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The homeobox transcription factor DUXBL controls exit from totipotency

Nature Genetics volume 56, pages 697–709 (2024)Cite this article

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Abstract

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.

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Fig. 1: The Duxbl gene generates two protein products upon DUX expression.
Fig. 2: DUXBL counteracts 2CLC conversion.
Fig. 3: DUXBL overexpression in zygotes halts development.
Fig. 4: DUXBL is essential for mouse embryo development.
Fig. 5: DUX-mediated chromatin opening facilitates DUXBL binding to DUX-bound regions.
Fig. 6: DUXBL interacts with the TRIM33/TRIM24 complex.
Fig. 7: DUXBL and TRIM24/TRIM33 colocalize with H3K9me3 deposition.

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Data availability

The proteomic data and search results associated with this study have been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the dataset identifier PXD047807, project doi:10.25345/C5XW4867R. The sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under accession codes GSE210892 and GSE213703. Datasets obtained from publicly available sources include GSE98149, GSE95517, GSE45719, GSE85627, GSE66582 and GSE97304 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE98149, GSE95517, GSE45719, GSE85627, GSE66582 and GSE97304, respectively). Additional data and/or reagents that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code used in this manuscript to analyze Cut&Run data and expression of TEs is available on Zenodo via the following links:

Cut&Run pipeline: https://doi.org/10.5281/zenodo.10658411 (ref. 61).

Gene/TE quantification pipeline: https://doi.org/10.5281/zenodo.10658421 (ref. 62).

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Acknowledgements

We thank principal investigators from the Laboratory of Genome Integrity for helpful comments and discussion on this work. We thank K. Tachibana and W. Kobayashi for their suggestions and critical reading of the manuscript. We also thank M. Malik, D. Goldstein and the CCR Genomics Core for sequencing support; F. Livak, S. Siddiqui and the CCR Flow Cytometry Core, G. Pegoraro and the High-Throughput Imaging Facility and M. Kruhlak and the Microscopy Core for experimental support at the NCI; the Epigenomics and DNA Sequencing, Fluorescence Microscopy and Imaging Core facilities at National Institute of Environmental Health Sciences (NIEHS) and to the core facilities in Max Planck Institute for Heart and Lung Research for support with mouse transgenics (S. Krüger, S. Kreutzer and D. Heil). Research in S.R. and C.J.W. laboratory is supported by the Intramural Research Program of the National Institutes of Health (NIH). T.O. is supported by a postdoctoral fellowship of the Helen Hay Whitney Foundation. Research in J.K. Laboratory was supported by the Max Planck Society and Deutsche Forschungsgemeinschaft (DFG, German Research Foundation): DFG project 496710327 and Excellence Cluster CPI (EXC2026). D.B. is supported by the R01 grant 1R01AR081228-01.

Author information

Author notes

  1. These authors contributed equally: Maria Vega-Sendino, Felipe F. Lüttmann.

  2. These authors jointly supervised this work: Johnny Kim, Sergio Ruiz.

Authors and Affiliations

  1. Laboratory of Genome Integrity, CCR, NCI, NIH, Bethesda, MD, USA

    Maria Vega-Sendino, Teresa Olbrich, Grace I. Carey, Bechara Saykali, Catherine N. Domingo & Sergio Ruiz

  2. Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany

    Felipe F. Lüttmann, Yanpu Chen, Jiasheng Zhong, Tianlin Lu, Leah Schüler, Stefan Günther & Johnny Kim

  3. Cardio-Pulmonary Institute, Frankfurt, Germany

    Felipe F. Lüttmann, Yanpu Chen, Tianlin Lu, Mario Looso & Johnny Kim

  4. GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macau Joint Laboratory for Cell Fate Regulation and Diseases, Department of Cardiology, Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China

    Yanpu Chen

  5. Bioinformatics Core Unit (BCU), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

    Carsten Kuenne & Mette Bentsen

  6. Reproductive and Developmental Biology Laboratory, NIEHS, NIH, Research Triangle Park, NC, USA

    Paula Stein, Virginia Savy, Lenka Radonova & Carmen J. Williams

  7. Genetics Branch, CCR, NCI, NIH, Bethesda, MD, USA

    Desiree Tillo

  8. German Cancer Research Center, Heidelberg, Germany

    Jiasheng Zhong

  9. Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany

    Kee-Pyo Kim & Hans Schöler

  10. Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea

    Kee-Pyo Kim

  11. German Center for Cardiovascular Research (DZHK), Partner Site Rhein/Main, Germany

    Stefan Günther, Mario Looso & Johnny Kim

  12. Lillehei Heart Institute, Department of Pediatrics, University of Minnesota, Minneapolis, USA

    Darko Bosnakovski & Michael Kyba

  13. Laboratory of Cell Biology, CCR, NCI, NIH, Bethesda, MD, USA

    Tapan K. Maity & Lisa M. Jenkins

  14. German Center for Lung Research (DZL), Partner Site Rhein/Main, Germany

    Johnny Kim

  15. Institute of Lung Health (ILH), Justus-Liebig-University Giessen, Giessen, Germany

    Johnny Kim

  16. The Center for Cardiovascular Regeneration and Immunology at TRON—Translational Oncology at the University Medical Center of the Johannes Gutenberg-University Mainz gGmbH, Mainz, Germany

    Johnny Kim

Authors
  1. Maria Vega-Sendino
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  2. Felipe F. Lüttmann
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Contributions

M.V.-S., F.F.L., T.O., J.K. and S.R. conceived the study. T.O., M.V.-S. and F.F.L. designed, performed and analyzed most of in vitro experiments. Y.C., J.Z., T.L. and L.S. designed and performed experiments. K.-P.K. and H.S. provided vectors. F.F.L., P.S., V.S. and L.R. performed microinjection and in vivo experiments. C.N.D. provided technical support. S.G. and D.T. analyzed sequencing data. C.K. and M.B. analyzed data and performed bioinformatic analyses under the supervision of M.L. G.I.C. provided support with high-throughput microscopy imaging. B.S. analyzed live confocal microscopy data. T.K.M. and L.M.J. performed and analyzed MS experiments. D.B. and M.K. provided critical input. S.R., J.K. and C.J.W. supervised the study and wrote the manuscript with comments and help from all authors.

Corresponding authors

Correspondence to Johnny Kim or Sergio Ruiz.

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The authors declare no competing interests.

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Peer review information

Nature Genetics thanks Haruhiko Siomi, Jianlong Wang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 DUX induces two protein products from the Duxbl gene.

a) High-throughput imaging quantification of GFP mean nuclear intensity in untreated BR1-GFP reporter ESCDUX or treated with DOX for 16 h. Centerlines indicate mean values. n = 537. b) Graph plots showing quantified mean intensity levels of RFP and GFP obtained from untreated and DOX-treated LTR-RFP reporter BR2- or BR3-GFP ESCDUX. c) DNA sequences surrounding exon 2 of Duxbl showing the deletions detected in representative DUXBLKO ESCDUX clones analyzed. DNA sequence from exon 2 is highlighted in bold. The alternative ATG at the end of the second Duxbl exon is highlighted in red. d) Schematic representation of the DUXBL protein products. e) Protein sequence alignment of DUX, DUXBLLG (large DUXBL isoform) and DUXBLSM (small DUXBL isoform). Highlighted in blue are the two homeodomains (note that DUXBLSM lacks most of the first homeodomains). Highlighted in dark gray are conserved residues and in light gray are similar residues. Highlighted in green is the peptide sequence used to generate our custom DUXBL antibody. f) Western blot analysis of DUXBL performed in lysates from a pool of approximately 488 embryos at the indicated stages of development. Increasing amounts of DOX-treated WT ESCDUX, as well as DOX-treated DUXBLKO ESCDUX lysates, were included as controls. Ponceau staining is shown as a loading control. For a, b and f, two independent experiments were performed but one representative experiment is shown.

Source data

Extended Data Fig. 2 DUXBL does not function as a transcriptional activator.

a) Volcano plots showing differentially expressed genes between DUX- (left panel) or DUXBL-expressing ESC (right panel) compared to control ESC. b) Heatmap showing the top 50 differentially expressed genes between DUX- and DUXBL-expressing ESC. c) Western blot analysis of the indicated proteins performed with V5 immunoprecipitates obtained from 293T cells transfected with the corresponding expression vectors. This experiment was repeated multiple times, but one is shown. Data in a and b were generated using three replicates per condition.

Source data

Extended Data Fig. 3 Duxbl-deficient ESC showed increased 2CLC conversion.

a) Time-lapse microscopy experiment performed in LTR-RFP reporter WT and DUXBLKO ESCDUX. Two independent experiments with two ESC lines per condition were performed, but one representative is shown. Time since the addition of DOX is indicated. Scale bar, 10 μm. b) High-throughput imaging (HTI) quantification of RFP+ cells in LTR-RFP reporter WT and DUXBLKO ESC. Centerlines indicate mean values. Percentages of RFP+ cells above the threshold (dotted line) are indicated. n = 2000; p value is from one-tailed unpaired t-test. Three independent experiments were performed but one representative is shown. c) Real-time PCR analysis shown in a box and whisker plot showing the relative fold change expression of nine 2C-associated genes/repeats (Dux, Zscan4c, Zfp352, Tcstv3, Sp110, Tdpoz1, Dub1, Eif1ad8 and MERVLs) in RFP negative, and RFP positive cells sorted from LTR-RFP reporter WT and DUXBLKO ESCDUX cultures. Gapdh expression was used to normalize. Centerline indicates the median, box extends from the 25th to 75th percentiles and whiskers show min to max values. P values are shown from one-tailed unpaired t-tests. Two independent experiments were performed, but one representative is shown. d) Unidimensional PCA plot of RNA-seq data from untreated or DOX-treated WT and DUXBLKO ESCDUX (two replicates each) together with WT DUX-expressing ESC from ref. 5. DUX-expressing ESCs were sorted into GFP+ or GFP− based on the activation of the LTR-GFP reporter. e) Box and whisker plot showing normalized fold change expression of the 244 genes downregulated as shown in Fig. 2b. RNA-seq data obtained from27. Centerline indicates the median, box extends from the 25th to 75th percentiles and whisker extends from the hinge to the largest or smallest value no further than 1.5-fold from the interquartile range. Data beyond whiskers were plotted individually. For c–e, data were generated using two independent ESC lines per genotype and condition. f) Venn diagram showing the overlap between the 210 genes described in Fig. 2d and the upregulated genes in LTR+ sorted DUX-expressing ESC (data obtained from ref. 5). P value was obtained from a two-tailed Fisher’s exact test. g) Genome browser tracks from individual samples showing RNA-seq RPKM read count in untreated or DOX-treated WT and DUXBLKO ESCDUX.

Source data

Extended Data Fig. 4 DUXBL expression limits 2CLC conversion.

a) Western blot analysis of DUXBL (FLAG) performed in lysates from untreated or indole-3-acetic acid (IAA)/DOX-treated WT or ESCCTCF-AID expressing DUXBL (FLAG). Tubulin levels are shown as a loading control. b) High-throughput imaging (HTI) quantification of RFP+ cells in LTR-RFP reporter WT and DUXBLKO ESC incubated with 0.53 mM RA for 48 hours. c) HTI quantification of RFP+ cells in untreated or DOX-treated LTR-RFP reporter ESCDUXBL incubated with 0.53 mM RA for 48 hours. d) Western blot analysis using lysates from untreated or DOX-treated ESCDUXBL incubated with 2.5 mM PlaB for 24 hours or 0.53 mM RA for 48 hours. e) Box and whisker plot showing the relative fold change (log2) expression of eight 2C-associated genes (Dux, Zscan4c, Zfp352, Tcstv3, Sp110, Tdpoz1, Dub1 and Eif1ad8) in untreated or DOX-treated ESCDUXBL incubated with 2.5 mM PlaB for 24 hours or 0.53 mM RA for 48 hours. Two independent experiments were performed, but one representative is shown. P values are shown from one-tailed unpaired t-tests. Centerline indicates the median, box extends from the 25th to 75th percentiles and whiskers show min to max values. f) Western blot analysis of CTCF performed in lysates from untreated or IAA-treated ESCCTCF-AID. Tubulin levels are shown as a loading control. g) HTI quantification of RFP+ cells in untreated LTR-RFP reporter ESCCTCF-AID expressing DUXBL or IAA/DOX-treated for 48 hours. h) HTI quantification of RFP+ cells in untreated LTR-RFP reporter WT or DUXBLKO ESCCTCF-AID or IAA-treated for 24 and 48 hours. For a,d and f, two independent experiments were performed, but one representative is shown. For b,c,g and h, centerlines indicate mean values. Percentages of RFP+ cells above the threshold (dotted line) are indicated. n = 2000. P values are shown from one-tailed unpaired t-tests. In all cases, three independent experiments were performed, but only one representative is shown.

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Extended Data Fig. 5 DUXBL expression in zygotes impairs proper ZGA.

a) Representative bright-field and fluorescence images of zygotes not injected or microinjected with mRNA encoding for mCherryNLS or DUXBLV5 plus mCherryNLS. Images were taken 6 hours post microinjection. Scale bar, 50 μm. Five independent experiments were performed, but one representative is shown. b) Representative immunofluorescence analysis of the V5 tag in zygotes not injected or microinjected with mRNA encoding DUXBLV5 plus mCherryNLS. DAPI was used to visualize the nuclei. Scale bars, 50 μm. At least 10 independent embryos per condition were stained, but one representative is shown. c) Heatmap and k-means clustering (3) showing the distribution of the indicated samples described in Fig. 3a. d,e) Volcano plots highlighting the differentially expressed genes observed by RNA-seq analysis between DUXBLV5 and mCherryNLS-overexpressing embryos (red: FC ≥2 DUXBL/mCherry; blue: FC ≤ −2 DUXBLV5/mCherry; padj < 0.05). f) Volcano plot showing differentially expressed genes between DUXBL-overexpressing embryos compared to mCherry-overexpressing embryos. Genes differentially induced by DUX are highlighted in red. ≤−1 log2 fold change ≥1; padj value < 0.05. For c–f, data were generated by using a total of 4–6 pools of 5 embryos/pool per stage and condition.

Extended Data Fig. 6 Downregulation of Duxbl in zygotes compromises development.

a) Plot showing mean ± standard error of the mean (SEM) summarizing four independent experiments with a total of 25–45 microinjected zygotes per group (non-microinjected, control morpholino (MO), or a second morpholino (MO2)-injected zygotes) per experiment. The percentage of embryos reaching each embryo stage is shown. Mor: morula, Blast: blastocyst. P values are shown from one-tailed unpaired t-tests. b) Plot showing mean ± standard error of the mean (SEM) summarizing four independent experiments with a total of 25–45 microinjected zygotes per group (non-microinjected, control or Duxbl siRNAs injected GV oocytes) per experiment. The percentage of embryos reaching each embryo stage is shown. Mor: morula, Blast: blastocyst. P values are shown from one-tailed unpaired t-tests. c) Relative fold change (log2) expression of Duxbl in uninduced or DOX-induced in ESCDUX transfected 36 hours prior to induction with the corresponding siRNAs. Reactions were performed by duplicate in two different ESCDUX lines. Two independent experiments were performed, but one representative is shown. P value is shown from one-tailed unpaired t-test. d) Pie chart showing percentages of genotypes from Duxbl+/Δ crossings at P14 after F1 generation. e) Gel showing PCR results from genotyping all 8C-stage embryos obtained from Duxbl+/Δ crossings. The identity of the bands obtained by PCR was confirmed by genotyping on positive and negative controls at least two times. f) Gene set enrichment analysis (PANTHER gene sets) according to differentially expressed genes observed in Duxbl Δ/Δ embryos compared to Duxbl+/Δ/Duxbl+/+ embryos (Kobas FDR < 0.2).

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Extended Data Fig. 7 DUXBL gains access at DUX-bound sites following DUX expression.

a) Predicted binding sites for DUX and DUXBL (Jaspar database). b) Venn diagrams showing the number of DUXBL peaks overlapping between those identified with DUXBL antibodies (blue) and FLAG antibodies (yellow) in ESCDUXBL. c) Genome browser tracks corresponding to individual samples from b showing DUXBL occupancy. Input (IgG) is shown as control. For b,c, two independent experiments with two independent ESC lines were performed. d) Pie chart showing the genomic distance of DUXBL peaks from gene TSS. e) Venn diagrams showing the number of DUXBL peaks overlapping with traditional ESC enhancers (left) and ESC SEs (right). Enhancer data obtained from31,32. P values shown are from two-tailed Fisher’s exact test. f) Gene ontology (GO) terms obtained from analyzing DUXBL peaks. P values were obtained from a binomial test. g) CUT&RUN read density plot (RPGC) showing DUXBL and DUX enrichment at MERVL-int elements in DOX-treated ESCDUXBL. Data from DUX-expressing ESC were obtained from5. Input (IgG) is shown as control. Data from one representative ESC line are shown. h) Venn diagrams showing the number of DUXBL (left panel, blue), and DUX peaks (right panel, blue) overlapping with accessible regions in ESC (yellow). ATAC-seq data were obtained from33. i) Genome browser tracks from individual samples showing DUXBL and DUX occupancy as well as ATAC-seq signal in DOX-treated WT ESC expressing DUX or DUXBL. Data from DUX-expressing ESC and ATAC-seq experiments were obtained from5,33. Input (IgG) is shown as control. j) Plot showing DUXBL enrichment over DUX-bound sites in the condition of DUX/DUXBL co-expression compared to only DUXBL expression. We used a log2(FC) cutoff of >1 and p-value < 0.01 to define differentially enriched sites. Representative genes in proximity to DUX-bound sites enriched for DUXBL binding are shown. Two independent experiments using one ESC line were performed, but data from one representative experiment are shown. k) Immunofluorescence analysis of DUXBL (FLAG) in untreated or DOX-treated LTR-RFP reporter ESCDUXBL/DUX. Scale bar, 20 μm. l) Immunofluorescence analysis of DUXBL in untreated or DOX-treated ESCDUXBL. In k and l, two independent experiments were performed, but one representative is shown. DAPI was used to visualize nuclei. Scale bar, 100 μm.

Extended Data Fig. 8 DUXBL interacts with the TRIM33/TRIM24 complex.

a, b) Western blot analysis of the indicated proteins performed with total protein extracts (input) and DUXBL immunoprecipitates (10% of total IP) obtained from untreated or DOX-treated ESCDUXBL (a) or untreated or DOX-treated ESCDUX (b). c) Volcano plot showing the enrichment of proteins obtained from endogenous DUXBL immunoprecipitation followed by mass spectrometry (IP-MS) analysis in two independent untreated or DOX-treated ESCDUX. d) Western blot analyses of the indicated proteins performed with total extracts (input) and DUXBL immunoprecipitates (IP) obtained from untreated and doxycycline treated ESCDUX. We also included the flowthrough (FT) following the immunoprecipitation to show the specific depletion of DUXBL. e) Schematic representation of the different TRIM24 mutants used in our immunoprecipitation assays. f, g) Western blot analysis of the indicated proteins performed with total protein extracts (input) and FLAG pulldowns obtained from DOX-treated ESC containing stably transfected Piggy-bac constructs encoding for HA-tagged DUXBL and/or each of the different FLAG-tagged TRIM24 mutants. h) Schematic representation of the different DUXBL protein forms used in our immunoprecipitation assays. i) Western blot analysis of the indicated proteins performed with total protein extracts (input) and FLAG pulldowns obtained from WT and DOX-treated DUXBLKO ESC containing stably transfected Piggy-bac constructs encoding for each of the different FLAG-tagged DUXBL protein forms. TRIM24 endogenous levels are shown. Two independent experiments were performed in a,b,d,f,g and i, but one representative is shown.

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Extended Data Fig. 9 DUXBL/TRIM33/TRIM24 colocalize at DUX-induced nuclear foci.

a) Immunofluorescence analysis of HA (DUX) and endogenous TRIM24 in untreated or DOX-treated ESCDUX-2XHA. Scale bars, 20 μm. b) Immunofluorescence analysis of endogenous TRIM24 in endogenous 2CLC observed in LTR-RFP reporter WT ESC. Scale bars, 20 μm. For a,b, DAPI was used to visualize nuclei and two independent experiments were performed but one representative is shown. c) High-throughput imaging (HTI) quantification of the number, total intensity and area of TRIM24 foci (upper, middle and lower panels, respectively) per cell in untreated or DOX-treated LTR-RFP reporter WT or DUXBLKO ESCDUX. d) Western blot analysis of TRIM24 performed in WT and TRIM24KO ESCDUX. e) Western blot analysis of TRIM33 performed in WT and TRIM33KO ESCDUX lysates. f) HTI quantification of the number, total intensity and area of DUXBL foci (upper, middle and lower panels, respectively) per cell in untreated or DOX-treated LTR-RFP reporter WT or TRIM33KO ESCDUX. For c and f, percentages of cells above the threshold (dotted line) are indicated. Centerlines indicate mean values. n = 2000; p values are shown from one-tailed unpaired t-tests. Two independent experiments using at least two WT, DUXBLKO ESCDUX or TRIM33KO ESCDUX clones were performed but one representative is shown. g) Graph showing 2CLC residency time evaluated by using the LTR-RFP reporter in DOX-treated WT and TRIM24KO ESCDUX. P value is shown from a two-tailed unpaired t-test. Plot was generated by using data from two independent ESC lines per genotype. h) Western blot analysis of TRIM24 performed in WT and TRIM24FKBP ESCDUX. i) Western blot analysis of TRIM24 and DUXBL performed in untreated and DOX/dTAG-treated for 16 hours TRIM24FKBP and DUXBLKO; TRIM24FKBP ESCDUX. For d,e,h,i, tubulin levels were used as loading control and two independent experiments were performed but one representative is shown. j) Graph showing 2CLC residency time evaluated by using the LTR-RFP reporter in dTAG-treated WT and DUXBLKO; TRIM24FKBP ESCDUX. P values are shown from two-tailed unpaired t-tests. Plot was generated by using data from two independent ESC lines per genotype.

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Extended Data Fig. 10 DUXBL/TRIM24/TRIM33 complex co-localizes with H3K9me3 at DUX-induced nuclear foci.

a) Immunofluorescence analysis of DUXBL in untreated or DOX-treated ESCDUX-FKBP. Treatments include DOX for 24 hours or DOX for 16 hours plus 8 hours with dTAG compounds. DAPI was used to visualize nuclei. Scale bar, 20 μm. Three independent experiments were performed but one representative is shown. b) High-throughput imaging (HTI) quantification of the number, total intensity and area of DUXBL foci (upper, middle and lower panels, respectively) per cell in ESCDUX-FKBP treated as in (a). c) HTI quantification of the number, total intensity and area of HA (DUX) foci (upper, middle and lower panels, respectively) per cell in untreated or DOX-treated ESCDUX-FKBP treated as in (a). d) HTI quantification of the number, total intensity and total area of TRIM24 foci (upper, middle and lower panels, respectively) per cell in ESCDUX-FKBP treated as in (a). For b,c, and d, centerlines indicate mean values; n = 2000. P values are shown from one-tailed unpaired t-tests. Percentages of cells above the threshold (dotted line) are indicated. At least, two independent experiments were performed but one representative experiment is shown. e,f) Plot showing the ratio for HDAC (e) and HP1 (f) between the fluorescence intensity found at TRIM24 foci and the mean nuclear intensity per cell detected in DOX-treated LTR-RFP reporter ESCDUX. Centerlines indicate mean values. n = 2000. Two independent experiments using two ESCDUX clones were performed but one representative is shown. g) HTI quantification of the number, total intensity and area of H3K9me3 foci (left, middle and right panels, respectively) per cell in untreated or DOX-treated LTR-RFP reporter ESCDUX. Centerlines indicate mean values. n = 2000; p values are shown from one-tailed unpaired t-tests. Three independent experiments using at least two ESCDUX clones were performed, but one representative is shown.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Methods and supporting data for Supplementary Fig. 4c.

Reporting Summary

Peer Review File

Supplementary Video 1

Live-cell imaging of DOX-treated LTR-RFP and BR2-EGFP reporter ESCDUX.

Supplementary Video 2

Live-cell imaging of DOX-treated LTR-RFP and BR3-EGFP reporter ESCDUX.

Supplementary Video 3

Live-cell imaging of LTR-RFP reporter DOX-induced ESCDUX endogenously tagged with a miRFP702 fluorescent protein at the Duxbl locus.

Supplementary Code

Custom code used to process Cut&Run data and analyze the expression of TEs.

Supplementary Tables

Supplementary Tables 1–14.

Supplementary Data

Statistical supporting for Supplementary Figs. 1 and 4.

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Vega-Sendino, M., Lüttmann, F.F., Olbrich, T. et al. The homeobox transcription factor DUXBL controls exit from totipotency. Nat Genet 56, 697–709 (2024). https://doi.org/10.1038/s41588-024-01692-z

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