A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Background & Summary:

After the first cell division that takes place in the mouse zygote, when the embryo consists of two cells, a critical event in development occurs: zygotic genome activation (ZGA). This transition coincides with the reprogramming of histone modifications, chromatin accessibility, as well as global DNA demethylation, and results in the transcriptional activation of thousands of genes. Some regulators of ZGA have been identified, but a global understanding of the regulation of this major event is still lacking.

The authors of this preprint use CRISPR-activation (CRISPRa) in a high-throughput screen in mouse embryonic stem cells (ESCs) to identify novel regulators of ZGA. This tool is based on the targeting of a dead Cas9 (dCas9) to gene promoters using short-guide RNAs (sgRNAs) to upregulate gene transcription. The authors targeted 230 candidate regulators of ZGA and after single-cell RNA-sequencing (scRNA-seq) of CRISPRa-perturbed cells, they identified 44 putative regulators that activated a ZGA signature. The authors then selected three of them: Dppa2, a DNA-binding factor already associated to ZGA by the same lab (Eckersley-Maslin et al. 2019), the chromatin remodeller Smarca5 and the top-ranking transcription factor Patz1; and confirmed that their activation resulted in a large fold-change in ZGA-signature genes. After analysing loss of function and rescue experiments using Dppa2 and Smarca5, the authors concluded that the chromatin remodeller Smarca5 regulates ZGA in a Dppa2-dependent manner.

 

Major experiments in the preprint.

• CRISPR-activation screening of candidate regulators of zygotic genome activation. The authors targeted 230 candidate genes which are expressed prior to and at the time of ZGA based on available datasets of mouse embryos or related to the ZGA-like state of mouse ESCs. Mouse ESCs were transduced with a lentiviral library containing selected sgRNAs and this was followed by scRNA-seq. After multi-omics factor analysis, they identified a set of 44 factors that promoted a ZGA-like response, including the upregulation of known ZGA associated elements such as MERVL or ERV1.

• Validation of candidate regulators of ZGA: Dppa2, Smarca5 and Patz1. The authors induced overexpression of their three candidate regulators either by individual CRISPRa again or by cDNA-eGFP overexpression followed by bulk RNA-seq. Induction of either of the three factors triggered a similar response and included genes highly expressed at the time of ZGA in mouse embryos.
• Smarca5 and Dppa2 are detected in the nuclei of 2-cell stage embryos. Smarca5 is expressed in mouse zygotes in both pronuclei, while Dppa2 is absent in the pronuclei. By the 2-cell stage, Dppa2 starts to be expressed in the nuclei co-localizing with Smarca5.

• Dppa2 is required for Smarca5-mediated regulation of ZGA. Lack of Smarca5 or Dppa2 in ESCs led to a downregulation of ZGA genes. Lack of Smarca5 could be rescued by Dppa2 In contrast, Smarca5 overexpression in Dppa2 mutant ESCs did not rescue the expression of ZGA-related genes

 

General comments and why I chose this preprint:

The use of a dead-Cas9 to perform CRISPR-activation assays is a very powerful technique and has been shown to activate gene expression at physiological levels (Chavez et al. 2015, Sanson et al. 2018). The authors use this method in a smart way by combining it with high-throughput screening. Also, they overcome the limitations of performing high-throughput screens in 2-cell embryos by successfully using embryonic stem cells. This work exemplifies how there can be major advantages of using a stem cell model system to find interesting candidate regulators for a process happening in embryos. After their initial experiments in ESCs, the authors point out clear candidates that may be studied further in vivo.

This strategy can be applied to multiple contexts to identify regulators of specific processes and complement loss of function perturbations.

 

Questions to the authors

• The authors use serum/LIF conditions to culture mouse embryonic stem cells (ESCs). Given the different possibilities described in the literature to grow ESCs and achieve a naïve state or a 2C-like state (Macfarlan et al. 2012, Ying et al. 2008), how did the authors decide which was the best option to culture their cells in order to identify regulators of zygotic genome activation? How do they think it could affect the identification of genes in other culture conditions?
• Transcriptional activation of Dppa2, Smarca5 and Patz1 lead to an upregulation of ZGA-related genes. Have the authors found if among the downregulated genes there are known or candidate genes that block ZGA?

References

Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12(4):326-8.

Eckersley-Maslin M, Alda-Catalinas C, Blotenburg M, Kreibich E, Krueger C, Reik W. (2019). Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcriptional program. Genes Dev 33(3-4):194-208.

Macfarlan TS, Gifford WD, Driscoll S, Lettieri K, Rowe HM, Bonanomi D, Firth A, Singer O, Trono D, Pfaff SL. (2012). Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487(7405):57-63.

Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9(1):5416.

Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453(7194):519-23.

Tags: crispr, dppa2, screening, smarca5, zygotic genome activation

doi: https://doi.org/10.1242/prelights.13748

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