3DPolyS-LE: an accessible simulation framework to model the interplay between chromatin and loop extrusion.

SUMMARY: Recent studies suggest that the loop extrusion activity of Structural Maintenance of Chromosomes complexes is central to proper organization of genomes in vivo. Polymer physics-based modeling of chromosome structure has been instrumental to assess which structures such extrusion can create. Only few laboratories however have the technical and computational expertise to create in silico models combining dynamic features of chromatin and loop extruders. Here, we present 3DPolyS-LE, a self-contained, easy to use modeling and simulation framework allowing non-specialists to ask how specific properties of loop extruders and boundary elements impact on 3D chromosome structure. 3DPolyS-LE also provides algorithms to compare predictions with experimental Hi-C data. AVAILABILITY AND IMPLEMENTATION: Software available at https://gitlab.com/togop/3DPolyS-LE; implemented in Python and Fortran 2003 and supported on any Unix-based operating system (Linux and Mac OS). SUPPLEMENTARY INFORMATION: Supplementary information are available at Bioinformatics online.

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal D.

Bicaudal D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. Given that BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize these components to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles and the tandem spindles during female meiosis II. Furthermore, BicD is required to localize clathrin and the microtubule-stabilizing factors transforming acidic coiled-coil protein (D-TACC/Tacc) and Mini spindles (Msps) correctly to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, immediately after the establishment of the female pronucleus, D-TACC and Caenorhabditis elegans BicD, tacc and Chc are also needed for pronuclear fusion, suggesting that the underlying mechanism might be more widely used across species.

Nonlinear control of transcription through enhancer-promoter interactions.

Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer-promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

Tissue-specific DamID protocol using nanopore sequencing.

DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA fragments, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.

Widespread gpa-4 promoter-driven expression during Caenorhabditis elegans development.

The gpa-4 promoter-driven expression is described as specific for ASIL and ASIR chemosensory neurons in the nematode Caenorhabditis elegans, yet this was mostly examined in adult animals. Here we used a recombination-mediated reporter to test the previously used gpa-4 promoter expression. This reporter highlights all cells in which the gpa-4 promoter has been active at one point or another during development. We show that the gpa-4 promoter is indeed active in ASI, yet to our surprise, thispromoter drives also expression in many other cell types, including the somatic gonad, the seam cells, a subset of tail and head neurons, and muscle cells, demonstrating a widespread activity of this transgenic gpa-4 promoter during embryonic and post-embryonic development.

Generation of Inexpensive, Highly Labeled Probes for Fluorescence In Situ Hybridization (FISH).

DNA-FISH remains the method of choice to visualize genomic regions in situ ranging from a single locus to entire chromosomes. Current methods to generate probes rely on expensive kits that vary in labeling efficiency and are limited by the size and/or amount of starting material and by the choice of fluorophores. Here we describe a protocol to prepare inexpensive ($20) DNA-FISH probes using an isothermal polymerase, incorporating labeled nucleotides while amplifying minute amounts of any template (PCR fragments/BAC/YAC/fosmids). For complete details on the use and execution of this protocol, please refer to Grosmaire et al. (2019) and Sharma et al. (2014).

Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans.

Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (RNA-seq) studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 3901 candidate genes with putatively active transcription in XXX cells, including the few previously known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

Males as somatic investment in a parthenogenetic nematode.

We report the reproductive strategy of the nematode Mesorhabditis belari This species produces only 9% males, whose sperm is necessary to fertilize and activate the eggs. However, most of the fertilized eggs develop without using the sperm DNA and produce female individuals. Only in 9% of eggs is the male DNA utilized, producing sons. We found that mixing of parental genomes only gives rise to males because the Y-bearing sperm of males are much more competent than the X-bearing sperm for penetrating the eggs. In this previously unrecognized strategy, asexual females produce few sexual males whose genes never reenter the female pool. Here, production of males is of interest only if sons are more likely to mate with their sisters. Using game theory, we show that in this context, the production of 9% males by M. belari females is an evolutionary stable strategy.

Polycomb and Notch signaling regulate cell proliferation potential during Caenorhabditis elegans life cycle.

Stable cell fate is an essential feature for multicellular organisms in which individual cells achieve specialized functions. Caenorhabditis elegans is a great model to analyze the determinants of cell fate stability because of its invariant lineage. We present a tractable cell fate challenge system that uses the induction of fate-specifying transcription factors. We show that wild-type differentiated animals are highly resistant to fate challenge. Removal of heterochromatin marks showed marked differences: the absence of histone 3 lysine 9 methylation (H3K9) has no effect on fate stability, whereas Polycomb homolog mes-2 mutants lacking H3K27 methylation terminally arrest larval development upon fate challenge. Unexpectedly, the arrest correlated with widespread cell proliferation rather than transdifferentiation. Using a candidate RNAi larval arrest-rescue screen, we show that the LIN-12Notch pathway is essential for hyperplasia induction. Moreover, Notch signaling appears downstream of food-sensing pathways, as dauers and first larval stage diapause animals are resistant to fate challenge. Our results demonstrate an equilibrium between proliferation and differentiation regulated by Polycomb and Notch signaling in the soma during the nematode life cycle.

Coupling 1D modifications and 3D nuclear organization: data, models and function.

Over the past decade, advances in molecular methods have strikingly improved the resolution at which nuclear genome folding can be analyzed. This revealed a wealth of conserved features organizing the one dimensional DNA molecule into tridimensional nuclear domains. In this review, we briefly summarize the main findings and highlight how models based on polymer physics shed light on the principles underlying the formation of these domains. Finally, we discuss the mechanistic similarities allowing self-organization of these structures and the functional importance of these in the maintenance of transcriptional programs.

DamID Analysis of Nuclear Organization in Caenorhabditis elegans.

The development of genomics and next generation sequencing platforms has dramatically improved our insight into chromatin structure and organization and its fine interplay with gene expression. The nuclear envelope has emerged as a key component in nuclear organization via extensive contacts between the genome and numerous proteins at the nuclear periphery. These contacts may have profound effects on gene expression as well as cell proliferation and differentiation. Indeed, their perturbations are associated with several human pathologies known as laminopathies or nuclear envelopathies. However, due to their dynamic behavior the contacts between nuclear envelope proteins and chromatin are challenging to identify, in particular in intact tissues. Here, we propose the DamID technique as an attractive method to globally characterize chromatin organization in the popular model organism Caenorhabditis elegans. DamID is based on the in vivo expression of a chromatin-associated protein of interest fused to the Escherichia coli DNA adenine methyltransferase, which produces unique identification tags at binding site in the genome. This marking is simple, highly specific and can be mapped by sensitive enzymatic and next generation sequencing approaches.

From single genes to entire genomes: the search for a function of nuclear organization.

The existence of different domains within the nucleus has been clear from the time, in the late 1920s, that heterochromatin and euchromatin were discovered. The observation that heterochromatin is less transcribed than euchromatin suggested that microscopically identifiable structures might correspond to functionally different domains of the nucleus. Until 15 years ago, studies linking gene expression and subnuclear localization were limited to a few genes. As we discuss in this Review, new genome-wide techniques have now radically changed the way nuclear organization is analyzed. These have provided a much more detailed view of functional nuclear architecture, leading to the emergence of a number of new paradigms of chromatin folding and how this folding evolves during development.

Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development.

C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes…). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest.

Dosage compensation and nuclear organization: cluster to control chromosome-wide gene expression.

In many species, male and female animals differ in the number of X chromosomes they possess. As a consequence, large scale differences in gene dosage exist between sexes; a phenomenon that is rarely tolerated by the organism for changes in autosome dosage. Several strategies have evolved independently to balance X-linked gene dosage between sexes, named dosage compensation (DC). The molecular basis of DC differs among the three best-studied examples: mammals, fruit fly and nematodes. In this short review, we summarize recent microscopic and chromosome conformation capture data that reveal key features of the compensated X chromosome and highlight the events leading to the establishment of a functional, specialized nuclear compartment, the X domain.

Linking dosage compensation and X chromosome nuclear organization in C. elegans.

Animal sex is determined by the number of X chromosomes in many species, creating unequal gene dosage (aneuploidy) between sexes. Dosage Compensation mechanisms equalize this dosage difference by regulating X-linked gene expression. In the nematode C. elegans the current model suggests that DC is achieved by a 2-fold transcriptional downregulation in hermaphrodites mediated by the Dosage Compensation Complex (DCC), which restricts access to RNA Polymerase II by an unknown mechanism. Taking a nuclear organization point of view, we showed that the male X chromosome resides in the pore proximal subnuclear compartment whereas the DCC bound to the X, inhibits this spatial organization in the hermaphrodites. Here we discuss our results and propose a model that reassigns the role of DCC from repression of genes to inhibition of activation.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

Modern tools to study nuclear pore complexes and nucleocytoplasmic transport in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is characterized by many features that make it highly attractive to study nuclear pore complexes (NPCs) and nucleocytoplasmic transport. NPC composition and structure are highly conserved in nematodes and being amenable to a variety of genetic manipulations, key aspects of nuclear envelope dynamics can be observed in great details during breakdown, reassembly, and interphase. In this chapter, we provide an overview of some of the most relevant modern techniques that allow researchers unfamiliar with C. elegans to embark on studies of nucleoporins in an intact organism through its development from zygote to aging adult. We focus on methods relevant to generate loss-of-function phenotypes and their analysis by advanced microscopy. Extensive references to available reagents, such as mutants, transgenic strains, and antibodies are equally useful to scientists with or without prior C. elegans or nucleoporin experience.

Modern techniques for the analysis of chromatin and nuclear organization in C. elegans.

In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.

Microscopic analysis of chromatin localization and dynamics in C. elegans.

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.