Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome.

We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well-studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single-stranded RNA fragments and one involving circular-template PCR (CircLigase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of CircLigase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double-strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the poly(A) junction. Using data sets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures.

Localization of the Drosophila pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression.

The eukaryotic genome is organized to enable the precise regulation of gene expression. This organization is established as the embryo transitions from a fertilized gamete to a totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. This non-uniform distribution is driven by binding to highly abundant GA repeats. At GA repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains essential for the robust regulation of gene expression.

Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos.

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the use of single-nucleus RNA sequencing in Drosophila embryos prior to cellularization, here we look at gene expression in control and insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we provide a resource of candidate differentially expressed genes that might show local changes in gene expression between control and maternal dCTCF null nuclei with no detectable differential expression in bulk. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo.

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos.

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo.

High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains.