Functional characterization of MEL-28/ELYS

The nuclear envelope (NE) is a highly regulated membrane barrier that separates the nucleus from the cytoplasm in eukaryotic cells. Although the NE enables complex levels of gene expression, it also poses a challenge during cell division. To allow access of the mitotic spindle to chromatin, the NE of metazoans completely disassembles during mitosis, generating the need to re-establish the nuclear compartment at the end of each cell division. MEL-28/ELYS is essential for proper NE assembly in various organisms, such as Xenopus, C. briggsae and C. elegans. Previous studies have shown that MEL-28/ELYS is located at nuclear pore complexes during interphase, to kinetochores in early mitosis and subsequently during late mitosis it is widely distributed on chromatin. C. elegans mel-28 mutant embryos are affected in various cellular processes, such as segregation of chromatin, nuclear-cytoplasmic transport and reassembly of the NE (Galy et al., 2006), whereas mutant zebrafish show defects in intestinal, liver, pancreas, and eye development. However, the mechanism of action of MEL-28 is not known, nor is it clear where and how MEL-28 binds to DNA during mitosis and NE assembly. In order to identify and characterize the location of protein domains that interact with chromatin we have constructed mutants expressing different fragments of MEL-28 fused to GFP protein. Likewise, DamID (van Steensel & Henikoff, 2000) assay was performed for identifying regions of chromatin to which joins MEL-28 and determining whether binding sites follow a specific pattern. Vincent Galy, Peter Askjaer, Cerstin Franz, Carmen López-Iglesias and Iain W. Mattaj. 2006. MEL-28, a Novel Nuclear-Envelope and Kinetochore Protein Essential for Zygotic Nuclear-Envelope Assembly in C. elegans. Current Biology, Vo 16. Pp 1748–1756.Bas van Steensel and Steven Henikoff. 2000. Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase. Nature Biotechnology, Vol 18. Pp 424-428

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, 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

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

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

MEL-28 N-terminal domains are required for NPC and kinetochore localization.

<p>(A) Still images from time-lapse recording of embryo carrying a GFP insertion into the endogenous <i>mel-28</i> locus. Time is indicated relative to anaphase onset (min:sec). (B) Metaphase plate of early embryo expressing GFP::MEL-28 (green in merge) analyzed by immunofluorescence with a specific antibody against HCP-3/CENP-A (red in merge) and Hoechst (blue in merge) to visualize chromosomes. MEL-28 localized to kinetochores, which appear as lines on both sides of the chromosomes. (C) Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28 and GFP::MEL-28^{Δ1140–1186} embryos, all embryos also expressed un-tagged endogenous MEL-28. Purple boxes in MEL-28 cartoons indicate a putative coiled-coil domain (aa. 1127–1160) whereas yellow (aa. 1630–1642) and orange (aa. 1746–1758) boxes indicate AT-hook sequences: their homology to the consensus AT-hook sequence is low and high, respectively. (D) Cropped images from metaphase embryos expressing GFP::MEL-28 or GFP::MEL-28^{Δ1140–1186}. Images were processed identically to facilitate visualization of full-length GFP::MEL-28 associated with the mitotic spindle. Signal intensities in boxed areas were quantified in raw images, normalized and plotted (n = 5, GFP::MEL-28; n = 2, GFP::MEL-28^{Δ1140–1186}). * p<0.05 by unpaired two-tailed t-test. Scale bars, 5 μm.</p

Mutation of MEL-28 loop2 impairs chromosome segregation.

<p>One-cell stage (A) and 4-cell stage (B) embryos from <i>mel-28</i> mutants expressing either GFP::MEL-28 or MEL-28^loop2mut::GFP were compared with wild type and <i>mel-28</i> embryos by immunofluorescence. Embryos were analyzed with Hoechst (blue in merge), a specific antibody against NPP-10C/NUP96 (green in merge) and mAb414 recognizing multiple nups (red in merge). Scale bars, 5 μm.</p

Identification of MEL-28 chromatin binding domain and nuclear localization signals.

<p>Cropped images from embryos expressing different MEL-28 truncations fused to GFP. Except GFP::MEL-28^1-1744 and GFP::MEL-28^1188-1784, fusion proteins were expressed from the <i>hsp-16</i>.<i>41</i> promoter in gastrulating embryos. Excluding GFP::MEL-28^1-1744 embryos, all embryos also expressed un-tagged endogenous MEL-28. Truncations containing MEL-28 residues 846–1071 and/or residues 1602–1784 (blue shading) were efficiently imported whereas truncations containing residues 1239–1601 (red shading) associated with chromatin in mitosis. Scale bars, 3 μm.</p

Overview of MEL-28 and ELYS localization domains.

<p>The N-terminal halves of MEL-28 and ELYS are sufficient to localize to NPCs (green shading) although less efficiently than full-length proteins. In the case of MEL-28, the N-terminus is also sufficient to localize to kinetochores. Both proteins contain central and C-terminal domains that are imported into nuclei (blue shading) and C-terminal domains that confer binding to chromatin (pink shading). A conserved loop2 motif in the N-terminal β-propeller is important for NPC localization in the context of truncated proteins. Both the loop2 motif and the AT-hook domain of MEL-28 are essential for embryonic viability.</p