prolonged post-reproductive lifespan (PRLS), during which the aging process continues. Reactive oxygen species (ROS) derived from mitochondria and from the NADPH oxidase (NOX) enzymes of innate immune cells are known to contribute to aging, with the former thought to be dominant. CD33-related-Siglecs are immune receptors that recognize self-associated-molecular-patterns and modulate NOX-derived-ROS. We herewith demonstrate a strong correlation of lifespan with CD33rSIGLEC gene number in 26 species, independent of body weight or phylogeny. The correlation is stronger when considering total CD33rSIGLEC gene number rather than those encoding inhibitory and activating subsets, suggesting that lifetime balancing of ROS is important. Combining independent lines of evidence including the short half-life and spontaneous activation of neutrophils, we calculate that even without inter-current inflammation, a major source of lifetime ROS exposure may actually be neutrophil NOX-derived. However, genomes of human supercentenarians (>110 years) do not harbor a significantly higher number of functional CD33rSIGLEC genes. Instead, lifespan correlation with CD33rSIGLEC gene number was markedly strengthened by excluding the post-reproductive lifespan of humans and orcas (R2  = 0.83; P < .0001). Thus, CD33rSIGLEC modulation of ROS likely contributes to maximum reproductive lifespan, but other unknown mechanisms could be important to PRLS.

A genome-wide association study for shoulder impingement and rotator cuff disease.

BACKGROUND: The purpose of the study was to identify genetic variants associated with rotator cuff disease by performing a genome-wide association study (GWAS) for shoulder impingement using the UK Biobank (UKB) cohort and then combining the GWAS data with a prior GWAS for rotator cuff tears. The loci identified by the GWAS and meta-analysis were examined for changes in expression following rotator cuff tearing using RNA sequencing. METHODS: A GWAS was performed using data from UKB with 3864 cases of shoulder impingement. The summary statistics from shoulder impingement and a prior study on rotator cuff tears were combined in a meta-analysis. Also, the previous association of 2 single-nucleotide polymorphisms (SNPs) with shoulder impingement from a published GWAS using the UKB was tested. Rotator cuff tendon biopsies were obtained from 24 patients with full-thickness rotator cuff tears who underwent arthroscopic rotator cuff repair (cases) and 9 patients who underwent open reduction internal fixation for a proximal humeral fracture (controls). Total RNA was extracted and differential gene expression was measured by RNA sequencing for genes with variants associated with rotator cuff tearing. RESULTS: The shoulder impingement GWAS identified 4 new loci: LOC100506457, LSP1P3, LOC100506207, and MIS18BP1/LINC00871. Combining data with a prior GWAS for rotator cuff tears in a meta-analysis resulted in the identification of an additional 7 loci: SLC39A8/UBE2D3, C5orf63, ASTN2, STK24, FRMPD4, ACOT9/SAT1, and LINC00890/ALG13. Many of the identified loci have known biologic functions or prior associations with diseases, suggesting possible biologic pathways leading to rotator cuff disease. RNA sequencing experiments show that expression of STK24 increases whereas expression of SAT1 and UBE2D3 decreases following rotator cuff tearing. Two SNPs previously reported to show an association with shoulder impingement from a prior UKB GWAS were not validated in our study. CONCLUSION: This is the first GWAS for shoulder impingement in which new data from UKB enabled the identification of 4 loci showing a genetic association. A meta-analysis with a prior GWAS for rotator cuff tearing identified an additional 7 loci. The known biologic roles of many of the 11 loci suggest plausible biologic mechanisms underlying the etiology of rotator cuff disease. The risk alleles from each of the genetic loci can be used to assess the risk for rotator cuff disease in individual patients, enabling preventative or restorative actions via personalized medicine.

Genetic variants associated with rotator cuff tearing utilizing multiple population-based genetic resources.

BACKGROUND: The etiology of rotator cuff tearing is likely multifactorial, including a potential genetic predisposition. The purpose of the study was to identify genetic variants associated with rotator cuff tearing utilizing the UK Biobank (UKB) cohort, confirm variants using a separate genetic database, and evaluate tissue expression of genes with associated variants following rotator cuff tearing using RNA sequencing. METHODS: Genome-wide association study (GWAS): A GWAS was performed using data from UKB with 5701 cases of rotator cuff injury. RNA sequencing analyses: rotator cuff biopsies were obtained from 24 patients with full-thickness rotator cuff tears who underwent arthroscopic rotator cuff repair (cases) and 9 patients who underwent open reduction internal fixation for a proximal humerus fracture (controls). Total RNA was extracted and differential gene expression was measured by RNAseq for genes with variants associated with rotator cuff tearing. RESULTS: The results of the UKB GWAS identified 3 loci that reached genome-wide statistical significance: 2 loci on chromosome 7 in GLCCI1 (rs4725069; P = 5.0E-09) and THSD7A (rs575224171; P = 5.3E-09), and 1 locus on chromosome 2 in ZNF804A (rs775583810; P = 3.9E-09). The association with rotator cuff injury of the GLCCI1 single-nucleotide polymorphism (SNP; rs4725069) was confirmed in the Kaiser Permanente Research Bank cohort (P = .008). Twenty previously reported SNPs in 12 genes were evaluated using summary statistics from the UKB GWAS, which confirmed 3 SNPs in TNC with rotator cuff injury (rs1138545, rs72758637, and rs7021589; all P < .0024). Of 17 genes with variants associated with rotator cuff injury (14 previously from literature plus 3 new genes from current UKB GWAS), TIMP2, Col5A1, TGFBR1, and TNC were upregulated (P < .001 for all) and THSD7A was downregulated (P = .005) in tears vs. controls in the RNA sequencing data set. CONCLUSION: The UKB GWAS has identified 3 novel loci associated with rotator cuff tearing (ZNF804A, GLCCI1, THSD7A). Expression of the THSD7A gene was significantly downregulated in rotator cuff tears vs. controls supporting a potential functional role. Three previously reported SNPs in the TNC gene were validated in the UKB GWAS, supporting a role for this gene in rotator cuff tearing. Finally, TIMP2, Col5A1, TGFBR1, and TNC genes were found to have significantly upregulated tissue expression in cases vs. controls supporting a biologic role in tearing for these genes.

Deactivation of the GATA Transcription Factor ELT-2 Is a Major Driver of Normal Aging in C. elegans.

To understand the molecular processes underlying aging, we screened modENCODE ChIP-seq data to identify transcription factors that bind to age-regulated genes in C. elegans. The most significant hit was the GATA transcription factor encoded by elt-2, which is responsible for inducing expression of intestinal genes during embryogenesis. Expression of ELT-2 decreases during aging, beginning in middle age. We identified genes regulated by ELT-2 in the intestine during embryogenesis, and then showed that these developmental genes markedly decrease in expression as worms grow old. Overexpression of elt-2 extends lifespan and slows the rate of gene expression changes that occur during normal aging. Thus, our results identify the developmental regulator ELT-2 as a major driver of normal aging in C. elegans.

Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans.

Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging.

The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney.

Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney.

Genome-Wide Scan Informed by Age-Related Disease Identifies Loci for Exceptional Human Longevity.

We developed a new statistical framework to find genetic variants associated with extreme longevity. The method, informed GWAS (iGWAS), takes advantage of knowledge from large studies of age-related disease in order to narrow the search for SNPs associated with longevity. To gain support for our approach, we first show there is an overlap between loci involved in disease and loci associated with extreme longevity. These results indicate that several disease variants may be depleted in centenarians versus the general population. Next, we used iGWAS to harness information from 14 meta-analyses of disease and trait GWAS to identify longevity loci in two studies of long-lived humans. In a standard GWAS analysis, only one locus in these studies is significant (APOE/TOMM40) when controlling the false discovery rate (FDR) at 10%. With iGWAS, we identify eight genetic loci to associate significantly with exceptional human longevity at FDR < 10%. We followed up the eight lead SNPs in independent cohorts, and found replication evidence of four loci and suggestive evidence for one more with exceptional longevity. The loci that replicated (FDR < 5%) included APOE/TOMM40 (associated with Alzheimer's disease), CDKN2B/ANRIL (implicated in the regulation of cellular senescence), ABO (tags the O blood group), and SH2B3/ATXN2 (a signaling gene that extends lifespan in Drosophila and a gene involved in neurological disease). Our results implicate new loci in longevity and reveal a genetic overlap between longevity and age-related diseases and traits, including coronary artery disease and Alzheimer's disease. iGWAS provides a new analytical strategy for uncovering SNPs that influence extreme longevity, and can be applied more broadly to boost power in other studies of complex phenotypes.

Two Genetic Variants Associated with Plantar Fascial Disorders.

Plantar fascial disorder is comprised of plantar fasciitis and plantar fibromatosis. Plantar fasciitis is the most common cause of heel pain, especially for athletes involved in running and jumping sports. Plantar fibromatosis is a rare fibrous hyperproliferation of the deep connective tissue of the foot. To identify genetic loci associated with plantar fascial disorders, a genome-wide association screen was performed using publically available data from the Research Program in Genes, Environment and Health including 21,624 cases of plantar fascial disorders and 80,879 controls. One indel (chr5:118704153:D) and one SNP (rs62051384) showed an association with plantar fascial disorders at genome-wide significance (p<5×10-8) with small effects (odds ratios=0.93 and 1.07 per allele, respectively). The indel chr5:118704153:D is located within TNFAIP8 (encodes a protein induced by TNF alpha) and rs62051384 is located within WWP2 (which is involved in proteasomal degradation). These DNA variants may be informative in explaining why some individuals are at higher risk for plantar fascial disorders than others.

The early bird catches the worm: new technologies for the Caenorhabditis elegans toolkit.

The inherent simplicity of Caenorhabditis elegans and its extensive genetic toolkit make it ideal for studying complex biological processes. Recent developments further increase the usefulness of the worm, including new methods for: altering gene expression, altering physiology using optogenetics, manipulating large numbers of worms, automating laborious processes and processing high-resolution images. These developments both enhance the worm as a model for studying processes such as development and ageing and make it an attractive model in areas such as neurobiology and behaviour.

A Genetic Marker Associated with De Quervain's Tenosynovitis.

De Quervain's tenosynovitis is a repetitive strain injury involving synovial inflammation of the tendons of the first extensor compartment of the wrist. It is relatively common in the general population, and is the most common radial-sided tendinopathy seen in athletes. Identifying a genetic marker associated with de Quervain's tenosynovitis could provide a useful tool to help identify those individuals with an increased risk for injury. A genome-wide association screen was performed using publically available data from the Research Program in Genes, Environment and Health (RPGEH) including 4,129 cases and 98,374 controls. rs35360670 on chromosome 8 showed an association with de Quervain's tenosynovitis at genome-wide significance (p=1.9×10-8; OR=1.46; 95% CI=1.38-1.59). This study is the first genome-wide screen for de Quervain's tenosynovitis and provides insights regarding its genetic etiology as well as a DNA marker with the potential to inform athletes and other high-risk individuals about their relative risk for injury.

A Genetic Marker Associated with Shoulder Dislocation.

Shoulder dislocations are common shoulder injuries associated with athletic activity in contact sports, such as football, rugby, wrestling, and hockey. Identifying genetic loci associated with shoulder dislocation could shed light on underlying mechanisms for injury and identify predictive genetic markers. To identify DNA polymorphisms associated with shoulder dislocation, a genome-wide association screen was performed using publically available data from the Research Program in Genes, Environment and Health including 662 cases of shoulder dislocation and 82 602 controls from the European ancestry group. rs12913965 showed an association with shoulder dislocation at genome-wide significance (p=9.7×10-9; odds ratio=1.6) from the European ancestry group. Individuals carrying one copy of the risk allele (T) at rs12913965 showed a 69% increased risk for shoulder dislocation in our cohort. rs12913965 is located within an intron of the TICRR gene, which encodes TOPBP1 interacting checkpoint and replication regulator involved in the cell cycle. rs12913965 is also associated with changes in expression of the ISG20 gene, which encodes an antiviral nuclease induced by interferons. This genetic marker may one day be used to identify athletes with a higher genetic risk for shoulder dislocation. It will be important to replicate this finding in future studies.

Two Genetic Loci associated with Medial Collateral Ligament Injury.

Medial collateral ligament (MCL) injuries are a common knee injury, especially in competitive athletes. Identifying genetic loci associated with MCL injury could shed light on its etiology. A genome-wide association screen was performed using data from the Research Program in Genes, Environment and Health (RPGEH) including 1 572 cases of MCL injury and 100 931 controls. 2 SNPs (rs80351309 and rs6083471) showed an association with MCL injury at genome-wide significance (p<5×10-8) with moderate effects (odds ratios=2.12 and 1.57, respectively). For rs80351309, the genotypes were imputed with only moderate accuracy, so this SNP should be viewed with caution until its association with MCL injury can be validated. The SNPs rs80351309 and rs6083471 show a statistically significant association with MCL injury. It will be important to replicate this finding in future studies.

Integrative analysis of C. elegans modENCODE ChIP-seq data sets to infer gene regulatory interactions.

The C. elegans modENCODE Consortium has defined in vivo binding sites for a large array of transcription factors by ChIP-seq. In this article, we present examples that illustrate how this compendium of ChIP-seq data can drive biological insights not possible with analysis of individual factors. First, we analyze the number of independent factors bound to the same locus, termed transcription factor complexity, and find that low-complexity sites are more likely to respond to altered expression of a single bound transcription factor. Next, we show that comparison of binding sites for the same factor across developmental stages can reveal insight into the regulatory network of that factor, as we find that the transcription factor UNC-62 has distinct binding profiles at different stages due to distinct cofactor co-association as well as tissue-specific alternative splicing. Finally, we describe an approach to infer potential regulators of gene expression changes found in profiling experiments (such as DNA microarrays) by screening these altered genes to identify significant enrichment for targets of a transcription factor identified in ChIP-seq data sets. After confirming that this approach can correctly identify the upstream regulator on expression data sets for which the regulator was previously known, we applied this approach to identify novel candidate regulators of transcriptional changes with age. The analysis revealed nine candidate aging regulators, of which three were previously known to have a role in longevity. We experimentally showed that two of the new candidate aging regulators can extend lifespan when overexpressed, indicating that this approach can identify novel functional regulators of complex processes.

Roles of the developmental regulator unc-62/Homothorax in limiting longevity in Caenorhabditis elegans.

The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.

An engineering approach to extending lifespan in C. elegans.

We have taken an engineering approach to extending the lifespan of Caenorhabditis elegans. Aging stands out as a complex trait, because events that occur in old animals are not under strong natural selection. As a result, lifespan can be lengthened rationally using bioengineering to modulate gene expression or to add exogenous components. Here, we engineered longer lifespan by expressing genes from zebrafish encoding molecular functions not normally present in worms. Additionally, we extended lifespan by increasing the activity of four endogenous worm aging pathways. Next, we used a modular approach to extend lifespan by combining components. Finally, we used cell- and worm-based assays to analyze changes in cell physiology and as a rapid means to evaluate whether multi-component transgenic lines were likely to have extended longevity. Using engineering to add novel functions and to tune endogenous functions provides a new framework for lifespan extension that goes beyond the constraints of the worm genome.