Cell type- and transcription-independent spatial proximity between enhancers and promoters.

Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell type-specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell type-specificity in enhancer function.

Allele-level visualization of transcription and chromatin by high-throughput imaging.

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants.

RNA-fluorescence in situ hybridization (RNA-FISH) is an essential and widely used tool for visualizing RNA molecules in intact cells. Recent advances have increased RNA-FISH sensitivity, signal detection efficiency, and throughput. However, detection of endogenous mRNA splice variants has been challenging due to the limits of visualization of RNA-FISH fluorescence signals and due to the limited number of RNA-FISH probes per target. HiFENS (high-throughput FISH detection of endogenous pre-mRNA splicing isoforms) is a method that enables visualization and relative quantification of mRNA splice variants at single-cell resolution in an automated high-throughput manner. HiFENS incorporates HCR (hybridization chain reaction) signal amplification strategies to enhance the fluorescence signal generated by low abundance transcripts or a small number of FISH probes targeting short stretches of RNA, such as single exons. The technique offers a significant advance in high-throughput FISH-based RNA detection and provides a powerful tool that can be used as a readout in functional genomics screens to discover and dissect cellular pathways regulating gene expression and alternative pre-mRNA splicing events.

Allele-level visualization of transcription and chromatin by high-throughput imaging.

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

Allele-level visualization of transcription and chromatin by high-throughput imaging.

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

Computational methods for analysing multiscale 3D genome organization.

Recent progress in whole-genome mapping and imaging technologies has enabled the characterization of the spatial organization and folding of the genome in the nucleus. In parallel, advanced computational methods have been developed to leverage these mapping data to reveal multiscale three-dimensional (3D) genome features and to provide a more complete view of genome structure and its connections to genome functions such as transcription. Here, we discuss how recently developed computational tools, including machine-learning-based methods and integrative structure-modelling frameworks, have led to a systematic, multiscale delineation of the connections among different scales of 3D genome organization, genomic and epigenomic features, functional nuclear components and genome function. However, approaches that more comprehensively integrate a wide variety of genomic and imaging datasets are still needed to uncover the functional role of 3D genome structure in defining cellular phenotypes in health and disease.

HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression.

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software for image analysis workflows does not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new algorithms for existing analysis pipelines and to adding new analysis pipelines through separate plugins. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI analysis platform for a variety of cell biology applications.

Identification of epigenetic modulators as determinants of nuclear size and shape.

The shape and size of the human cell nucleus is highly variable among cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, and eliminating cell cycle effectors, we identified a set of novel determinants of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.

Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2.

One of the major cellular mechanisms to ensure cellular protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is triggered by accumulation of misfolded proteins in the ER lumen. The ER stress response is also activated in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we explore the mechanism of activation of the ER stress response in HGPS. We find that aggregation of the diseases-causing progerin protein at the nuclear envelope triggers ER stress. Induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nucleoplasmic protein aggregates can be sensed, and signaled to the ER lumen, via clustering of SUN2. These results identify a mechanism of communication between the nucleus and the ER and provide insight into the molecular disease mechanisms of HGPS.

Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression.

Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located âˆ¼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.

HiFENS: high-throughput FISH detection of endogenous pre-mRNA splicing isoforms.

Splicing factors play an essential role in regulation of alternative pre-mRNA splicing. While much progress has been made in delineating the mechanisms of the splicing machinery, the identity of signal transduction pathways and upstream factors that regulate splicing factor activity is largely unknown. A major challenge in the discovery of upstream regulatory factors of pre-mRNA splicing is the scarcity of functional genomics screening methods to monitor splicing outcomes of endogenous genes. Here, we have developed HiFENS (high throughput FISH detection of endogenous splicing isoforms), a high-throughput imaging assay based on hybridization chain reaction (HCR) and used HiFENS to screen for cellular factors that regulate alternative splicing of endogenous genes. We demonstrate optimized detection with high specificity of endogenous splicing isoforms and multiplexing of probes for accurate detection of splicing outcomes with single cell resolution. As proof-of-principle, we perform an RNAi screen of 702 human kinases and identify potential candidate upstream splicing regulators of the FGFR2 gene. HiFENS should be a useful tool for the unbiased delineation of cellular pathways involved in alternative splicing regulation.

Nuclear position modulates long-range chromatin interactions.

The human genome is non-randomly organized within the cell nucleus. Spatial mapping of genome folding by biochemical methods and imaging has revealed extensive variation in locus interaction frequencies between cells in a population and between homologs within an individual cell. Commonly used mapping approaches typically examine either the relative position of genomic sites to each other or the position of individual loci relative to nuclear landmarks. Whether the frequency of specific chromatin-chromatin interactions is affected by where in the nuclear space a locus is located is unknown. Here, we have simultaneously mapped at the single cell level the interaction frequencies and radial position of more than a hundred locus pairs using high-throughput imaging to ask whether the location within the nucleus affects interaction frequency. We find strong enrichment of many interactions at specific radial positions. Position-dependency of interactions was cell-type specific, correlated with local chromatin type, and cell-type-specific enriched associations were marked by increased variability, sometimes without a significant decrease in mean spatial distance. These observations demonstrate that the folding of the chromatin fiber, which brings genomically distant loci into proximity, and the position of that chromatin fiber relative to nuclear landmarks, are closely linked.

Mesoscale structure-function relationships in mitochondrial transcriptional condensates.

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed in vitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates.

High-Throughput DNA FISH (hiFISH).

High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.

HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells.

DNA damage is a prominent biomarker for numerous diseases, including cancer, as well as for the aging process. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, quantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and [Formula: see text]-H2AX. We demonstrate sensitive detection with low inter-assay variability of DNA damage in various types of freshly isolated and cryopreserved primary human immune cells, including CD4 + and CD8 + T cells, B cells and monocytes. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

Large-scale mapping of positional changes of hypoxia-responsive genes upon activation.

Chromosome structure and nuclear organization are important factors in the regulation of gene expression. Transcription of a gene is influenced by local and global chromosome features such as chromatin condensation status. The relationship between the 3D position of a gene in the nucleus and its activity is less clear. Here we used high-throughput imaging to perform a large-scale analysis of the spatial location of nearly 100 hypoxia-responsive genes to determine whether their location and activity state are correlated. Radial distance analysis demonstrated that the majority of Hypoxia-Inducible Factor (HIF)- and CREB-dependent hypoxia-responsive genes are located in the intermediate region of the nucleus, and some of them changed their radial position in hypoxia. Analysis of the relative distances among a subset of HIF target genes revealed that some gene pairs altered their relative location to each other on hypoxic treatment, suggesting higher-order chromatin rearrangements. While these changes in location occurred in response to hypoxic activation of the target genes, they did not correlate with the extent of their activation. These results suggest that induction of the hypoxia-responsive gene expression program is accompanied by spatial alterations of the genome, but that radial and relative gene positions are not directly related to gene activity.