Maternal SETDB1 is essential for development beyond the 8-cell stage

(A) Quantification of Setdb1mat-/+ (KO) and Setdb1fl/+ (WT) embryo stages from the following number of total recovered embryos: KO (n=638), WT (n=484) at 1.5 dpc, and KO (n=310) and WT (n=80) at 2.5 dpc. (B) Principal component analysis of single-embryo total RNA-seq data from 2cWT (n=6), 2cKO (n=15), 8cWT (n=8), and 8cKO (n=8) embryos. (C) Schematic of four pair-wise comparisons defining requirements for normalcy and development. (D) Volcano plots highlighting DEGs using |log₂FC| > 1 and adjusted P < 0.05. (E–F) Four-way DEG comparisons visualized by Venn diagrams: (E) downregulated; (F) upregulated. (G–J) Heatmaps of DEGs from Venn compartments, showing stage- and genotype-specific patterns.

Maternal SETDB1 extinguishes 2-cell transient gene expression

(A) Bubble plot showing overrepresentation analysis of DBTMEE [34]-defined transcript sets among differentially expressed genes (DEGs) identified in the four pairwise comparisons. (B) IGV browser snapshots of representative transcripts from the DBTMEE-defined minor ZGA to MGA, 2-cell transient, and 4-cell transient gene sets across five biological replicates. Venn diagram compartments are indicated above each track. Unit in brackets represent normalized counts per million (CPM). Bigwig tracks are shown in the transcriptional direction matching the depicted gene. (C) Boxplots of selected DEGs *(|log₂FC| > 1, adjusted P < 0.05) from each Venn compartment, based on data from 2cWT (n=6), 2cKO (n=15), 8cWT (n=8), and 8cKO (n=8) embryos.

Maternal SETDB1 regulates MERVL-driven chimeric transcripts

(A) Boxplots showing normalized counts of multimapped MERVL-int and MT2_Mm elements. (B) Heatmap from Setdb1 KO embryos at MERVL chimeric transcripts classified by Macfarlan [9]. (C–D) Volcano plots marking Macfarlan-defined chimeric transcripts in 2cWT vs. 2cKO and 8cWT vs. 8cKO pairwise comparisons. (E) IGV browser images of MT2B1 LTR-driven MERVL-chimeric transcripts. (F) Venn diagram showing differentially expressed (P < 0.05) known and novel TE-driven chimeric transcripts.

Maternal SETDB1 suppresses MT2 LTR-regulated genes

(A–B) Heatmaps of H3K9me3 deposition [26] at MT2-controlled [23] DEGs (A) and their MT2 elements (B) across early embryonic stages. (C–E) IGV browser views of a representative MERVL and MT2-regulated loci. (F) Heatmap of maternal Setdb1 KO embryos at DEGs classified by MT2i data. (G–H) Volcano plots highlighting MT2-regulated genes in 2cWT vs. 2cKO and 8cWT vs. 8cKO pairwise comparisons.

SETDB1 regulates MT2-activated genes across time

(A) Heatmaps comparing MT2i-responsive [23] early two cell (E2c) DEGs with the Setdb1 KO transcriptomes. (B, C) Volcano plots highlighting MT2-regulated E2c DEGs in 2cWT vs. 2cKO and 8cWT vs. 8cKO pairwise comparisons. (D) Heatmaps of MT2i late two cell (L2c) DEGs. (E) Volcano plot highlighting MT2-regulated L2c DEGs.

SETDB1 represses DUXBL-responsive transcripts

(A) Heatmap of top 50 DEGs identified in Duxbl KO embryos [25] analyzed in the Setdb1 KO RNA-seq dataset. (B) Volcano plots marking up-/downregulated DUXBL targets in 2cWT vs. 2cKO and 8cWT vs. 8cKO pairwise comparisons. (D–E) IGV browser examples of DUXBL-responsive DEGs with aligned H3K9me3 ChIP-seq data.

SETDB1 collaborates with DUXBL to repress totipotency programs

(A) Bubble plot of overrepresentation analysis showing enrichment of previously identified MERVL-chimeric, MT2i-responsive and Duxbl KO-responsive gene sets [9, 23, 25] in Setdb1 KO pairwise DEGs. (B) Summary model: Maternal SETDB1 deposits H3K9me3 at MT2 elements to silence MERVL-driven 2-cell transcripts in coordination with DUXBL, enabling exit from totipotency.

Normal features of Setdb1 KO embryos

(A) Morphology. Brightfield images of 2c and 8c WT and KO embryos at 1.5 and 2.5 dpc. Scale bar 50 µM. (B) Transcription. Single embryo total RNA sequencing results of 2c and 8c WT and KO embryos at 1.5 and 2.5 dpc (n=5). IGV browser views of selected transcripts, with normalized CPM scales shown in brackets.

Gene set enrichment analyis (GSEA) of Setdb1 KO DEGs

(A–D) Bubble plots of GSEA for 2cWT vs. 2cKO, 8cWT vs. 8cKO, 8cWT vs. 2cWT, and 8cKO vs. 2cKO pairwise comparisons.

Developmental transcriptional changes independent of maternal SETDB1

(A, C) Heatmaps of DEGs commonly downregulated (n=1722) or upregulated (n=828) from 2c to 8c stages. (B, D) GO analysis of those DEGs.

Developmental misregulation in Setdb1 KO embryos

(A, C, E) Heatmaps showing DEGs uniquely changed in KO embryos. (B, D, F) GO enrichment analyses of these misregulated gene sets.

SETDB1 suppresses transposable elements at cleavage stages

(A) PCA of multimapped TE profiles. (B) Venn diagram of DE TE families (adjusted P < 0.05). (C–D) DE TE tallies across pairwise comparisons by TE class. (E-F) DE TE heatmaps across pairwise comparisons by TE class.

Examples of DUXBL targets derepressed in Setdb1 KO embryos

(A–D) IGV images of Duxbl KO DEGs (e.g., Antxr1, Aqr, Nelfa, Zscan4d) with associated H3K9me3 profiles. MERVL insertions upstream or intronic are indicated.

Sample Information

Details of all single embryos analyzed in the study, including genotype, developmental stage, and sequencing sample ID.