The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene ...expression in many organisms. However, new biotechnological tools—specifically CRISPR-based technologies—have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: “Which technology should I use in my experiment?” This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.
Boettcher and McManus provide a practical guide to choosing the right tool for your gene silencing experiment—RNAi, TALEN, or CRISPR.
Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of ...transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in ...microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
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► Ultracomplex shRNA library minimizes false positives/negatives in genome-wide screens ► Pooled double-shRNA strategy systematically maps genetic interactions between hits ► Application of two-step strategy identifies pathways controlling ricin susceptibility ► The resulting map uncovers functionally distinct mammalian TRAPP complexes
A high-throughput method that relies on the use of ultracomplex shRNA libraries makes it possible to create genetic interaction maps in mammalian cells. This approach will be applicable to many cellular processes and conditions, as illustrated by the discovery of distinct TRAPP complexes involved in endocytosis.
Alzheimer's disease is characterized by the accumulation of amyloid-beta in plaques, aggregation of hyperphosphorylated tau in neurofibrillary tangles and neuroinflammation, together resulting in ...neurodegeneration and cognitive decline
. The NLRP3 inflammasome assembles inside of microglia on activation, leading to increased cleavage and activity of caspase-1 and downstream interleukin-1β release
. Although the NLRP3 inflammasome has been shown to be essential for the development and progression of amyloid-beta pathology in mice
, the precise effect on tau pathology remains unknown. Here we show that loss of NLRP3 inflammasome function reduced tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. Tau activated the NLRP3 inflammasome and intracerebral injection of fibrillar amyloid-beta-containing brain homogenates induced tau pathology in an NLRP3-dependent manner. These data identify an important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer's disease, demonstrating that neurofibrillary tangles develop downstream of amyloid-beta-induced microglial activation.
Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the ...survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.
Previously, the contribution of peripheral infection to cognitive decline was largely overlooked however, the past 15 years have established a key role for infectious pathogens in the progression of ...age-related neurodegeneration. It is now accepted that the immune privilege of the brain is not absolute, and that cells of the central nervous system are sensitive to both the inflammatory events occurring in the periphery and to the infiltration of peripheral immune cells. This is particularly relevant for the progression of Alzheimer's disease, in which it has been demonstrated that patients are more vulnerable to infection-related cognitive changes. This can occur from typical infectious challenges such as respiratory tract infections, although a number of specific viral, bacterial, and fungal pathogens have also been associated with the development of the disease. To date, it is not clear whether these microorganisms are directly related to Alzheimer's disease progression or if they are opportune pathogens that easily colonize those with dementia and exacerbate the ongoing inflammation observed in these individuals. This review will discuss the impact of each of these challenges, and examine the changes known to occur with age in the peripheral immune system, which may contribute to the age-related vulnerability to infection-induced cognitive decline.
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived ...macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe−/− mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.
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•M2 macrophage exosomes reprogram inflammation and energy metabolism in recipient cells•Specific miRNAs enriched in M2 macrophage exosomes reduce TNF-α and NF-κB signaling•M2 exosomes control atherosclerosis by reducing hematopoiesis and myeloid inflammation
Anti-inflammatory properties of M2 macrophages can be communicated as miRNA packaged into exosomes. Bouchareychas et al. show that when tested in the Apoe−/− mouse model of hyperlipidemia, M2 macrophage exosomes reduced hematopoiesis and the inflammatory state of circulating monocytes and macrophages in atherosclerotic plaques.
Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and ...diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.
Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, ...validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson's R
of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder causing memory loss, language problems and behavioural disturbances. AD is associated with the accumulation of fibrillar ...amyloid‐β (Aβ) and the formation of neurofibrillary tau tangles. Fibrillar Aβ itself represents a danger‐associated molecular pattern, which is recognized by specific microglial receptors. One of the key players is formation of the NOD‐, LRR‐ and pyrin domain‐containing 3 (NLRP3) inflammasome, whose activation has been demonstrated in AD patient brains and transgenic animal models of AD. Here, we investigated whether Aβ oligomers or protofibrils that represent lower molecular aggregates prior to Aβ deposition are able to activate the NLRP3 inflammasome and subsequent interleukin‐1 beta (IL‐1β) release by microglia. In our study, we used Aβ preparations of different sizes: small oligomers and protofibrils of which the structure was confirmed by atomic force microscopy. Primary microglial cells from C57BL/6 mice were treated with the respective Aβ preparations and NLRP3 inflammasome activation, represented by caspase‐1 cleavage, IL‐1β production, and apoptosis‐associated speck‐like protein containing a CARD speck formation was analysed. Both protofibrils and low molecular weight Aβ aggregates induced a significant increase in IL‐1β release. Inflammasome activation was confirmed by apoptosis‐associated speck‐like protein containing a CARD speck formation and detection of active caspase‐1. The NLRP3 inflammasome inhibitor MCC950 completely inhibited the Aβ‐induced immune response. Our results show that the NLRP3 inflammasome is activated not only by fibrillar Aβ aggregates as reported before, but also by lower molecular weight Aβ oligomers and protofibrils, highlighting the possibility that microglial activation by these Aβ species may initiate innate immune responses in the central nervous system prior to the onset of Aβ deposition.
Cover Image for this issue: https://doi.org/10.1111/jnc.14773.
Amyloid‐beta (Aβ) is recognized as a danger‐associated molecular pattern by pattern recognition receptors present on microglial cells. The aim of the study was to analyse whether low molecular weight Aβ oligomers that are more neurotoxic in comparison with fibrils, could activate NLRP3 inflammasome in microglial cells. Our results confirm that NLRP3 inflammasome is activated not only by fibrils forming Aβ aggregates but also by small oligomers. The inflammasome activation could be completely blocked by the specific inhibitor, MCC950. These results highlight that microglia activated by Aβ oligomers and protofibrils are potent drivers of neuroinflammatory processes.
Cover Image for this issue: https://doi.org/10.1111/jnc.14773.
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