Periodic Reporting for period 5 - TotipotentZygotChrom (Mechanisms of chromatin organization and reprogramming in totipotent mammalian zygotes)
Período documentado: 2024-11-01 hasta 2026-01-31
Our project addressed the question of how life starts, which is of fundamental importance for continuation of a species and human reproductive health. Fertilization of egg by sperm generates a one-cell embryo that has the potential to generate an entire organism. Initially, the one-cell embryo is not competent to express its genome. Instead, this large cell is stockpiled with maternal RNA and protein that control diverse biological processes. A key transition is the handover of control from the egg genome to the embryo genome. This genome “awakening” known as zygotic genome activation (ZGA) is essential for life to proceed and is controlled by largely unidentified maternal factors in mammals. We focused on identifying novel regulators of ZGA and studied their mechanisms in vivo and in vitro. Specifically, we discovered a transcription factor called NR5A2 that functions as the “spark of life” because it activates expression of the majority of genes that are expressed in the early mouse embryo. We determined a cryo-electron microscopy structure of NR5A2 bound to a nucleosome, revealing the mechanism by which it unwraps DNA from its packaging and facilitates transcription. We also discovered that the three-dimensional folding of the genome is influenced by molecular machines that are important for DNA replication. Together, our studies identified key regulators of genome awakening and folding, two processes that are fundamental to life. The manipulation of reprogramming factors like NR5A2 in other cell types has the potential to revolutionize regenerative medicine.
The key aim was to identify novel reprogramming factors that activate the embryonic genome. We hypothesized that motifs for these transcription factors are enriched in the regulatory regions of ZGA genes. Bioinformatics motif searches identified an enrichment of SINE B1 retrotransposable elements upstream of ZGA genes. To study transcription factors such as NR5A2 that potentially bind to SINE B1, we optimized low-input genomic profiling approaches (Methods in Mol Biol 2025) and determined that NR5A2 binds to SINE B1. Using knockdown, Trim-Away and chemical inhibition, we demonstrated that NR5A2 perturbation reduced ZGA, suggesting that NR5A2 and potentially other orphan nuclear receptors are regulators of this process (Science 2022; Nat Rev Mol Cell Biol 2024). To understand NR5A2’s mechanism, we determined a cryo-EM structure of NR5A2 bound to the nucleosome, the DNA packaging unit. NR5A2 promotes partial unwrapping of nucleosomal DNA, which can contribute to generating accessible chromatin and facilitating transcription (Nat Struct Mol Biol 2024). How NR5A2 regulates distinct transcriptional programs during development remained a mystery. To address this, we profiled NR5A2 binding at multiple stages of development and found that its preferential binding to SINE B1 decreases with each embryonic cell division. NR5A2 regulates gene expression of lineage determining genes, whose products in turn co-regulate transcriptional networks during the totipotency-to-pluripotency transition (Development 2026).
Another aim was to test how chromatin-bound complexes affect genome folding. DNA is folded into loops by a process of loop extrusion that is mediated by the cohesin complex in interphase cells. Our previous work showed that cohesin is required for loops in early embryos. The progressive growth of loops is stopped when the loop extrusion machinery encounters a chromatin-bound protein called CTCF. This was the only known barrier to loop extrusion in vertebrates. Our work revealed that the replicative helicase MCM complex forms randomly placed extrusion barriers that hinder loop extrusion and affect gene expression (Nature 2022; Curr Opin Genet Dev 2024). These findings opened up the possibility to investigate the molecular properties that confer barrier function to proteins and understand more comprehensively how genome folding occurs in the complex chromatin environment.
Another aim was to test how chromatin-bound complexes affect genome folding. DNA is folded into loops by a process of loop extrusion that is mediated by the cohesin complex in interphase cells. Our previous work showed that cohesin is required for loops in early embryos. The progressive growth of loops is stopped when the loop extrusion machinery encounters a chromatin-bound protein called CTCF. This was the only known barrier to loop extrusion in vertebrates. Our work revealed that the replicative helicase MCM complex forms randomly placed extrusion barriers that hinder loop extrusion and affect gene expression (Nature 2022; Curr Opin Genet Dev 2024). These findings opened up the possibility to investigate the molecular properties that confer barrier function to proteins and understand more comprehensively how genome folding occurs in the complex chromatin environment.
The work resulted in two discoveries that each moved forward the respective fields. Our work showed for the first time that SINE B1-binding transcription factors regulate ZGA. Chemical inhibition of NR5A2 and potentially other orphan nuclear receptors resulted in a 2-cell arrest or delay and a strong downregulation of ZGA. The identification of NR5A2 as a reprogramming factor with pioneer activity therefore provided an important step towards understanding mammalian ZGA. Based on this, we proposed a “Rise and SINE” model for the mechanism of ZGA in which the pioneer factor activity of maternal NR5A2 opens chromatin and allows other transcription factors to bind to their motifs in SINE B1 (Nat Rev Mol Cell Biol 2024). In addition, our work also showed that other chromatin-bound complexes like the MCM replicative helicase can have barrier functions to loop extrusion, suggesting that the loop extrusion machinery encounters many different types of obstacles on chromatin.
From a technical point of view, we were able to optimize ultra-low input CUT&Tag to generate the first chromatin binding profiles of transcription factors in 2-cell embryos. To complement ATAC-seq data based on several embryos, we also developed an imaging-based approach to study accessible chromatin in individual nuclei that we termed Chromatin Accessibility Revealed by Microscopy (ChARM). We established a single-molecule RNA FISH assay to quantify nascent ZGA transcripts (ZGA FISH) and utilized this readout to establish a screen for novel regulators of ZGA in mouse embryos.
From a technical point of view, we were able to optimize ultra-low input CUT&Tag to generate the first chromatin binding profiles of transcription factors in 2-cell embryos. To complement ATAC-seq data based on several embryos, we also developed an imaging-based approach to study accessible chromatin in individual nuclei that we termed Chromatin Accessibility Revealed by Microscopy (ChARM). We established a single-molecule RNA FISH assay to quantify nascent ZGA transcripts (ZGA FISH) and utilized this readout to establish a screen for novel regulators of ZGA in mouse embryos.