Project description:The molecular regulation of zygotic genome activation (ZGA) in mammals remains poorly understood. Primed mouse embryonic stem cells contain a rare subset of “2C-like” cells that are epigenetically and transcriptionally similar to the two cell embryo and thus represent an ideal system for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed exclusively in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA either in a Dux dependent or independent manner. Here we performed a candidate-based overexpression screen, identifying, amongst others, Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as positive regulators of 2C-like cells and ZGA transcription. In the germ line, promoter DNA demethylation coincides with upregulation of Dppa2 and Dppa4 which remain expressed until E7.5 when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during iPSC reprogramming at the time 2C-like ZGA transcription transiently peaks. Through a combination of overexpression, knockdown, knockout and rescue experiments, together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we tease apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilised. Dppa2 and Dppa4 require Dux to initiate 2C-like ZGA transcription, suggesting they act upstream by directly regulating Dux. Supporting this, ChIP-seq analysis revealed Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild type cells, but, in contrast to Dux, can no longer do so in Dppa2/4 double knockout cells, suggesting it may act to stabilise rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux upregulation and activation of the 2C-like transcriptional program which is subsequently reinforced by Zscan4c.

Project description:Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics and applied this method to systematically probe candidate regulators of ZGA-like transcription. We screened 230 epigenetic and transcriptional factors by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq), we generated approximately 200,000 single-cell transcriptomes of CRISPRa-perturbed cells, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using multi-omics factor analysis (MOFA) of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. We further validated nine candidate genes by arrayed CRISPRa analysed by bulk transcriptomics, which demonstrates that the combination of CRISPRa with scRNA-seq is a powerful and valid approach to identify regulators of ZGA-like transcription. Additional cDNA overexpression assays for three top hits, the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, confirmed these factors as ZGA-like regulators by alternative methods. Supporting these findings, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes and functional experiments revealed that Smarca5’s regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.

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Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

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