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Automated RNAi screening reveals MRG-1/MRG15 as a barrier for germline to neuron reprogramming in C. elegans

ABSTRACT: Abstract: Chromatin-regulating factors safeguard cell identities by suppressing ectopic gene expression thereby imposing barriers for transcription factor-induced reprogramming of cell fates. Identifying factors opposing the induction of ectopic cell fates is important to better understand developmental differentiation processes and may improve cellular reprogramming for therapeutic approaches. Reprogramming barriers have previously been studied mostly in tissue cultures. Hence, knowledge about chromatin regulators acting as reprogramming barriers in vivo needs improvement. We therefore made use of the nematode C. elegans as an in vivo gene discovery tool and interrogated its chromatin-regulating factors for being involved in protecting germ cells against reprogramming to neurons by performing automated RNAi screening. We identified the conserved chromodomain factor MRG-1 (MRG15 in humans) as a barrier for converting germ cells into neurons and characterized its protein-protein and genome interactions using IP-MS and ChIP-seq in wild-type animals versus mutants lacking the germline. We found that MRG-1 associates predominantly with genes carrying active histone modifications but it also cooperates with the histone H3K9 methyltransferase SET-26 in order to prevent conversion of germ cells into neurons. Conserved chromatin regulators such as MRG-1 potentially have similar functions in higher organisms as shown previously for the histone chaperone LIN-53 (CAF-1p48/RBBP7). LIN-53 was initially identified as a barrier of C. elegans germ cell reprogramming and was later shown to also block reprogramming in mammals. Such remarkable conservation of reprogramming barriers illustrates that understanding cell fate protection in C. elegans could help to improve the generation of new cell types for therapeutic approaches in humans.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE110969 | GEO | 2019/02/21

REPOSITORIES: GEO

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Project description:The Rb/E2F tumor suppressor regulates gene expression to control the onset and timing of differentiation in many different cell types during development. This critical function makes Rb/E2F a frequent target for inactivation in tumors of diverse tissue origin. However, the mechanisms by which Rb/E2F governs tissue-specific gene regulation in vivo are poorly understood. We have determined the genome-wide binding profiles for components of the C. elegans Rb/E2F transcriptional regulatory pathway in the germ line, the intestine, and broadly in somatic tissues. Rb/E2F exhibits highly tissue-specific regulation, with unique sets of target genes that are activated or repressed depending on whether cells are in a progenitor (germ line) or terminally differentiated (somatic) state. In particular, LIN-35 binding is impaired in the germ line relative to the soma. Moreover, in the intestine, LIN-35 binds independently of EFL-1/DPL-1 at broad intergenic sites at which the heterochromatin protein HPL-2 also binds. Finally, recently defined âHOTâ sites, which are non-specifically bound by many unrelated factors, appear to be restricted primarily to somatic tissues, suggesting that germ line chromatin is in a state that globally restricts promiscuous association of regulatory factors. In sum, these tissue-specific binding profiles led to significant mechanistic insights into tissue-specific properties of Rb/E2F function in C. elegans that are likely relevant to other organisms. Staged C. elegans worms carrying different transgenes expressing GFP-transcription factors under different promoters were treated to ChIP-seq in biological replicate. A total of eleven different transgenic lines were analyzed. Three transcription factors (LIN-35, EFL-1 and DPL-1) were each tagged with GFP and expressed under the pie-1 promoter (germline), the ges-1 promoter (intestine), or their endogenous promoter (somatic expression), for a total of nine lines. In addition, LIN-35:GFP was expressed under the mex-5 promoter (germline), and HPL-2 was expressed under the ges-1 promoter, for an additional two lines. All strains with germline expression were analyzed as young adults, while the intestine or somatic expressed lines were analyzed as L1s. For each replicate of each transgenic line at the appropriate stage, anti-GFP was used to immunoprecipitate the tagged transcription factor, and the factor-enriched chromatin was subjected to Illumina sequencing. As a control, for every ChIP sample, the input (without immunoprecipitation) was also sequenced.

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Automated RNAi screening reveals MRG-1/MRG15 as a barrier for germline to neuron reprogramming in C. elegans

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