Active H3K27me3 demethylation by KDM6B is required for normal development of bovine preimplantation embryos

Abstract

The substantial epigenetic remodeling that occurs during early stages of mammalian embryonic development likely contributes to reprogramming the parental genomes from a differentiated to a totipotent state and activation of the embryonic genome. Trimethylation of lysine 27 of histone 3 (H3K27me3) is a repressive mark that undergoes global dynamic changes during preimplantation development of several species. To ascertain the role of H3K27me3 in bovine preimplantation development we perturbed the activity of KDM6B, which demethylates H3K27me3. Knockdown of maternal KDM6B mRNA inhibited the reduction in global levels of H3K27me3 from 2-cell to 8-cell embryo stages and compromised development to the blastocyst stage; embryos that developed to the blastocyst stage had fewer inner cell mass (ICM) and trophectoderm (TE) cells. In addition, the transcriptome of KDM6B knockdown embryos was altered at the 8-cell stage and characterized by downregulation of transcripts related to transcriptional regulation, chromatin remodeling, and protein catabolism. Inhibiting the catalytic activity of KDM6B with a specific small molecule inhibitor also prevented the global decrease in H3K27me3 and compromised development to the blastocyst stage. These results indicate that histone demethylation activity, mediated by KDM6B, is required for the global decrease in H3K27me3, correct activation of the embryonic genome, and development to the blastocyst stage in bovine embryos.

Keywords: Cattle; H3K27me3; JMJD3; embryonic genome activation; histone demethylation; preimplantation development; reprogramming; totipotency.

Figure 1.

Efficient knockdown of KDM6B mRNA in bovine embryos derived by IVF. The abundance of KDM6B mRNA relative to RPL15 in: A) 4-cell stage embryos [2 days post-insemination (dpi)], and B) morulae (5 dpi), was assayed by qPCR in three experimental groups: non-injected, control-siRNA, and KDM6B-siRNA injected embryos. Data are shown as mean ± s.e.m.^a,b Different letters indicate significant differences between groups (P value <0.05).

Figure 2.

KDM6B mRNA knockdown affects global H3K27me3 dynamics from 2-cell to 8-cell stage embryos. A) H3K27me3 nuclear fluorescence intensity was normalized against fluorescence intensity of the 2-cell embryo group within the same treatment group (black bars: non-injected group, dark grey: control siRNA group, light gray: KDM6B siRNA group). B) Representative pictures for H3K27me3 staining. The inset at the left down corner shows DNA stained with Hoechst 33342. Data are shown as mean ± s.e.m. ^{a, b} Different letters indicate significant differences between groups (P value <0.05).

Figure 3.

KDM6B mRNA knockdown affects blastocysts development. A) Incidence of development to blastocyst stage determined at 7 days post-insemination in non-injected, control-siRNA, and KDM6B-siRNA embryos. B) Total cell number; C) ICM cell number; and D) TE cell number of day 7 blastocysts from each treatment group. Data are shown as mean ± s.e.m.^a,b Different letters indicate significant differences between groups (P value <0.05). E) Representative images of SOX2 and CDX2 immunostained blastocysts for determination of ICM and TE cell numbers, respectively. DNA stained with Hoechst33342 for determination of total cell number.

Figure 4.

KDM6B siRNA affects the transcriptome at the 8-cell stage. A) Number of differentially expressed genes between 8-cell embryos from non-injected, control si-RNA, and KDM6B-siRNA treatments. EdgeR was used to find differentially expressed genes between groups using FDR<0.05 and FC>2 as cutoffs. Red square indicates number of downregulated genes. B) Gene Ontology analysis of 82 genes downregulated in 8-cell KDM6B siRNA treated embryos and sensitive to α-amanitin.

Figure 5.

H3K27me3 demethylase activity is important for removal of H3K27me3 after fertilization and for development to the blastocyst stage. A) Experimental design for treatment of bovine embryos with GSK-J4, an H3K27me3 demethylase inhibitor. B) Incidence of development to blastocyst stage for embryos supplemented with different GSK-J4 concentrations and exposure periods. Number of replicates and embryos treated is indicated under each graph. Data are shown as mean ± s.e.m.^a,b,c Different letters indicate significant differences between groups (P value <0.05). C) H3K27me3 nuclear fluorescence intensity in control and GSK-J4 treatment at different stages of bovine embryo development. Data are shown as mean ± s.e.m., * indicates significant differences between treatment groups (P value <0.05).