The interaction of regulatory proteins with the complex nucleoprotein structures that are found in mammalian cells involves chromatin reorganization at multiple levels. Mechanisms that support these ...transitions are complex on many timescales, which range from milliseconds to minutes or hours. In this Review, we discuss emerging concepts regarding the function of regulatory elements in living cells. We also explore the involvement of these dynamic and stochastic processes in the evolution of fluctuating transcriptional activity states that are now commonly reported in eukaryotic systems.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Genomes are arranged non-randomly in the 3D space of the cell nucleus. Here, we have developed HIPMap, a high-precision, high-throughput, automated fluorescent in situ hybridization imaging pipeline, ...for mapping of the spatial location of genome regions at large scale. High-throughput imaging position mapping (HIPMap) enabled an unbiased siRNA screen for factors involved in genome organization in human cells. We identify 50 cellular factors required for proper positioning of a set of functionally diverse genomic loci. Positioning factors include chromatin remodelers, histone modifiers, and nuclear envelope and pore proteins. Components of the replication and post-replication chromatin re-assembly machinery are prominently represented among positioning factors, and timely progression of cells through replication, but not mitosis, is required for correct gene positioning. Our results establish a method for the large-scale mapping of genome locations and have led to the identification of a compendium of cellular factors involved in spatial genome organization.
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•Development of a method for accurate mapping of gene position in the 3D nuclear space at large scale•siRNA screen identifies nuclear factors involved in genome organization•Replication is a critical step in determining spatial gene positioning
HIPMap is a FISH-based, high-throughput platform that can be applied to determine the spatial position of a gene in the 3D nuclear space and to discover factors that determine genome organization.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chromosome translocations are a hallmark of cancer cells. We have developed an experimental system to visualize the formation of translocations in living cells and apply it to characterize the ...spatial and dynamic properties of translocation formation. We demonstrate that translocations form within hours of the occurrence of double-strand breaks (DSBs) and that their formation is cell cycle—independent. Translocations form preferentially between prepositioned genome elements, and perturbation of key factors of the DNA repair machinery uncouples DSB pairing from translocation formation. These observations generate a spatiotemporal framework for the formation of translocations in living cells.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Progression through the cell cycle is one of the most fundamental features of cells. Studies of the cell cycle have traditionally relied on the analysis of populations, and they often require ...specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unperturbed cells. We describe a protocol, suitable for use in high-resolution imaging approaches, for determining cell cycle staging of individual cells by measuring their DNA content by fluorescence microscopy. The approach is based on the accurate quantification by image analysis of the integrated nuclear intensity of cells stained with a DNA dye, and it can be used in combination with several histochemical methods. We describe and provide the algorithms for two automated image analysis pipelines and the derivation of cell cycle profiles with both commercial and open-source software. This 1-2-d protocol is applicable to adherent cells, and it is adaptable for use with several DNA dyes.
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.
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•Impaired activity of the NRF2 antioxidative pathway is a driver mechanism in HGPS•Suppressed NRF2 activity and oxidative stress recapitulate HGPS aging defects•Reactivation of NRF2 decreases oxidative stress and reverses cellular HGPS defects
Impairment of the NRF2 pathway is a key contributor to premature aging in Hutchinson-Gilford progeria syndrome (HGPS) via increasing chronic oxidative stress that recapitulates HGPS aging defects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living ...cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.
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► Many transcription factors exchange with response elements on a time scale of seconds ► Proteins with identical recognition sites fail to exhibit competitive binding in vivo ► Models support dynamic, temporally sparse, factor/response element interactions ► “Assisted loading” results from chromatin remodeling that creates transient access
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BACKGROUND: Cancer cachexia is a multifactorial syndrome that is poorly defined. OBJECTIVE: Our objective was to evaluate whether a 3-factor profile incorporating weight loss (>=10%), low food intake ...(<=1500 kcal/d), and systemic inflammation (C-reactive protein >= 10 mg/L) might relate better to the adverse functional aspects of cachexia and to a patient's overall prognosis than will weight loss alone. DESIGN: One hundred seventy weight-losing (>=5%) patients with advanced pancreatic cancer were screened for nutritional status, functional status, performance score, health status, and quality of life. Patients were followed for a minimum of 6 mo, and survival was noted. Patients were characterized by using the individual factors, >=2 factors, or all 3 factors. RESULTS: Weight loss alone did not define a population that differed in functional aspects of self-reported quality of life or health status and differed only in objective factors of physical function. The 3-factor profile identified both reduced subjective and objective function. In the overall population, the 3 factors, >=2 factors, and individual profile factors (except weight loss) all carried adverse prognostic significance (P < 0.01). Subgroup analysis showed that the 3-factor profile carried adverse prognostic significance in localized (hazard ratio: 4.9; P < 0.001) but not in metastatic disease. CONCLUSIONS: Weight loss alone does not identify the full effect of cachexia on physical function and is not a prognostic variable. The 3-factor profile (weight loss, reduced food intake, and systemic inflammation) identifies patients with both adverse function and prognosis. Shortened survival applies particularly to cachectic patients with localized disease, thereby reinforcing the need for early intervention.
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CMK, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
IntroductionAffective episodes often emerge in adolescence and young adulthood. Identification of factors subjectively associated with their onset may improve aetiological models and targeted ...intervention.ObjectivesTo examine precipitating conditions of (hypo-)manic and depressive episodes in adolescents and young adults from the general population.MethodsA random sample of 14-21 year-olds was drawn from the population registry of Dresden, Germany, and N=1180 were assessed in 2015/2016 (response rate: 21.7%). Lifetime depressive and (hypo-)manic symptoms as well as full-threshold depressive and (hypo-)manic episodes (DSM-5) were identified using standardized interview. Participants reporting depressive or (hypo-)manic symptoms were asked whether and which events or conditions they associate with episode onset. Besides responses on a list providing potential triggering conditions a free answer was possible. Qualitative content analysis preceded quantitative logistic regression analyses (significance level p<.05). Considered categories were: negative life events (further divided for depression into loss/danger events, burdensome life conditions, and interpersonal factors), events requiring adaptation, positive life events, internal factors, and other factors.ResultsThe vast majority of participants reporting depressive (n=682) respectively (hypo-)manic (n=200) symptoms also reported a precipitating condition (94.7%, 83.1%). There was no significant association between any triggering condition and the occurrence of a full-threshold depressive (n=206) or (hypo-)manic (n=25) episode. However, the number of reported categories of precipitating conditions was associated with full-threshold depressive and (hypo-)manic episodes. Among those with depressive or (hypo-)manic symptoms and at least one reported precipitating condition, multiple regression models including all condition categories showed that in particular internal factors, interpersonal problems and other factors were associated with the occurrence of a full-threshold depressive episode (n=199) and positive life events as well as internal factors were associated with the occurrence of a full-threshold (hypo-)manic episode (n=21).ConclusionsAdolescents and young adults from the general population usually associate the onset of phases with affective symptoms with precipitating conditions but these do not necessarily signal the emergence of a diagnostically relevant episode. Nevertheless, a greater number of and the presence of particular precipitating conditions may indicate the emergence of full-blown depressive or (hypo-)manic episodes. Thus, asking for subjective triggers appears relevant and may guide early identification and intervention.Disclosure of InterestNone Declared
Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form ...amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Image segmentation pipelines are commonly used in microscopy to identify cellular compartments like nucleus and cytoplasm, but there are few standards for comparing segmentation accuracy across ...pipelines. The process of selecting a segmentation assessment pipeline can seem daunting to researchers due to the number and variety of metrics available for evaluating segmentation quality. Here we present automated pipelines to obtain a comprehensive set of 69 metrics to evaluate segmented data and propose a selection methodology for models based on quantitative analysis, dimension reduction or unsupervised classification techniques and informed selection criteria. We show that the metrics used here can often be reduced to a small number of metrics that give a more complete understanding of segmentation accuracy, with different groups of metrics providing sensitivity to different types of segmentation error. These tools are delivered as easy to use python libraries, command line tools, Common Workflow Language Tools, and as Web Image Processing Pipeline interactive plugins to ensure a wide range of users can access and use them. We also present how our evaluation methods can be used to observe the changes in segmentations across modern machine learning/deep learning workflows and use cases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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