Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. Based on de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse 2-cell embryos. Nr5a2 is required for progression beyond the 2-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis -regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

Oocyte cytoplasm is able to reprogram somatic nuclei to totipotency, suggesting that maternally provided factors reprogram and trigger zygotic genome activation (ZGA) in the embryo. The transcription factors that initiate ZGA are poorly conserved between species and largely unknown in mammals. Pioneer factors bind nucleosomal DNA and are recruited to closed chromatin in vivo, eliciting chromatin opening and ZGA in fruit flies and zebrafish, although it is unknown whether these act in mammals as well.This study aimed to search for orphan nuclear receptors that may function as pioneer factors for ZGA in mice. Transcription profiles of 2 mouse strains during the oocyte-to-embryo transition were studied. A total of 2508 extended ZGA genes were upregulated in 2-cell embryos of the 2 strains, with 985 genes common to both (core ZGA genes). A consensus sequence was identified that was enriched within 8 kb upstream of ~70% of core and extended ZGA gene and contained a sequence correlating to higher chromatin accessibility and histone acetylation during major ZGA. The orphan nuclear receptor Nr5a2 was contained in the CA version of this motif.The Nr5a2 sequence was maternally deposited before ZGA as transcripts in metaphase II eggs and zygotes, and was also strongly expressed in 2-cell embryos. To test its function, Nr5a2 was inhibited by a chemical compound (SR1848) and embryos failed to form blastocysts and fragmented or died at 108 hours postfertilization. This suggested the function of Nr5a2 may be to maintain naive pluripotency in mouse embryonic stem and may be required for pluripotency establishment in vivo. Administration of SR1848 on Nr5a2 after the 2-cell stage resulted in a less severe phenotype. Using single-molecule fluorescence in situ hybridization (ZGA-FISH), it was observed that treatment of zygotes with SR1848 resulted in a dose-dependent reduction of nascent ZGA transcripts, suggesting that Nr5a2 regulates ZGA. Single-embryo RNA sequencing (RNA-seq) of 2-cell embryos resulted in a strong decrease in Nr5a2 abundance in embryos treated with SR1848. In addition, Nr5a2 inhibition by SR1848 resulted in downregulation of several orphan nuclear receptors including its own gene, suggesting that Nr5a2 is required for their expression during ZGA.To establish whether Nr5a2 is required for chromatic accessibility in 2-cell embryos, a microscopy approach was used to quantify open chromatin in single cells. Knockdown and inhibition with siRNA and SR1848, respectively, of Nr5a2 revealed chromatin accessibility was reduced following knockdown compared with controls. Using SeEN-seq (selected engagement on nucleosome sequencing), it was determined that Nr5a2 bound at the entry-exit sites on the nucleosome, very similarly to how the pioneer factors Oct4-Sox2 and GATA3 bind to nucleosomal DNA.Taken together, the results of this study suggest that the orphan nuclear receptor Nr5a2 activates up to 72% of major ZGA genes in mouse embryos and exhibits many properties consistent with pioneer factor activity in vivo and i...