Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.
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.
Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA) in totipotent embryos. 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 embryos. Nr5a2 binds to its motif within a subtype of SINE B1/Alu transposable 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 family receptors. Consistent with a role in ZGA, Nr5a2 is required for progression beyond the 2-cell stage. Nr5a2 promotes chromatin accessibility during ZGA and binds to entry/exit sites of nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that distinctly regulates totipotency and pluripotency during mammalian development.One-Sentence SummaryNr5a2 is an essential pioneer transcription factor that activates expression of zygotic genes in mouse embryos.
The fusion of two transcriptionally silent gametes, egg and sperm, generates a totipotent zygote that activates zygotic transcription to support further development. Although the molecular details of zygotic genome activation (ZGA) are not well understood in most species, an emerging concept is that one or more pioneer transcription factors trigger zygotic transcription. Concomitantly, extensive changes in 3D chromatin organization occur during development. In this review, we discuss recent advances in understanding when and how genome architecture emerges in early metazoan embryos, how the zygotic genome is activated, and how these events might be coordinated. We also highlight some of the unknowns that may be critical to address in the future.
The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.
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...
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