A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program

Abstract

Summary Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2 , the chromatin remodeler Smarca5 , and the transcription factor Patz1 , 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 both system-level and molecular insights into the mechanisms that orchestrate ZGA.

Host Gene Regulation by Transposable Elements: The New, the Old and the Ugly

“…Elegant work has identified that the transcription factors DUX, ZSCAN4, DPPA2 and DPPA4 activate 2C specific genes by binding to MT2 LTR promoters [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Mechanistically, MERVL LTRs, therefore, regulate genes by acting as poised promoters ( Figure 2 ).…”

Keeping your options open: insights from Dppa2/4 into how epigenetic priming factors promote cell plasticity

“…Aspects of zygotic genome activation can be studied in vitro using serum-grown mouse embryonic stem cells which contain a rare subpopulation of 2C-like cells that share features of the two-cell embryo [ 37–40 ]. Using this model, several studies have revealed an essential role for Dppa2/4 in regulating 2C-like cells and the ZGA-associated transcripts, including the Zscan4 cluster, MERVL endogenous retrovirus and LINE1 repeats, expressed in these cells [ 32 , 35 , 41 , 42 ].…”

“…Dppa2/4 were initially discovered as strong inducers of the 2C-like state through a candidate-based screen where oocyte/zygotic epigenetic factors were overexpressed in mESCs and the effects on ZGA-like transcription assessed [ 32 ]. This screen approach was recently extended using CRISPR-activation coupled with a single-cell transcriptomic read out, confirming Dppa2 as a potent activator of ZGA-like transcription in ESCs [ 41 ]. Mechanistically, ChIP-seq experiments revealed that Dppa2/4 bind the Dux gene, but not other 2C-like genes or repeats, regulating its expression [ 32 , 35 , 42 ].…”

Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells

“…Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 10× Genomics scRNA-seq as a readout. For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020) .…”

“…For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020) .…”