RefCell: multi-dimensional analysis of image-based high-throughput screens based on 'typical cells'.

BACKGROUND: Image-based high-throughput screening (HTS) reveals a high level of heterogeneity in single cells and multiple cellular states may be observed within a single population. Currently available high-dimensional analysis methods are successful in characterizing cellular heterogeneity, but suffer from the "curse of dimensionality" and non-standardized outputs. RESULTS: Here we introduce RefCell, a multi-dimensional analysis pipeline for image-based HTS that reproducibly captures cells with typical combinations of features in reference states and uses these "typical cells" as a reference for classification and weighting of metrics. RefCell quantitatively assesses heterogeneous deviations from typical behavior for each analyzed perturbation or sample. CONCLUSIONS: We apply RefCell to the analysis of data from a high-throughput imaging screen of a library of 320 ubiquitin-targeted siRNAs selected to gain insights into the mechanisms of premature aging (progeria). RefCell yields results comparable to a more complex clustering-based single-cell analysis method; both methods reveal more potential hits than a conventional analysis based on averages.

Blank spots on the map: some current questions on nuclear organization and genome architecture.

The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.

A Cell-Intrinsic Interferon-like Response Links Replication Stress to Cellular Aging Caused by Progerin.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease caused by a truncated lamin A protein (progerin) that drives cellular and organismal decline. HGPS patient-derived fibroblasts accumulate genomic instability, but its underlying mechanisms and contribution to disease remain poorly understood. Here, we show that progerin-induced replication stress (RS) drives genomic instability by eliciting replication fork (RF) stalling and nuclease-mediated degradation. Rampant RS is accompanied by upregulation of the cGAS/STING cytosolic DNA sensing pathway and activation of a robust STAT1-regulated interferon (IFN)-like response. Reducing RS and the IFN-like response, especially with calcitriol, improves the fitness of progeria cells and increases the efficiency of cellular reprogramming. Importantly, other compounds that improve HGPS phenotypes reduce RS and the IFN-like response. Our study reveals mechanisms underlying progerin toxicity, including RS-induced genomic instability and activation of IFN-like responses, and their relevance for cellular decline in HGPS.

Shared molecular and cellular mechanisms of premature ageing and ageing-associated diseases.

Ageing is the predominant risk factor for many common diseases. Human premature ageing diseases are powerful model systems to identify and characterize cellular mechanisms that underpin physiological ageing. Their study also leads to a better understanding of the causes, drivers and potential therapeutic strategies of common diseases associated with ageing, including neurological disorders, diabetes, cardiovascular diseases and cancer. Using the rare premature ageing disorder Hutchinson-Gilford progeria syndrome as a paradigm, we discuss here the shared mechanisms between premature ageing and ageing-associated diseases, including defects in genetic, epigenetic and metabolic pathways; mitochondrial and protein homeostasis; cell cycle; and stem cell-regenerative capacity.

Repression of the Antioxidant NRF2 Pathway in Premature Aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.

A high-content imaging-based screening pipeline for the systematic identification of anti-progeroid compounds.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery.

Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins.

Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin.

A BRCA1-interacting lncRNA regulates homologous recombination.

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.

Restoring SIRT6 Expression in Hutchinson-Gilford Progeria Syndrome Cells Impedes Premature Senescence and Formation of Dysmorphic Nuclei.

OBJECTIVES: Mice overexpressing SIRT6 live longer than wild-type mice while SIRT6 knockout mice exhibit similar degenerative phenotypes as individuals with Hutchinson-Gilford progeria syndrome (HGPS). Thus, we sought to test whether levels of SIRT6 are reduced in cells from individuals with HGPS and whether restored SIRT6 expression may impede premature aging phenotypes. METHODS: Levels of endogenous SIRT6 and progerin in HGPS and normal fibroblasts were assessed by Western blotting and immunofluorescence. A tetracycline-inducible system was utilized to test whether progerin causes a rapid reduction in SIRT6 protein. SIRT6 was overexpressed in HGPS cells via lentiviral infection with biological endpoints including senescence-associated ß-galactosidase (SA-ß-gal) positivity, frequency of nuclear atypia, the number of 53BP1-positive DNA damage foci and growth rates. RESULTS: Typical HGPS fibroblasts express lower levels of SIRT6 than fibroblasts from normal and atypical HGPS donors. Experimental induction of progerin did not cause a detectable reduction of SIRT6 protein. However, overexpression of SIRT6 in HGPS cells was associated with a reduced frequency of SA-ß-gal positivity, fewer misshapen nuclei, fewer DNA damage foci, and increased growth rates. CONCLUSIONS: Typical HGPS fibroblasts exhibit reduced levels of SIRT6 protein via a mechanism that remains to be elucidated. Our findings suggest that restoring SIRT6 expression in HGPS cells may partially impede senescence and the formation of dysmorphic nuclei. © 2015 S. Karger AG, Basel.

Systematic identification of pathological lamin A interactors.

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype-phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A-binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome-wide ORFeome library, we identified and validated 337 lamin A-binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson-Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)-like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein-protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.

Mapping of lamin A- and progerin-interacting genome regions.

Mutations in the A-type lamins A and C, two major components of the nuclear lamina, cause a large group of phenotypically diverse diseases collectively referred to as laminopathies. These conditions often involve defects in chromatin organization. However, it is unclear whether A-type lamins interact with chromatin in vivo and whether aberrant chromatin-lamin interactions contribute to disease. Here, we have used an unbiased approach to comparatively map genome-wide interactions of gene promoters with lamin A and progerin, the mutated lamin A isoform responsible for the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS) in mouse cardiac myoytes and embryonic fibroblasts. We find that lamin A-associated genes are predominantly transcriptionally silent and that loss of lamin association leads to the relocation of peripherally localized genes, but not necessarily to their activation. We demonstrate that progerin induces global changes in chromatin organization by enhancing interactions with a specific subset of genes in addition to the identified lamin A-associated genes. These observations demonstrate disease-related changes in higher order genome organization in HGPS and provide novel insights into the role of lamin-chromatin interactions in chromatin organization.

Post-natal myogenic and adipogenic developmental: defects and metabolic impairment upon loss of A-type lamins.

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNA(GT-/-)) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.

Syndecan-1 amplifies angiotensin II-induced cardiac fibrosis.

Syndecan-1 (Synd1) is a transmembrane heparan sulfate proteoglycan that functions as a coreceptor for various growth factors and modulates signal transduction. The present study investigated whether Synd1, by affecting growth factor signaling, may play a role in hypertension-induced cardiac fibrosis and dysfunction. Expression of Synd1 was increased significantly in mouse hearts with angiotensin II-induced hypertension, which was spatially related to cardiac fibrosis. Angiotensin II significantly impaired fractional shortening and induced cardiac fibrosis in wild-type mice, whereas these effects were blunted in Synd1-null mice. Angiotensin II significantly increased cardiac expression of connective tissue growth factor and collagen type I and III in wild-type mice, which was blunted in Synd1-null mice. These findings were confirmed in vitro, where angiotensin II induced the expression of both connective tissue growth factor and collagen I in fibroblasts. The absence of Synd1 in either Synd1-null fibroblasts, after knockdown of Synd1 by short hairpin RNA, or after inhibition of heparan sulfates by protamine attenuated this increase, which was associated with reduced phosphorylation of Smad2. In conclusion, loss of Synd1 reduces cardiac fibrosis and dysfunction during angiotensin II-induced hypertension.

Mapping of protein- and chromatin-interactions at the nuclear lamina.

The nuclear envelope and the lamina define the nuclear periphery and are implicated in many nuclear processes including chromatin organization, transcription and DNA replication. Mutations in lamin A proteins, major components of the lamina, interfere with these functions and cause a set of phenotypically diverse diseases referred to as laminopathies. The phenotypic diversity of laminopathies is thought to be the result of alterations in specific protein- and chromatin interactions due to lamin A mutations. Systematic identification of lamin A-protein and -chromatin interactions will be critical to uncover the molecular etiology of laminopathies. Here we summarize and critically discuss recent technology to analyze lamina-protein and-chromatin interactions.

Identification of differential protein interactors of lamin A and progerin.

The nuclear lamina is an interconnected meshwork of intermediate filament proteins underlying the nuclear envelope. The lamina is an important regulator of nuclear structural integrity as well as nuclear processes, including transcription, DNA replication and chromatin remodeling. The major components of the lamina are A- and B-type lamins. Mutations in lamins impair lamina functions and cause a set of highly tissue-specific diseases collectively referred to as laminopathies. The phenotypic diversity amongst laminopathies is hypothesized to be caused by mutations affecting specific protein interactions, possibly in a tissue-specific manner. Current technologies to identify interaction partners of lamin A and its mutants are hampered by the insoluble nature of lamina components. To overcome the limitations of current technologies, we developed and applied a novel, unbiased approach to identify lamin A-interacting proteins. This approach involves expression of the high-affinity OneSTrEP-tag, precipitation of lamin-protein complexes after reversible protein cross-linking and subsequent protein identification by mass spectrometry. We used this approach to identify in mouse embryonic fibroblasts and cardiac myocyte NklTAg cell lines proteins that interact with lamin A and its mutant isoform progerin, which causes the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identified a total of 313 lamina-interacting proteins, including several novel lamin A interactors, and we characterize a set of 35 proteins which preferentially interact with lamin A or progerin.

Ageing-related chromatin defects through loss of the NURD complex.

Physiological and premature ageing are characterized by multiple defects in chromatin structure and accumulation of persistent DNA damage. Here we identify the NURD chromatin remodelling complex as a key modulator of these ageing-associated chromatin defects. We demonstrate loss of several NURD components during premature and normal ageing and we find an ageing-associated reduction in HDAC1 activity. Silencing of individual NURD subunits recapitulated chromatin defects associated with ageing and we provide evidence that structural chromatin defects precede DNA damage accumulation. These results outline a molecular mechanism for chromatin defects during ageing.

Lysosomal integral membrane protein 2 is a novel component of the cardiac intercalated disc and vital for load-induced cardiac myocyte hypertrophy.

The intercalated disc (ID) of cardiac myocytes is emerging as a crucial structure in the heart. Loss of ID proteins like N-cadherin causes lethal cardiac abnormalities, and mutations in ID proteins cause human cardiomyopathy. A comprehensive screen for novel mechanisms in failing hearts demonstrated that expression of the lysosomal integral membrane protein 2 (LIMP-2) is increased in cardiac hypertrophy and heart failure in both rat and human myocardium. Complete loss of LIMP-2 in genetically engineered mice did not affect cardiac development; however, these LIMP-2 null mice failed to mount a hypertrophic response to increased blood pressure but developed cardiomyopathy. Disturbed cadherin localization in these hearts suggested that LIMP-2 has important functions outside lysosomes. Indeed, we also find LIMP-2 in the ID, where it associates with cadherin. RNAi-mediated knockdown of LIMP-2 decreases the binding of phosphorylated beta-catenin to cadherin, whereas overexpression of LIMP-2 has the opposite effect. Collectively, our data show that LIMP-2 is crucial to mount the adaptive hypertrophic response to cardiac loading. We demonstrate a novel role for LIMP-2 as an important mediator of the ID.

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Oxidized quercetin reacts with thiols rather than with ascorbate: implication for quercetin supplementation.

When an antioxidant scavenges a reactive species, i.e., when it exerts its antioxidant activity, the antioxidant is converted into potentially harmful oxidation products. In this way, the antioxidant quercetin might yield an ortho-quinone, denoted as QQ, which has four tautomeric forms, i.e., the ortho-quinone and three quinonmethides. We evaluated the interaction of QQ with ascorbate or glutathione (GSH). Ascorbate recycles QQ to the parent compound quercetin, while GSH forms two adducts with QQ, i.e., 6-GSQ and 8-GSQ. When both GSH and ascorbate are present, QQ is converted exclusively into GSQ. In the absence of GSH, protein thiols will be arylated by QQ. This protein arylation is not prevented by ascorbate. Thiol arylation by quinones and quinonmethides can impair several vital enzymes. This implies that the product formed when quercetin displays its antioxidant scavenging effect is toxic in the absence of GSH. Therefore, an adequate GSH level should be maintained when quercetin is supplemented.