Meister, P. | bioRxiv Science

bioRxiv2020

Novel roles for BicD in pronuclear fusion and meiosis II progression via localization of the CHC/TACC/Msps complex to MII spindles

Drosophila Clathrin heavy chain (Chc) is transported by the dynein/dynactin microtubule motor through its interaction with the adaptor protein Bicaudal-D (BicD). Here we show that Drosophila BicD and Chc localize to centrosomes and spindles during mitosis and to the tandem spindles during female meiosis II. Reducing the activity of BicD::GFP specifically in freshly laid eggs revealed that BicD is essential for the production of normal female meiosis II products and for pronuclear fusion. Chc interacts with BicD and D-TACC, and BicD is needed to correctly localize the microtubule-stabilizing factors D-TACC, clathrin, and Msps to the meiosis II spindles, suggesting that BicD acts by localizing these proteins. In unfertilized eggs, reduced BicD levels cause the female meiotic products to re-enter the cell cycle. As 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 them to control metaphase arrest of polar bodies. Finally, Drosophila and C. elegans orthologs of BicD and tacc are also needed for pronuclear fusion.

developmental biology↗

bioRxiv2020

Tissue-specific transcription footprinting using RNA PoI DamID (RAPID) in C. elegans

Differential gene expression across cell types underlies the development and cell physiology in multicellular organisms. C. elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates, however, 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 PoI DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit in order 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, sorted embryonic blastomeres and in different tissues from intact young adults by driving Dam fusion expression tissue-specifically. We obtained meaningful transcriptional footprints in line with 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 2362 candidate genes with putatively active transcription in XXX cells, among which the few 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 and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging. Article summaryGene expression is a major determinant of cell fate and physiology, yet it is notoriously difficult to characterize in individual cell types for the widely used model system C. elegans. Here, we introduce a method based on the in vivo covalent modification of DNA by transcribing RNA polymerases to determine genome-wide transcription patterns in single tissues of embryos or young adult animals. We show that the method is able to identify actively transcribed genes in tissues representing down to 0.2% of the somatic cells in adult animals. Additionally, this method can be fully performed in a single laboratory by using third generation sequencing methods (ONT).

genetics↗