introduction
Embryo implantation is an extremely critical biological event in early embryonic development. During this period, the embryo is implanted into the maternal uterus and the mother-fetal communication interface is established, which is the basis for subsequent development. However, this process is fraught with risks, and fertility problems due to implantation failure often occur clinically. Due to the transient and dynamic nature of the process, embryo implantation research remains a "black box".
miRNAs are a conserved post-transcriptional regulatory mechanism in eukaryotes and play an important role in the implantation process. DGCR8 is the core protein of the miRNA synthesis pathway, and together with DROSHA, it forms a Microprocessor complex, which cleaves the stem-loop structure on the pri-miRNA to form pre-miRNA, which is further processed into mature miRNA. [1] After DCGR8 knockout, the embryo could not develop to the post-implantation stage, suggesting that DGCR8 played an important role in the implantation process, but this lethal phenotype could not be fully explained by miRNA defects. 【2, 3】
In contrast to the unclear implantation process, preimplantation embryos and postimplantation embryos have been extensively studied, mainly due to the establishment of in vitro stem cell models. Stem cells isolated from pre-implantation embryos are called naïve mESCs (mouse embryonic stem cells) and stem cells isolated from post-implantation embryos are called Formative/Primed mESCs, and these cells have been widely used in the study of various developmental problems. [4, 5] Peng Du's group captured an intermediate pluripotent stem cell called Poised mESCs in 2018 with naive and formative/primary pluripotency. [6, 7] This state represents an embryo in implantation and is regulated by a specific set of miRNAs.
2024年7月1日，北京大学杜鹏课题组在Molecular cell杂志在线发表了题为：A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation的研究论文，发现由DGCR8/FLII/JUN介导的瞬时转录激活事件调控着床期胚胎的形态转变。
In this study, the researchers used stem cell models such as naïve-poised-formative/primed, combined with the laboratory's previous research on miRNA regulation, to explain the molecular driving forces that drive the formation of embryo shape at implantation stage, and the main contents are summarized as follows:
1. DGCR8, as the core protein of the miRNA synthesis pathway, can play a non-canonical function that does not depend on miRNA transcriptional repression. In naïve mESCs (representing pre-bed embryos), DGCR8 can bind to a short stem-loop structure located on mRNA to recruit and isolate the transcriptional activator FLII, inhibiting transcriptional progression. Transient activation of the ERK pathway in Poised mESCs (representing embryos in the bed) allows FLII to be phosphorylated and dissociated from DGCR8, which in turn binds to the transcription factor JUN and activates transcription of cell migration genes. After implantation, the ERK pathway is rapidly inactivated (Formative mESCs), and DGCR8 rebinds and inhibits FLII. Therefore, DGCR8/FLII/JUN specifically mediated a transient transcriptional activation event at the implantation stage under the mediation of the ERK pathway (Fig. 1).
2. This transcriptional activation event controls the morphological transition from disordered to polarized state during embryonic implantation. and the event was conserved in humans and mice (Figure 1).
图1: 在着床前(naive)-着床中(Poised)-着床后(Formative)发育过程中，DGCR8/FLII/JUN在ERK通路调控下介导瞬时转录激活事件的发生，进而调控着床期胚胎形态转变。 (Credit: Molecular cell)
The specific conclusions of this study are as follows:
I: In naïve mESCs, DGCR8 recruits and inhibits the transcriptional activator FLII by binding to the stem-loop structure on the mRNA.
Based on the previous data/Co-IP/Pull down of DGCR8 interaction protein in naïve mouse embryonic stem cells, the authors found that DGCR8 has a direct protein interaction with the transcriptional activator FLII. However, FLII is not involved in the miRNA synthesis pathway. By comparing the transcriptomes of multiple cell lines, the authors found that the traditional miRNA regulatory genes were upregulated due to the overall loss of miRNA after knocking out Dgcr8 and Dicer, two genes essential for miRNA synthesis. Another large group of genes was only up-regulated after Dgcr8 knockout and had no phenotype after Dicer knockout, which proves that these genes are not regulated by miRNAs. Flii (DF-dKO mESCs) were knocked out on the basis of Dgcr8 knockout, and the upregulation of this part of the gene was eliminated. The results showed that DGCR8 and FLII jointly controlled a group of genes that were not regulated by miRNA at the transcriptional level, which were mainly involved in cell migration and other activities, and were called non-miRNA-targeting genes. DGCR8 and FLII play opposite roles in regulating these genes, with DGCR8 inhibiting and FLII activating the expression of these genes.
To further investigate how DGCR8 inhibits transcription of these genes, the authors identified the RNA-binding target of DGCR8/FLII in naïve mouse embryonic stem cells by eCLIP assay, and also incorporated S4U into the eCLIP assay to simultaneously identify nascent RNA (nascent RNA). The authors found that DGCR8 and FLII bind a large number of nascent mRNAs of target genes at the same time, and the DGCR8 binding region tended to form a stem-loop structure. The stem-loop structure is significantly shorter than that on pri-miRNA and cannot be cleaved by the complex formed by DGCR8 and DROSHA. By knocking out the stem-loop structure located on the mRNA of the target gene bound by DGCR8, the transcriptional repression of the target gene was resolved. This proves that DGCR8 does inhibit transcription by binding to the stem-loop structure on mRNA.
In summary, this indicates that DGCR8 is responsible for the cleavage of pri-miRNA by binding to DROSHA on the one hand, and on the other hand, it binds to the stem-loop structure of the target gene mRNA to recruit and inhibit the transcriptional activator FLII, thereby inhibiting the transcription.
2. FLII promotes the activation of a group of cell migration-related genes and the establishment of Poised pluripotency during the naïve-poised-formative pluripotency transition.
In the naïve-poised-formative (pre-implantation→ implantation→ post-implantation) transition system, the authors found that cells lacking FLII gradually died during the process compared to normal cells, suggesting that FLII is necessary for this process. Transcriptome analysis showed that the deletion of FLII did not affect the withdrawal of naïve pluripotency, but specifically affected the activation of specific cell migration-related genes during the Poised period. Further, cells are unable to continue to develop normally to formative pluripotency. In summary, although FLII had no transcriptional activating activity in the naïve period, it regained transcriptional activating activity during the Poised period and activated the expression of a group of cell migration-related genes, which promoted the establishment of Poised pluripotency. This process is also necessary for the subsequent transition to Formative pluripotency.
III: FLII dissociates from DGCR8 in Poised mESCs (representing bed-stage embryos) and binds to the transcription factor JUN, activating Poised-specific cell migration genes.
Specifically, FLII dissociates its interaction with DGCR8 during the Poised period, and at the same time, it no longer binds to the mRNA of the target gene. This indicates that the dissociation of FLII from DGCR8 is a key condition for FLII to have transcriptional activating activity. Through a series of biochemical experiments, the authors demonstrated that during the naïve-poised transition, the ERK pathway is transiently activated and phosphorylates FLII, so that FLII no longer binds to DGCR8. At the same time, phosphorylated FLII interacts with the transcription factor JUN. By inhibiting the MEK/ERK pathway, the dissociation of DGCR8/FLII is prevented, while the formation of FLII/JUN is inhibited.
In order to further explore whether JUN and FLII promote the establishment of Poised pluripotency, the authors constructed Jun knockout and overexpression cell lines to explore the role of JUN in the transition of pluripotency. The results showed that, similar to Flii, Jun's knockout also affected the activation of cell migration genes, resulting in the inability to establish Poised pluripotency, which further affected the establishment of formative pluripotency. Conversely, Jun overexpression significantly enhanced the expression of cell migration genes and prolonged their expression time, ultimately leading to a longer duration of Poised pluripotency and preventing further development to formative pluripotency.
IV: FLII promotes the binding of JUN to DNA, which in turn activates cell migration genes and establishes Poised pluripotency.
The authors further explored the role of FLII in the expression of JUN activated genes, and performed CUT&Tag experiments in a variety of cell lines, and found that JUN was widely bound to the promoter/enhancer of cell migration genes activated during the Poised period, and after the deletion of Flii, JUN almost completely lost its DNA-binding ability, and in addition, FLII overexpression could significantly enhance the binding ability of JUN. These results indicate that FLII achieves transcriptional activation by promoting the binding of JUN to DNA.
Five: Poised pluripotent stem cells represent the epidermal cells of mouse and human implantation embryos, which are undergoing morphological transitions from disordered to polarized.
By comparing the transcriptome with in vivo embryos, the authors found that the transcriptome of Poised ESCs was most similar to that of implantation embryos in mice and humans (E4.75 days for mice and E8 days for humans). During this period, the epidermal cells of the embryo gradually changed from a disordered state to a rosette-like polarized structure. In addition, the expression of JUN and other resting-state pluripotency marker genes can be observed in implantation mouse embryos cultured in vitro, which further proves the existing conclusion. By simulating the disorder-to-polarized morphological transition system in vitro, the authors found that inhibition of this transcriptional activation event would lead to the failure of the morphological transition from disordered to polarized structures. Therefore, the transcriptional activation events identified in this paper correspond to implantation embryos, while the activation genes are dominated by cell migration-related genes, which correspond to the morphological transformation of cells during this period.
In summary, this study found that DGCR8 has a transcriptional repression function independent of miRNA, and together with FLII/JUN, under the control of the ERK pathway, it controls transient transcriptional activation events in the implantation phase, which in turn controls morphogenesis in this period.
bibliography
1. DeVeale, B., Swindlehurst-Chan, J., and Blelloch, R. (2021). The roles of microRNAs in mouse development. Nat Rev Genet 22, 307-323. 10.1038/s41576-020-00309-5.2. Greve, T.S., Judson, R.L., and Blelloch, R. (2013). microRNA control of mouse and human pluripotent stem cell behavior. Annu Rev Cell Dev Biol 29, 213-239. 10.1146/annurev-cellbio-101512-122343.3. Wu, Q., Song, R., Ortogero, N., Zheng, H., Evanoff, R., Small, C.L., Griswold, M.D., Namekawa, S.H., Royo, H., Turner, J.M., and Yan, W. (2012). The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis. J Biol Chem 287, 25173-25190. 10.1074/jbc. M112.362053.4. Kinoshita, M., Barber, M., Mansfield, W., Cui, Y., Spindlow, D., Stirparo, G.G., Dietmann, S., Nichols, J., and Smith, A. (2021). Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency. Cell Stem Cell 28, 2180. 10.1016/j.stem.2021.11.002.5. Wang, X., Xiang, Y., Yu, Y., Wang, R., Zhang, Y., Xu, Q., Sun, H., Zhao, Z.A., Jiang, X., Wang, X., et al. (2021). Formative pluripotent stem cells show features of epiblast cells poised for gastrulation. Cell Res 31, 526-541. 10.1038/s41422-021-00477-x.6. Du, P., Pirouz, M., Choi, J., Huebner, A.J., Clement, K., Meissner, A., Hochedlinger, K., and Gregory, R.I. (2018). An Intermediate Pluripotent State Controlled by MicroRNAs Is Required for the Naive-to-Primed Stem Cell Transition. Cell Stem Cell 22, 851-864 e855. 10.1016/j.stem.2018.04.021.7. Du, P., Wang, L., Sliz, P., and Gregory, R.I. (2015). A Biogenesis Step Upstream of Microprocessor Controls miR-17∼92 Expression. Cell 162, 885-899. 10.1016/j.cell.2015.07.008.https://doi.org/10.1016/j.molcel.2024.06.005
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