Nucleolar-based Dux repression is essential for embryonic two-cell stage exit

In this study, Xie et al. investigated the mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation in mammalian embryos, and report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. Their findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.


Figure S1
A) Representative flow cytometry plots of 2C-GFP/CD4 ESCs before and after MACS purification B) Flow cytometry plots showing increased purity of 2C-GFP+ cells after two rounds of MACS purification. C) qRT-PCR analysis of 2C-specific transcripts after MACS purification. Note that 2C gene induction with MACS is lower than CD4 column-free, bead-based purification (StemCell Inc) ( Figure 1D). CD4 columnfree isolation was therefore used in all subsequent experiments throughout this study. Data are mean +/s.e.m, 3 independent experiments, P values, unpaired Student's t-test.  A) Representative confocal microscopy images and quantification of nascent RNA levels (EU incorporation) following 2h RNA Pol I inhibition. Data are mean +/-s.e.m, P values, one-way ANOVA with Dunnett multiple comparisons correction. Scale bars, 25 µm. B) Representative confocal microscopy images of ESC nucleolar proteins and morphology 8h following the indicated Pol I/II inhibitors. Nucleolar caps are indicated with arrowheads. Scale bars, 20 µm. C) Western blots of Ncl/Fbl protein after 8h iPol I treatment, representative of 3 experiments. D) Quantification of relative amounts of Ncl/Fbl protein relative to Gapdh/Tubulin loading controls, data are mean +/-s.e.m from 3 experiments. E) Western blot of MERVL gag protein induction upon overnight iPol I treatment. F) Z-scores for 2C-GFP cell induction following nucleolar protein knockdown, at d3 following transfection. G) qPCR expression data for the indicated genes 72h after siRNA-mediated knockdowns. Data are mean +/s.e.m, n =3 biological replicates, representative of 2 independent experiments. P values, 2-way ANOVA with Dunnett's multiple comparisons test.

Figure S4
A  there is some evidence of partial reduction in inhibition by CX-5461 (CX) by 8h; this may explain a lower induction of Dux compared to BMH-21 (BMH) at this timepoint ( Figure 4A) and could be due to reduced stability or feedback mechanisms. Conversely, 2C-specific gene upregulation is lower at 16-24h BMH-21 ( Figure 3D), which may be due to apoptosis caused by abnormally high Dux expression. C) Normalised log2 RNA-seq expression data showing upregulation of TEs belonging to the MERVL family at 8h following iPol I. CPM, counts per million. D) Relative expression of Dux and 2C-like genes in wt E14 ESCs compared to E14 Dux-/-ESCs (Grow et al., 2021) after 16-24h iPol I. All data are shown relative to E14 wt control and are mean +/-s.e.m, n=3 biological replicates, representative of 2 experiments. E) Proportion of embryos showing absent, faint, or bright Nucleolin (Ncl) staining before and after 8h iPol I, from 2 experiments. F) Ncl immunofluorescence in mid-2C embryos fixed immediately or cultured for 24h with the indicated inhibitors. N=number of embryos with the representative staining, from one experiment, scale, 20µm. G) Developmental progression rates past 2-cell before and 8h after the indicated treatments. P values, Fisher's exact test, >50 embryos from 4 experiments per condition.  Browser screenshot of the indicated region of chr10 containing the Dux locus (highlighted) and neighbouring genes with overlayed RNA-seq tracks, confirming that Dux is situated in a NAD as defined in (Bizhanova et al., 2020). Dux is considered within a "neither" NAD, because its NAD does not overlap a cLAD or ciLAD -meaning in some cell types it is also lamina associated. This is consistent with some Dux loci positioning at the lamina in ESCs (Fig 5).

Figure S8 Xie et al
Gcc2

RNA-seq
NAD key

Nascent RNA foci quantification
For identification of nascent RNA, integrated intensity measurements were initially taken for all identified RNA probes, in Imaris. After calculation of the population mean integrated intensity, multiple intensity thresholds were calculated in increments of population standard deviation above the mean. RNA probe intensities that fell within calculated thresholds were visually highlighted in the raw image data for further validation and classification. For scoring of the location of putative nascent foci, these were identified as the ≤ 2 brightest/largest spots per nucleus in cells with multiple foci. In cells with ≤ 2 foci present per cell, for example after 4h iPol I (where very few cells have >2 foci), these loci were treated as "putative nascent".

Embryo DNA-FISH
The genomic region of Dux locus was detected using fosmid probes (WI1-1484C16; Genomic Coordinates: chr10:57682337-57718286) from BACPAC Resources Center (https://bacpacresources.org/; California, USA). Probes were labelled with tetramethyl-rhodamine-5-dUTP (Roche) by nick translation (Roche), and purified from unincorporated nucleotides using MicroBioSpin P-30 chromatography columns (Bio-Rad). Different stages of embryos were attached on poly-l lysine coated coverslips first, fixed with 4%PFA + 0.1% Triton in PBS (30 min), permeabilized with 0.5% triton in PBS (30min), and then processed for FISH as for ESCs. Embryo Immuno-DNA-FISH was performed directly on fresh fixed embryos or on embryos previously immunolabelled with antibody against B23 to mark the nucleolus as above. Hybridization was carried out as described, and hybridizaiton mixtures contained 1 μg mouse Cot1 DNA, 10 μg salmon sperm DNA and the appropriate 4 ul tetramethyl-rhodamine-5-dUTP nick-translated Dux fosmid probe, respectively. Before hybridization, probes were precipitated and resuspended in 6 μl of hybridisation buffer, then were denatured (10 min) at 70°C and re-annealed (30 min) at 37°C. The signal of rhodaminelabelled Dux was amplified with rabbit anti-rhodamine antibodies (2h; 1:250; Invitrogen) and Alexa555 donkey antibodies raised against rabbit IgG (1h; 1:500; Invitrogen). Nuclei were stained with DAPI in in PBS, washed in PBS, before coverslips mounted with VectaShield, immediately before imaging, which was carried out on Olympus spinning disk confocal system, taking Z stacks every 250nm. Individual images were prepared for presentation ( Figure 5A) using Max Projections (FIJI) of selected slices to show the location of Dux DNA loci.