Dialing down SUN1 for laminopathies.

Laminopathies, caused by mutations in A-type nuclear lamins, encompass a range of diseases, including forms of progeria and muscular dystrophy. In this issue, Chen et al. provide evidence that elevated expression of the nuclear inner membrane protein SUN1 drives pathology in multiple laminopathies.

An eQTL-based approach reveals candidate regulators of LINE-1 RNA levels in lymphoblastoid cells.

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition, the overwhelming majority of copies are degenerate and immobile. Nevertheless, both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability, inflammation, or cellular senescence) on their hosts, and L1's contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSD17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels.

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

Ultrafine mapping of chromosome conformation at hundred basepair resolution reveals regulatory genome architecture.

The resolution limit of chromatin conformation capture methodologies (3Cs) has restrained their application in detection of fine-level chromatin structure mediated by cis-regulatory elements (CREs). Here, we report two 3C-derived methods, Tri-4C and Tri-HiC, which utilize multirestriction enzyme digestions for ultrafine mapping of targeted and genome-wide chromatin interaction, respectively, at up to one hundred basepair resolution. Tri-4C identified CRE loop interaction networks and quantitatively revealed their alterations underlying dynamic gene control. Tri-HiC uncovered global fine-gauge regulatory interaction networks, identifying >20-fold more enhancer:promoter (E:P) loops than in situ Hi-C. In addition to vastly improved identification of subkilobase-sized E:P loops, Tri-HiC also uncovered interaction stripes and contact domain insulation from promoters and enhancers, revealing their loop extrusion behaviors resembling the topologically associating domain boundaries. Tri-4C and Tri-HiC provide robust approaches to achieve the high-resolution interactome maps required for characterizing fine-gauge regulatory chromatin interactions in analysis of development, homeostasis, and disease.

Chromosome instability and aneuploidy in the mammalian brain.

This review investigates the role of aneuploidy and chromosome instability (CIN) in the aging brain. Aneuploidy refers to an abnormal chromosomal count, deviating from the normal diploid set. It can manifest as either a deficiency or excess of chromosomes. CIN encompasses a broader range of chromosomal alterations, including aneuploidy as well as structural modifications in DNA. We provide an overview of the state-of-the-art methodologies utilized for studying aneuploidy and CIN in non-tumor somatic tissues devoid of clonally expanded populations of aneuploid cells.CIN and aneuploidy, well-established hallmarks of cancer cells, are also associated with the aging process. In non-transformed cells, aneuploidy can contribute to functional impairment and developmental disorders. Despite the importance of understanding the prevalence and specific consequences of aneuploidy and CIN in the aging brain, these aspects remain incompletely understood, emphasizing the need for further scientific investigations.This comprehensive review consolidates the present understanding, addresses discrepancies in the literature, and provides valuable insights for future research efforts.

Aging: a sirtuin shake-up?

The yeast sirtuin (Sir2) is a histone deacetylase that modulates yeast replicative life span by suppressing genome instability through chromatin modification. In this issue, Oberdoerffer et al. (2008) report that SIRT1, the mammalian ortholog of Sir2, is involved in DNA damage-induced chromatin reorganization, which promotes genome stability in mammalian cells.

Signal Transduction, Ageing and Disease.

Ageing is defined by the loss of functional reserve over time, leading to a decreased tissue homeostasis and increased age-related pathology. The accumulation of damage including DNA damage contributes to driving cell signaling pathways that, in turn, can drive different cell fates, including senescence and apoptosis, as well as mitochondrial dysfunction and inflammation. In addition, the accumulation of cell autonomous damage with time also drives ageing through non-cell autonomous pathways by modulation of signaling pathways. Interestingly, genetic and pharmacologic analysis of factors able to modulate lifespan and healthspan in model organisms and even humans have identified several key signaling pathways including IGF-1, NF-κB, FOXO3, mTOR, Nrf-2 and sirtuins. This review will discuss the roles of several of these key signaling pathways, in particular NF-κB and Nrf2, in modulating ageing and age-related diseases.

mTOR inhibitors may benefit kidney transplant recipients with mitochondrial diseases.

Mitochondrial diseases represent a significant clinical challenge. Substantial efforts have been devoted to identifying therapeutic strategies for mitochondrial disorders, but effective interventions have remained elusive. Recently, we reported attenuation of disease in a mouse model of the human mitochondrial disease Leigh syndrome through pharmacological inhibition of the mechanistic target of rapamycin (mTOR). The human mitochondrial disorder MELAS/MIDD (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes/Maternally Inherited Diabetes and Deafness) shares many phenotypic characteristics with Leigh syndrome. MELAS/MIDD often leads to organ failure and transplantation and there are currently no effective treatments. To examine the therapeutic potential of mTOR inhibition in human mitochondrial disease, four kidney transplant recipients with MELAS/MIDD were switched from calcineurin inhibitors to mTOR inhibitors for immunosuppression. Primary fibroblast lines were generated from patient dermal biopsies and the impact of rapamycin was studied using cell-based end points. Metabolomic profiles of the four patients were obtained before and after the switch. pS6, a measure of mTOR signaling, was significantly increased in MELAS/MIDD cells compared to controls in the absence of treatment, demonstrating mTOR overactivation. Rapamycin rescued multiple deficits in cultured cells including mitochondrial morphology, mitochondrial membrane potential, and replicative capacity. Clinical measures of health and mitochondrial disease progression were improved in all four patients following the switch to an mTOR inhibitor. Metabolomic analysis was consistent with mitochondrial function improvement in all patients.

CRISPR-based strategies for studying regulatory elements and chromatin structure in mammalian gene control.

The development of high-throughput methods has enabled the genome-wide identification of putative regulatory elements in a wide variety of mammalian cells at an unprecedented resolution. Extensive genomic studies have revealed the important role of regulatory elements and genetic variation therein in disease formation and risk. In most cases, there is only correlative evidence for the roles of these elements and non-coding changes within these elements in pathogenesis. With the advent of genome- and epigenome-editing tools based on the CRISPR technology, it is now possible to test the functional relevance of the regulatory elements and alterations on a genomic scale. Here, we review the various CRISPR-based strategies that have been developed to functionally validate the candidate regulatory elements in mammals as well as the non-coding genetic variants found to be associated with human disease. We also discuss how these synthetic biology tools have helped to elucidate the role of three-dimensional nuclear architecture and higher-order chromatin organization in shaping functional genome and controlling gene expression.

In vivo epigenome editing and transcriptional modulation using CRISPR technology.

The rapid advancement of CRISPR technology has enabled targeted epigenome editing and transcriptional modulation in the native chromatin context. However, only a few studies have reported the successful editing of the epigenome in adult animals in contrast to the rapidly growing number of in vivo genome editing over the past few years. In this review, we discuss the challenges facing in vivo epigenome editing and new strategies to overcome the huddles. The biggest challenge has been the difficulty in packaging dCas9 fusion proteins required for manipulation of epigenome into the adeno-associated virus (AAV) delivery vehicle. We review the strategies to address the AAV packaging issue, including small dCas9 orthologues, truncated dCas9 mutants, a split-dCas9 system, and potent truncated effector domains. We discuss the dCas9 conjugation strategies to recruit endogenous chromatin modifiers and remodelers to specific genomic loci, and recently developed methods to recruit multiple copies of the dCas9 fusion protein, or to simultaneous express multiple gRNAs for robust epigenome editing or synergistic transcriptional modulation. The use of Cre-inducible dCas9-expressing mice or a genetic cross between dCas9- and sgRNA-expressing flies has also helped overcome the transgene delivery issue. We provide perspective on how a combination use of these strategies can facilitate in vivo epigenome editing and transcriptional modulation.

Network analysis of mitonuclear GWAS reveals functional networks and tissue expression profiles of disease-associated genes.

While mitochondria have been linked to many human diseases through genetic association and functional studies, the precise role of mitochondria in specific pathologies, such as cardiovascular, neurodegenerative, and metabolic diseases, is often unclear. Here, we take advantage of the catalog of human genome-wide associations, whole-genome tissue expression and expression quantitative trait loci datasets, and annotated mitochondrial proteome databases to examine the role of common genetic variation in mitonuclear genes in human disease. Through pathway-based analysis we identified distinct functional pathways and tissue expression profiles associated with each of the major human diseases. Among our most striking findings, we observe that mitonuclear genes associated with cancer are broadly expressed among human tissues and largely represent one functional process, intrinsic apoptosis, while mitonuclear genes associated with other diseases, such as neurodegenerative and metabolic diseases, show tissue-specific expression profiles and are associated with unique functional pathways. These results provide new insight into human diseases using unbiased genome-wide approaches.

Genome and Epigenome Editing in Mechanistic Studies of Human Aging and Aging-Related Disease.

The recent advent of genome and epigenome editing technologies has provided a new paradigm in which the landscape of the human genome and epigenome can be precisely manipulated in their native context. Genome and epigenome editing technologies can be applied to many aspects of aging research and offer the potential to develop novel therapeutics against age-related diseases. Here, we discuss the latest technological advances in the CRISPR-based genome and epigenome editing toolbox, and provide insight into how these synthetic biology tools could facilitate aging research by establishing in vitro cell and in vivo animal models to dissect genetic and epigenetic mechanisms underlying aging and age-related diseases. We discuss recent developments in the field with the aims to precisely modulate gene expression and dynamic epigenetic landscapes in a spatial and temporal manner in cellular and animal models, by complementing the CRISPR-based editing capability with conditional genetic manipulation tools including chemically inducible expression systems, optogenetics, logic gate genetic circuits, tissue-specific promoters, and the serotype-specific adeno-associated virus. We also discuss how the combined use of genome and epigenome editing tools permits investigators to uncover novel molecular pathways involved in the pathophysiology and etiology conferred by risk variants associated with aging and aging-related disease. A better understanding of the genetic and epigenetic regulatory mechanisms underlying human aging and age-related disease will significantly contribute to the developments of new therapeutic interventions for extending health span and life span, ultimately improving the quality of life in the elderly populations.

Genome instability and aging.

Genome instability has long been implicated as the main causal factor in aging. Somatic cells are continuously exposed to various sources of DNA damage, from reactive oxygen species to UV radiation to environmental mutagens. To cope with the tens of thousands of chemical lesions introduced into the genome of a typical cell each day, a complex network of genome maintenance systems acts to remove damage and restore the correct base pair sequence. Occasionally, however, repair is erroneous, and such errors, as well as the occasional failure to correctly replicate the genome during cell division, are the basis for mutations and epimutations. There is now ample evidence that mutations accumulate in various organs and tissues of higher animals, including humans, mice, and flies. What is not known, however, is whether the frequency of these random changes is sufficient to cause the phenotypic effects generally associated with aging. The exception is cancer, an age-related disease caused by the accumulation of mutations and epimutations. Here, we first review current concepts regarding the relationship between DNA damage, repair, and mutation, as well as the data regarding genome alterations as a function of age. We then describe a model for how randomly induced DNA sequence and epigenomic variants in the somatic genomes of animals can result in functional decline and disease in old age. Finally, we discuss the genetics of genome instability in relation to longevity to address the importance of alterations in the somatic genome as a causal factor in aging and to underscore the opportunities provided by genetic approaches to develop interventions that attenuate genome instability, reduce disease risk, and increase life span.

A common variant in pre-miR-146 is associated with coronary artery disease risk and its mature miRNA expression.

miRNAs are small non-coding RNAs that play an important role in numerous physiological processes. Common single nucleotide polymorphisms (SNPs) in pre-miRNAs may change their property through altering miRNAs expression and/or maturation, resulting in diverse functional consequences. To date, the role of genetic variants in pre-miRNAs on coronary artery disease (CAD) risk remains poorly understood. Here we aimed to evaluate the influence of three common SNPs in pre-miRNAs (miR-146a rs2910164 G>C, miR-196a2 rs11614913 C>T, miR-499 rs3746444 T>C) on individual susceptibility to CAD in a Chinese population of 295 CAD patients and 283 controls. Genotyping was performed using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method. In a logistic regression analysis, we detected an association of rs2910164 in pre-miR-146a with the CAD risk; compared with the GG homozygotes, the GC heterozygotes [odds ratio (OR)=1.89, 95% confidence interval (CI)=1.06-3.36, P=0.029] and the CC homozygotes (OR=1.83, 95% CI=1.01-3.32, P=0.046) genotype were statistically significantly associated with the increased risk for CADs. As we used further genotype association models, we found a similar trend of the association in recessive model (OR=1.86, 95% CI=1.09-3.19, P=0.023). We also found that the genotypes of miR-146a rs2910164 were associated with its mature miRNA expression by analyzing 23 PBMC samples from CAD patients. Individuals carrying rs11614913 GC or CC genotypes showed 3.2-fold higher expression compared to GG genotype carriers (P<0.05). We observed no association of the other two SNPs in miR-196a2 (rs11614913) and miR-499 (rs3746444) with the CAD incidence. Our data provide the first evidence that the miR-146a rs2910164 polymorphism is associated with increased risk of CAD in Chinese Han population, which may be through influencing the expression levels of the miRNA.

In vitro heterochronic parabiosis identifies pigment epithelium-derived factor as a systemic mediator of rejuvenation by young blood.

Several decades of heterochronic parabiosis (HCPB) studies have demonstrated the restorative impact of young blood, and deleterious influence of aged blood, on physiological function and homeostasis across tissues, although few of the factors responsible for these observations have been identified. Here we develop an in vitro HCPB system to identify these circulating factors, using replicative lifespan (RLS) of primary human fibroblasts as an endpoint of cellular health. We find that RLS is inversely correlated with serum donor age and sensitive to the presence or absence of specific serum components. Through in vitro HCPB, we identify the secreted protein pigment epithelium-derived factor (PEDF) as a circulating factor that extends RLS of primary human fibroblasts and declines with age in mammals. Systemic administration of PEDF to aged mice reverses age-related functional decline and pathology across several tissues, improving cognitive function and reducing hepatic fibrosis and renal lipid accumulation. Together, our data supports PEDF as a systemic mediator of the effect of young blood on organismal health and homeostasis and establishes our in vitro HCPB system as a valuable screening platform for the identification of candidate circulating factors involved in aging and rejuvenation.

A systems biology-based identification and in vivo functional screening of Alzheimer's disease risk genes reveal modulators of memory function.

Genome-wide association studies (GWASs) have uncovered over 75 genomic loci associated with risk for late-onset Alzheimer's disease (LOAD), but identification of the underlying causal genes remains challenging. Studies of induced pluripotent stem cell (iPSC)-derived neurons from LOAD patients have demonstrated the existence of neuronal cell-intrinsic functional defects. Here, we searched for genetic contributions to neuronal dysfunction in LOAD using an integrative systems approach that incorporated multi-evidence-based gene mapping and network-analysis-based prioritization. A systematic perturbation screening of candidate risk genes in Caenorhabditis elegans (C. elegans) revealed that neuronal knockdown of the LOAD risk gene orthologs vha-10 (ATP6V1G2), cmd-1 (CALM3), amph-1 (BIN1), ephx-1 (NGEF), and pho-5 (ACP2) alters short-/intermediate-term memory function, the cognitive domain affected earliest during LOAD progression. These results highlight the impact of LOAD risk genes on evolutionarily conserved memory function, as mediated through neuronal endosomal dysfunction, and identify new targets for further mechanistic interrogation.

Recruitment of CTCF to the SIRT1 promoter after Oxidative Stress mediates Cardioprotective Transcription.

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

Evolution in health and medicine Sackler colloquium: Genetic variation in human telomerase is associated with telomere length in Ashkenazi centenarians.

Telomere length in humans is emerging as a biomarker of aging because its shortening is associated with aging-related diseases and early mortality. However, genetic mechanisms responsible for these associations are not known. Here, in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls, we studied the inheritance and maintenance of telomere length and variations in two major genes associated with telomerase enzyme activity, hTERT and hTERC. We demonstrated that centenarians and their offspring maintain longer telomeres compared with controls with advancing age and that longer telomeres are associated with protection from age-related diseases, better cognitive function, and lipid profiles of healthy aging. Sequence analysis of hTERT and hTERC showed overrepresentation of synonymous and intronic mutations among centenarians relative to controls. Moreover, we identified a common hTERT haplotype that is associated with both exceptional longevity and longer telomere length. Thus, variations in human telomerase gene that are associated with better maintenance of telomere length may confer healthy aging and exceptional longevity in humans.