SETDB1 enables development beyond cleavage stages by extinguishing the MERVL-driven two-cell totipotency transcriptional program in the mouse embryo

Abstract

Loss of maternal SETDB1, a histone H3K9 methyltransferase, leads to developmental arrest prior to implantation, with very few mouse embryos advancing beyond the eight-cell stage, which is currently unexplained. We genetically investigate SETDB1's role in the epigenetic control of the transition from totipotency to pluripotency-a process demanding precise timing and forward directionality. Through single-embryo total RNA sequencing of two-cell and eight-cell embryos, we find that Setdb1^mat-/+ embryos fail to extinguish one-cell and two-cell transient genes-alongside persistent expression of MERVL retroelements and MERVL-driven chimeric transcripts that define the totipotent state in mouse two-cell embryos. Comparative bioinformatics reveals that SETDB1 acts at MT2 LTRs and MERVL-driven chimeric transcripts, which normally acquire H3K9me3 during early development. The dysregulated targets substantially overlap with DUXBL-responsive genes, indicating a shared regulatory pathway for silencing the two-cell transcriptional program. We establish maternal SETDB1 as a critical chromatin regulator required to extinguish retroelement-driven totipotency networks and ensure successful preimplantation development.

Keywords: MERVL; SETDB1; genetics; genomics; maternal effect; mouse; preimplantation; single embryo total RNAseq; totipotency.

Figure 1.. Maternal SETDB1 is essential for development beyond the eight-cell stage.

(A) Quantification of Setdb1^mat-/+ (KO) and Setdb1^fl/+ (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 (Figure 1—source data 1). (C) Schematic of four pairwise comparisons defining requirements for normalcy and development. (D) Volcano plots highlighting differentially expressed genes (DEGs) using |log₂FC|>1 and adjusted p<0.05. (E, F) Four-way DEG comparisons (Figure 1—source data 2) visualized by Venn diagrams: (E) downregulated; (F) upregulated. (G–J) Heatmaps of DEGs from Venn compartments, showing stage- and genotype-specific patterns.

Figure 1—figure supplement 1.. 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.

Figure 1—figure supplement 2.. Gene set enrichment analysis (GSEA) of Setdb1 KO differentially expressed genes (DEGs).

(A–D) Bubble plots of GSEA for 2cWT vs. 2cKO, 8cWT vs. 8cKO, 8cWT vs. 2cWT, and 8cKO vs. 2cKO pairwise comparisons (Figure 1—figure supplement 2—source data 1).

Figure 1—figure supplement 3.. Developmental transcriptional changes independent of maternal SETDB1.

(A, C) Heatmaps of differentially expressed genes (DEGs) commonly downregulated (n=1722) or upregulated (n=828) from 2c to 8c stages. (B, D) Gene ontology (GO) analysis of those DEGs (Figure 1—figure supplement 3—source data 1).

Figure 1—figure supplement 4.. Developmental misregulation in Setdb1 KO embryos.

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

Figure 1—figure supplement 5.. SETDB1 suppresses transposable elements at cleavage stages.

(A) Principal component analysis (PCA) of multimapped TE profiles. (B) Venn diagram of DE TE families (adjusted p<0.05) (Figure 1—figure supplement 5—source data 1). (C, D) DE TE tallies across pairwise comparisons by TE class. (E, F) DE TE heatmaps across pairwise comparisons by TE class.

Figure 2.. Maternal SETDB1 extinguishes two-cell transient gene expression.

(A) Bubble plot showing overrepresentation analysis of Database of Transcriptome in Mouse Early Embryos (DBTMEE) (Park et al., 2015)-defined transcript sets among differentially expressed genes (DEGs) identified in the four pairwise comparisons (Figure 2—source data 1). (B) IGV browser snapshots of representative transcripts from the DBTMEE-defined minor ZGA to MGA, two-cell transient, and four-cell transient gene sets across five biological replicates. Venn diagram compartments are indicated above each track. Units 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.

Figure 3.. 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 et al., 2012. (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 (Figure 3—source data 1).

Figure 4.. Maternal SETDB1 suppresses MT2 LTR-regulated genes.

(A, B) Heatmaps of H3K9me3 deposition (Wang et al., 2018) at MT2-controlled (Yang et al., 2024) differentially expressed genes (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.

Figure 5.. SETDB1 regulates MT2-activated genes across time.

(A) Heatmaps comparing MT2i-responsive (Yang et al., 2024) early two-cell (E2c) differentially expressed genes (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.

Figure 6.. SETDB1 represses DUXBL-responsive transcripts.

(A) Heatmap of top 50 differentially expressed genes (DEGs) identified in Duxbl KO embryos (Vega-Sendino et al., 2024) analyzed in the Setdb1 KO RNA-seq dataset. (B) Volcano plots marking upregulated/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.

Figure 6—figure supplement 1.. 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.

Figure 7.. 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 (Macfarlan et al., 2012; Vega-Sendino et al., 2024; Yang et al., 2024) in Setdb1 KO pairwise differentially expressed genes (DEGs) (Figure 7—source data 1). (B) Summary model: Maternal SETDB1 deposits H3K9me3 at MT2 elements to silence MERVL-driven two-cell transcripts in coordination with DUXBL, enabling exit from totipotency.