Approaches to characterize chromatin subcompartment organization in the cell nucleus.

The mechanism of self-organization of chromatin subcompartments on the 0.1-1 µm scale and their impact on genome-associated activities has long been a key aspect of research on nuclear organization. Understanding the underlying structure-function relationship, however, remains challenging due to the complex hierarchical structure of chromatin and the polymorphic organization of subcompartments that assemble around it. Towards this goal, approaches to measure local properties and compositional dynamics of chromatin in its endogenous cellular environment are instrumental. Here, we discuss recent advancements in studying these features and their functional implications in protein and RNA enrichment and genome accessibility.

Transcription activation is enhanced by multivalent interactions independent of phase separation.

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.

PITX1 Is a Regulator of TERT Expression in Prostate Cancer with Prognostic Power.

The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT, transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT, we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length.

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats.

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.

Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators.

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.

Data for the assessment of vulnerability and resilience in the field of environmental health in the north of France.

The integration of multidimensional data is necessary to improve the understanding of environmental and social inequalities in health. The challenge is to define a dataset that provides the most holistic description possible of the territory. This article presents a relevant dataset to characterize the territorial accumulation of health determinants in the second most densely populated region of metropolitan France (Hauts-de-France Region, in the north of France). The multidimensional dataset combines data related to the economic, social, environment, services, health and policy dimensions at fine scale (i.e., each municipality). Data outlining a negative impact on health inequalities (e.g. anthropogenic pressures, socioeconomics factors related to vulnerability, etc.) are considered to be as important as data outlining a positive impact on health inequalities (e.g. natural resources, diversity and economic drive, etc.). The proposed theoretical framework relies on data reuse. Over one hundred variables covering a time frame from 2008 to 2017 were collected from a dozen public and national database providers. The use of official organizations ensured the quality of the collected data. The Geographic Information System, designed to map and catalogue ready-to-use data, was used to generate new data or to deal with missing data. Finally, 50 variables, including mostly quantitative but also qualitative data, were selected after application of inclusion and exclusion criteria. The resulting dataset provides a broad characterisation of the 3,817 municipalities in the Hauts-de-France Region. These data will help to discriminate the distribution pattern of vulnerability and resilience levels in this region. This novel approach is described in the paper "How can we analyse environmental health resilience and vulnerability? A joint analysis with composite indices applied to the north of France", which provides a detailed description of the methodology used to develop composite indices. This research could therefore be of use to researchers, policy makers and stakeholders in the field of environmental health seeking to identify the weaknesses but also the strengths of municipalities.

How can we analyze environmental health resilience and vulnerability? A joint analysis with composite indices applied to the north of France.

In environmental health, vulnerability reflecting the cumulative harmful constraints and nuisances to which populations are subjected and resilience defined as the capacity of a territory to cope with health inequalities have been little extensively investigated together with the same importance. Besides the diversity of factors involved, there is no consensual framework to develop composite indices, one recognized methodology to deal with a multifaceted issue. Therefore, this research aims to establish a new transferable approach to assess the spatial heterogeneity of territorial inequalities. This new strategy relies on the simultaneous evaluation of resilience and vulnerability and the joint analysis based on the cross-interpretation of the spatialized composite indices of resilience and vulnerability. A case study was conducted to demonstrate the feasibility of this methodology, using the municipality as a spatial unit of analysis within a region in the north of France. To provide the most holistic description possible of the 3817 studied municipalities, 50 variables related to the economic, environment, policy, health, services and social dimensions were used to develop the composite indices. The vulnerability Index has a median value of 0.151 with an IQR of [0.126-0.180] and the Resilience Index has a median value of 0.341 with an IQR of [0.273-0.401]. The joint analysis was conducted to classify each municipality among four defined typologies: 1687 municipalities (44.2%) belong to the "To monitor" category, 1646 (43.1%) to the "Resilient" category, 329 (8.6%) to the "Have resources" category and 155 (4.1%) to the "Territorial blackspot" category. The methodology herein may be a diagnostic tool to identify and prioritize municipalities that could benefit from the implementation of specifically tailored public health policies.

Energy Metabolism Disturbances in Cell Models of PARK2 CNV Carriers with ADHD.

The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics.

MeCP2 gates spatial learning-induced alternative splicing events in the mouse hippocampus.

Long-term memory formation is supported by functional and structural changes of neuronal networks, which rely on de novo gene transcription and protein synthesis. The modulation of the neuronal transcriptome in response to learning depends on transcriptional and post-transcriptional mechanisms. DNA methylation writers and readers regulate the activity-dependent genomic program required for memory consolidation. The most abundant DNA methylation reader, the Methyl CpG binding domain protein 2 (MeCP2), has been shown to regulate alternative splicing, but whether it establishes splicing events important for memory consolidation has not been investigated. In this study, we identified the alternative splicing profile of the mouse hippocampus in basal conditions and after a spatial learning experience, and investigated the requirement of MeCP2 for these processes. We observed that spatial learning triggers a wide-range of alternative splicing events in transcripts associated with structural and functional remodeling and that virus-mediated knockdown of MeCP2 impairs learning-dependent post-transcriptional responses of mature hippocampal neurons. Furthermore, we found that MeCP2 preferentially affected the splicing modalities intron retention and exon skipping and guided the alternative splicing of distinct set of genes in baseline conditions and after learning. Lastly, comparative analysis of the MeCP2-regulated transcriptome with the alternatively spliced mRNA pool, revealed that MeCP2 disruption alters the relative abundance of alternatively spliced isoforms without affecting the overall mRNA levels. Taken together, our findings reveal that adult hippocampal MeCP2 is required to finetune alternative splicing events in basal conditions, as well as in response to spatial learning. This study provides new insight into how MeCP2 regulates brain function, particularly cognitive abilities, and sheds light onto the pathophysiological mechanisms of Rett syndrome, that is characterized by intellectual disability and caused by mutations in the Mecp2 gene.

Repetitive RNAs as Regulators of Chromatin-Associated Subcompartment Formation by Phase Separation.

Repetitive RNA (repRNA) sequences emerge as important regulators of the dynamic organization of genomic loci into membrane-less subcompartments with distinct nuclear functions. These domains include sites of active transcription like the nucleolus as well as (peri)centromeric and telomeric satellite repeats. Recent studies point to an important role of repRNAs in complex with proteins to promote a phase separation-driven formation of chromatin domains. We review how key features of the phase separation process can be revealed by different experimental approaches and discuss the associated structure-function relationships for chromatin subcompartments that involve repRNA.

Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation.

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.

The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells.

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.

Validation of the AgraStrip® gluten G12 test kit.

Romer Labs , Inc. developed an immunochromatographic lateral flow assay for the qualitative detection of gluten in raw ingredients, processed foods, finished food products, and environmental surfaces, using the G12 antibody developed by Belén Morón. The G12 antibody targets a 33-mer peptide which is resistant to enzymatic digestion and heat denatiuration, as well as being the fragment of the gliadin protein.to which celiac disease sufferers react, making it a reliable analytical marker. This study was performed to validate the AgraStrip® GlutenG12 assay method under the guidance of the AOAC Peiformance Tested MethodsSM (PTM) program against AOAC Official Method of AnalysisSM 2012.01 in rice flour, bread, cookie, ice cream, and chocolate matrixes spiked with either purified gliadin or wheat gluten standard at 0, 3, 8, 15, and 25 ppm concentrations and tested at the 5, 10, and 20 ppm assay thresholds, as well as on, environmental surfaces. Stability, robustness, variation, and lot consistency studies were performed by spiking wheat gluten into a rice flour matrix at 0 and 15 ppm concentrations. The AgraStrip Gluten G12 assay was rigorously evaluated during this study and demonstrates its suitability as an AOAC PTM-certified gluten detection method.

A dual role for autophagy in a murine model of lung cancer.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.