Obox4 promotes zygotic genome activation upon loss of Dux
- Department of Molecular Biology, Keio University School of Medicine, Japan
- Bioresource Engineering Division, Bioresource Center, RIKEN, Japan
- Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan
- Human Biology Microbiome Quantum Research Center (WPI-Bio2Q), Keio University, Japan
Figures
Figure 1 with 1 supplement
OBOX4 is expressed during zygotic genome activation (ZGA).
(A) Mouse homeobox genes that are specifically expressed during ZGA. The genes were identified by means of statistically determined k-means clustering based on their expression in preimplantation …
Figure 1—figure supplement 1
Production of anti-OBOX4 monoclonal antibodies.
(A) Schematic of the production of monoclonal antibodies. Mice were immunized with the GST-OBOX4 fusion protein. After fusing B-cells with myelomas, the antibodies produced by hybridomas were …
Figure 2 with 2 supplements
Obox4 and Dux induce 2C-gene expression in mouse embryonic stem cells (mESCs).
(A) Diagram of the 2C::tdTomato reporter assay. mESCs bearing the tdTomato expression cassette under the control of the MERVL long terminal repeat (LTR) promoter showed red fluorescence upon …
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Figure 2—source data 1
Original and uncropped blot for Figure 2C.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig2-data1-v2.zip
Figure 2—figure supplement 1
k-means clustering of reimplantation gene expression and MERVL induction by ectopic Obox4 and Dux expression.
(A) Expression profiles of k-means clustered gene sets during preimplantation development based on z-scored expression of scRNA-seq and bulk RNA-seq. Genes appeared in both scRNA-seq cluster 1 and …
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Figure 2—figure supplement 1—source data 1
Original and uncropped blot for Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Ectopic expression of Obox4 and Dux activates MERVL reporter.
(A) FACS analysis of 2C::tdTomato cells co-transfected with candidate gene expression plasmids and EGFP. (B) Fluorescence microscopy of 2C::tdTomato mESCs transfected with empty plasmid, plasmid …
Figure 3 with 3 supplements
OBOX4 binds and activates 2C-specific long terminal repeat (LTR) elements.
(A) Left panel: pie chart displaying proportions of annotated genomic regions of the OBOX4 binding sites. Right panel: heatmap showing the OBOX4 binding site distribution near 2C-gene promoters. (B) …
Figure 3—figure supplement 1
CUT&RUN captures DNA binding profiles of DUX and OBOX4.
(A) Schematic diagram of the cleavage under targets and release using nuclease (CUT&RUN) workflow using mouse embryonic stem cells (mESCs) with induced expression of FLAG-tagged DUX. mESCs bearing a …
Figure 3—figure supplement 2
Knockout strategy of Obox4 and Dux clusters.
(A) Schematic diagram of producing Obox4 knockout mice using CRISPR-Cas9 (not drawn to scale). sgRNAs flanking the Obox4 cluster as well as the internal sequence of individual Obox4 were introduced …
Figure 3—figure supplement 3
Validation of Obox4 and Dux knockout mouse embryonic stem cell lines.
(A) Agarose gel image of PCR-based genotyping of the Obox4 knockout allele in founder mouse embryonic stem cell (mESC) lines. The red arrows indicate cell lines bearing biallelic deletions of the Obo…
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Figure 3—figure supplement 3—source data 1
Original and uncropped gels for Figure 3—figure supplement 3A.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig3-figsupp3-data1-v2.zip
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Figure 3—figure supplement 3—source data 2
Original and uncropped gels for Figure 3—figure supplement 3B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig3-figsupp3-data2-v2.zip
Figure 4 with 5 supplements
Concomitant loss of OBOX4 and DUX severely hinders zygotic genome activation (ZGA).
(A) Schematic representation of the somatic cell nuclear transfer (SCNT) experiment. The nuclei of knockout mouse embryonic stem cells (mESCs) were transferred into enucleated oocytes to generate …
Figure 4—figure supplement 1
Genotyping of Obox4 and Dux knockout alleles in the F1 mice.
(A) Agarose gel image of PCR-based genotyping of the Obox4 knockout allele in the F1 mice. (B) Agarose gel image of PCR-based genotyping of the Dux knockout allele in the F1 mice. (C) Agarose gel …
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Figure 4—figure supplement 1—source data 1
Original and uncropped gels of F1 mice 108–152 for Figure 4—figure supplement 1A–E.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Original and uncropped gels of F1 mice 200–204 for Figure 4—figure supplement 1A–E.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp1-data2-v2.zip
Figure 4—figure supplement 2
Genotyping of Obox4 knockout allele in the F2 mice.
(A) Agarose gel image of PCR-based genotyping of the Obox4 knockout and Obox4 cluster 5’ cutting site wild-type allele in the F2 mice produced by intercrossing Obox4Het F1 mice. (B) Bar plot showing …
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Figure 4—figure supplement 2—source data 1
Original and uncropped gels of Figure 4—figure supplement 2A.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp2-data1-v2.zip
Figure 4—figure supplement 3
Genotyping of Obox4 and Dux knockout alleles in the F2 mice.
(A) Bar and dot plot showing litter sizes of intercross used to produce Dux/Obox4 double knockout mice. Each dot represents individual litter size. Six and three litters were analyzed for DuxKO/Obox4…
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Figure 4—figure supplement 3—source data 1
Original and uncropped gels of F2 mice 187s–201s for Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp3-data1-v2.zip
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Figure 4—figure supplement 3—source data 2
Original and uncropped gels of F2 mice 202s–206s for Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp3-data2-v2.zip
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Figure 4—figure supplement 3—source data 3
Original and uncropped gels of F2 mice 207s–215s for Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp3-data3-v2.zip
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Figure 4—figure supplement 3—source data 4
Original and uncropped gels of F2 mice 216s–240s for Figure 4—figure supplement 3B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp3-data4-v2.zip
Figure 4—figure supplement 4
Genotyping of Obox4 and Dux knockout alleles in the embryos.
(A) Schematic diagram of knockout embryo production for development monitoring. DuxHet/Obox4Het female mice were mated with DuxKO/Obox4Het male mice. Embryos were collected at 0.5 days post coitum …
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Figure 4—figure supplement 4—source data 1
Original and uncropped gels for Figure 4—figure supplement 4B.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp4-data1-v2.zip
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Figure 4—figure supplement 4—source data 2
Original and uncropped gels for Figure 4—figure supplement 4E.
- https://cdn.elifesciences.org/articles/95856/elife-95856-fig4-figsupp4-data2-v2.zip
Figure 4—figure supplement 5
Obox4/Dux double knockdown embryos showed developmental defects.
(A) Transcript levels of Dux and Obox4 in antisense oligonucleotide (ASO)-injected embryos, as measured using RNA-seq. CPM, counts per million. (B) Representative images of immunofluorescence …
Figure 5
OBOX4 promotes zygotic genome activation (ZGA) in the absence of DUX.
(A) Schematic of the double knockdown rescue experiment. Male pronuclei of zygotes were injected with antisense oligonucleotide (ASO) targeting Obox4 and Dux transcripts as well as in …
Figure 6
OBOX4-mediated DUX redundancy is distinct among the OBOX family.
(A) Heatmaps of the expression of 2C-genes in preimplantation embryos, non-induced mouse embryonic stem cells (mESCs), and Dux/Obox4/Obox3/Obox5-induced mESCs. (B) Venn diagram showing overlap of …
Author response image 1
Comparison of transcriptome data in different KO and KD experiments.
a) Correlations between biological replicates of Dux/Obox4 DKO pseudo-bulk scRNA-seq (left panel) and ASO mediated Dux/Obox4 DKD RNA-seq (right panel). DKO samples bear higher inter-sample variance …
Author response image 2
Comparison of Obox3/Obox4/Obox5 induced transcriptome.
a) Venn diagram showing overlap of 2C genes induced by ectopic expression of Obox3, Obox4, and Obox5 in mESCs. b) Scatterplot showing per-gene expression changes in Obox3 versus Obox5 induced mESCs. …
Tables
Author response table 1
Expression levels of all Obox family members in mouse pluripotent stem cell (PSC) and totipotent blastomere-like cell (TBLC).
| PSC (TPM) | TBLC (TPM) | ZGA Expression | |
|---|---|---|---|
| Oboxl Obox2 Obox3 Obox4 Obox5 Obox6 Obox7 Obox8 | 0.00 0.00 0.00 0.09 0.00 0.78 0.00 0.90 | 0.00 0.00 0.26 2.09 0.11 7.24 0.00 1.02 | TRUE TRUE TRUE TRUE TRUE FALSE TRUE FALSE |
Additional files
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Supplementary file 1
List of Z-score and k-means clustering of genes based on preimplantation expression profiling data by Wu et al., 2016 (GSE66390) and Deng et al., 2014 (GSE45719).
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp1-v2.xlsx
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Supplementary file 2
Deferential expression analysis results of genes and transposable elements in Obox4 and Dux overexpressing mESCs.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp2-v2.xlsx
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Supplementary file 3
List OBOX4 and DUX bond genes.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp3-v2.xlsx
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Supplementary file 4
Total numbers of annotated loci in each transposable element family and numbers of loci bond by OBOX4.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp4-v2.xlsx
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Supplementary file 5
List of mating pairs and genotyping results of Dux/Obox4 knockout mice.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp5-v2.xlsx
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Supplementary file 6
Genotyping and development monitoring results of preimplantation embryos produced by DuxKO/Obox4Het × DuxHet/Obox4Het mating pair.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp6-v2.xlsx
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Supplementary file 7
Deferential expression analysis results of genes and transposable elements in DuxKO/Obox4Het and DuxKO/Obox4KO versus DuxKO/Obox4WT 2C embryos.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp7-v2.xlsx
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Supplementary file 8
Combined deferential expression analysis results of genes and transposable elements in Obox3/4/5 overexpressing mESCs.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp8-v2.xlsx
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Supplementary file 9
Combined deferential expression analysis results of genes and transposable elements in Dux/Obox4 knockdown and Obox family knockout 2C embryos.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp9-v2.xlsx
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Supplementary file 10
Primer sequences used in this study.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp10-v2.xlsx
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Supplementary file 11
Antisense oligonucleotides sequences used in this study.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp11-v2.xlsx
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Supplementary file 12
sgRNA sequences used in this study.
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp12-v2.xlsx
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Supplementary file 13
- https://cdn.elifesciences.org/articles/95856/elife-95856-supp13-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/95856/elife-95856-mdarchecklist1-v2.pdf
