DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans

Walle, Pieter Van de; Muñoz-Jiménez, Celia; Askjaer, Peter; Schoofs, Liliane; Temmerman, Liesbet

doi:10.1371/journal.pone.0242939

Cited by 7 publications

(9 citation statements)

References 85 publications

(149 reference statements)

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“…Previously, a series of efficient tissue-specific FLP-lines were generated by Mos1-mediated Single Copy Insertion (MosSCI) [20,22,23,51]. To expand the existing toolkit, we created additional MosSCI lines that express FLP in specific cell types and evaluated recombination efficiency with the heat-inducible dual color reporter bqSi294 that produces green nuclei (GFP::HIS-58) upon successful recombination and red nuclei (mCherry::HIS-58) elsewhere (Figure 1A) [20].…”

Section: Resultsmentioning

confidence: 99%

“…An increasing number of specific and efficient FLP and Cre drivers generated by the C. elegans community are facilitating a variety of precise functional assays to interrogate neural circuits [10,13,24,12], cell proliferation and differentiation [11], gene expression programs [14,51] and chromatin organization [15,16]. In this study, we report four new FLP lines that reliably induce recombination specifically in either distal tip cells, the somatic gonad, coelomocytes or in epithelial Pn.p cells.…”

Section: Discussionmentioning

confidence: 99%

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Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

Fragoso-Luna

Ayuso

Eibl

et al. 2021

Preprint

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Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify nascent transcripts in a tissue of interest. We have adapted thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue (SLAM-ITseq) for C. elegans. By focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by SLAM-ITseq are known to be expressed in this tissue, but with the added value of temporal resolution. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

Fragoso-Luna

Ayuso

Eibl

et al. 2021

Preprint

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“…DNA adenine methyltransferase identification (DamID) offers an alternative way to reveal targets by fusing a TF of interest to the Dam methyltranferase from Escherichia coli ( 19 ). This enzyme mediates site-specific methylation in the adenine of GATC sequences ( 20 , 21 ). A key requirement in DamID methods is keeping the levels of Dam expression very low to avoid toxicity and saturated methylation of DNA ( 19 , 22 ).…”

Section: Introductionmentioning

confidence: 99%

Targeted DamID in C. elegans reveals a direct role for LIN-22 and NHR-25 in antagonizing the epidermal stem cell fate

Katsanos

Barkoulas

2022

Sci. Adv.

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Transcription factors are key players in gene networks controlling cell fate specification during development. In multicellular organisms, they display complex patterns of expression and binding to their targets, hence, tissue specificity is required in the characterization of transcription factor–target interactions. We introduce here targeted DamID (TaDa) as a method for tissue-specific transcription factor target identification in intact Caenorhabditis elegans animals. We use TaDa to recover targets in the epidermis for two factors, the HES1 homolog LIN-22, and the NR5A1/2 nuclear hormone receptor NHR-25. We demonstrate a direct link between LIN-22 and the Wnt signaling pathway through repression of the Frizzled receptor lin-17 . We report a direct role for NHR-25 in promoting cell differentiation via repressing the expression of stem cell–promoting GATA factors. Our results expand our understanding of the epidermal gene network and highlight the potential of TaDa to dissect the architecture of tissue-specific gene regulatory networks.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Tissue-specific DamID protocol using nanopore sequencing

2021

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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.

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DamID identifies targets of CEH-60/PBX that are associated with neuron development and muscle structure in Caenorhabditis elegans

Cited by 7 publications

References 85 publications

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

Targeted DamID in C. elegans reveals a direct role for LIN-22 and NHR-25 in antagonizing the epidermal stem cell fate

Tissue-specific DamID protocol using nanopore sequencing

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