Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes.

Recent advances in microscopy have enabled studying chromosome organization at the single-molecule level, yet little is known about inherited chromosome organization. Here we adapt single-molecule chromosome tracing to distinguish two C. elegans strains (N2 and HI) and find that while their organization is similar, the N2 chromosome influences the folding parameters of the HI chromosome, in particular the step size, across generations. Furthermore, homologous chromosomes overlap frequently, but alignment between homologous regions is rare, suggesting that transvection is unlikely. We present a powerful tool to investigate chromosome architecture and to track the parent of origin.

Chromosome organization in 4D: insights from C. elegans development.

Eukaryotic genome organization is ordered and multilayered, from the nucleosome to chromosomal scales. These layers are not static during development, but are remodeled over time and between tissues. Thus, animal model studies with high spatiotemporal resolution are necessary to understand the various forms and functions of genome organization in vivo. In C. elegans, sequencing- and imaging-based advances have provided insight on how histone modifications, regulatory elements, and large-scale chromosome conformations are established and changed. Recent observations include unexpected physiological roles for topologically associating domains, different roles for the nuclear lamina at different chromatin scales, cell-type-specific enhancer and promoter regulatory grammars, and prevalent compartment variability in early development. Here, we summarize these and other recent findings in C. elegans, and suggest future avenues of research to enrich our in vivo knowledge of the forms and functions of nuclear organization.

Gene family evolution underlies cell-type diversification in the hypothalamus of teleosts.

Hundreds of cell types form the vertebrate brain but it is largely unknown how similar cellular repertoires are between or within species or how cell-type diversity evolves. To examine cell-type diversity across and within species, we performed single-cell RNA sequencing of ~130,000 hypothalamic cells from zebrafish (Danio rerio) and surface and cave morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which diverged from a common ancestor over 150 million years ago. Shared cell types displayed shifts in paralogue expression that were generated by subfunctionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes and characterized by the neofunctionalization of expression patterns of members of recently expanded or contracted gene families. Comparisons between surface and cave morphs revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types and reveal how gene family evolution and shifts in paralogue expression contribute to cellular diversity.

Multiplexed Sequential DNA FISH in Caenorhabditis elegans Embryos.

This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization). For complete details on the use and execution of this protocol, please refer to Sawh et al. (2020).

A Family of Argonaute-Interacting Proteins Gates Nuclear RNAi.

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.

Lamina-Dependent Stretching and Unconventional Chromosome Compartments in Early C. elegans Embryos.

Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.

A truncated form of dicer tilts the balance of RNA interference pathways.

The RNase III enzyme Dicer is responsible for key steps in the biogenesis of small RNA species in multiple RNA interference pathways. Here, we show that, in the adult C. elegans soma, half of the total DCR-1 protein is expressed as a truncated, stable C-terminal fragment named small DCR-1 (sDCR-1). sDCR-1 operates independently of full-length DCR-1 in two distinct RNAi pathways; it enhances exogenous RNAi (exoRNAi) and concurrently acts as a negative regulator of microRNA (miRNA) biogenesis. Enhancement of exoRNAi relies on sDCR-1 catalytic activity, whereas impinging on miRNA processing does not. Instead, sDCR-1 competes with pre-miRNA processing by interacting with the miRNA-dedicated Argonautes ALG-1 and ALG-2. Finally, triggering a strong exoRNAi response in the presence of elevated levels of sDCR-1 exacerbates the miRNA processing defect. Our results unveil a surprising role for a truncated form of DCR-1 in the modulation of multiple RNAi activities and in the regulation of mechanistic boundaries between pathways.

Turning Dicer on its head.

Recent structural analysis of full-length human Dicer supports a new model of the enzyme's domain arrangement and provides a structural basis for many of Dicer's biochemical attributes.

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1.

Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.