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Abstract
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.
Highlights
Differential gene expression across cell types encompasses both key determinants and markers of the cells’ identity
Using a fusion between a small subunit present in all three RNA polymerases, we show that the technique can be used on both fluorescently sorted blastomere cells, or DNA
The RAPID approach relies on the expression of trace levels of the E. coli DNA adenine methyltransferase (Dam) fused to a subunit of the RNA polymerases, and the analysis of its DNA occupancy to evaluate the transcriptional state of genes
Summary
Differential gene expression across cell types encompasses both key determinants and markers of the cells’ identity. Cataloging these differences can provide critical insights and entry points in research aiming at elucidating the mechanisms controlling fundamental biological processes, such as organismal development and cell/tissue physiology. C. elegans is a widely used model animal, well suited for integrative studies bridging our understanding across the molecular, cellular and organismal levels. In contrast to the versatility of the model for individual gene expression analysis, more holistic approaches such as tissue-specific transcriptomics still remain relatively challenging, in particular in postembryonic animals due to the tough cuticle and the difficulty to isolate intact tissue or cell types.
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