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

Abstract

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 RNAPol 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.

Tissue-specific DamID protocol using nanopore sequencing

“…Notably, DamID is highly versatile and has been applied in many organisms, ranging from fission yeast [ 6-10 ], C. elegans [ 11-17 ], Drosophila [ 18-23 ], Medaka [ 24 ], Arabidopsis [ 25-27 ], and mice [ 28-31 ] to human cells [ 32-35 ]. Additionally, DamID has been adapted to evaluate an extensive range of chromatin features in vivo such as chromatin accessibility (CATaDA) [ 36 ], chromatin dynamics visualization (m6A-tracer) [ 37 ], transcription factor binding (MaTaDa) [ 38 ] and co-binding (SpDamID) [ 39 ], RNA-DNA interactions (RNA-DamID) [ 40 ], three-dimensional genome organization (DamC) [ 41 ], transcriptional profiling (TaDa [ 42 ] and RAPID [ 43 ]), or simultaneous transcription and protein-DNA interaction profiling in single cells (scDam&T-seq) [ 44 , 45 ].…”

“…We recently developed RAPID (RNA PoI DamID) to profile genome-wide, tissue-specific RNA Polymerase occupancy in vivo without cell isolation or nuclei purification [ 43 ]. In contrast to other methods, which sequence transcripts from specific cell types, RAPID is based on the methylation footprinting by an RNA polymerase subunit fused to a Dam methyltransferase, expressed only in the target tissue ( Fig.…”

“…We further showed that this method can be applied for rare cell types by profiling cell types representing only two cells per animal or 0.2% of the somatic cells. The specific advantages and limitations of RAPID as compared to RNA-seq-based methods were extensively discussed in this previous work [ 43 ].…”

“…C. elegans has 269049 GATC motifs per haploid genome [ 48 ], corresponding to a mean distance between sites of 374 bp and a median of 210 bp [ 49 ]. With this density, DamID was successfully applied to obtain the genomic footprint of several transcription factors such as DAF-16 [ 13 ], CEH-60 [ 14 ] and CRH-1 (our unpublished data), as well as RNA Polymerases [ 43 ]. However, for most genomic regions the binding sites are defined as relatively large windows of several hundred bases, sometimes much larger in some regions with low GATC density.…”

Targeted DamID in C. elegans reveals a role for LIN-22 and NHR-25 in epidermal cell differentiation

“…An additional challenge in multicellular systems like C. elegans is to characterise DNA-protein interactions with tissue-specific resolution. This has been previously achieved using recombinase-based systems to allow expression of Dam fusions at low levels defined by the basal transcription from non-induced heatshock promoters (Gómez-Saldivar et al, 2020;Harr et al, 2020;Pindyurin et al, 2016). Alternatively, Targeted DamID (TaDa) allows tissue-specific target identification (Southall et al, 2013) and has been successfully employed in Drosophila to dissect mechanisms of neuronal fate determination (Sen et al, 2019;Vissers et al, 2018), as well as in mammalian stem cell lines to characterise the binding profile of pluripotency factors (Cheetham et al, 2018).…”