Abstract
Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.
Graphical Abstract In oogenesis, SETD1B and CXXC1 target H3K4me3 to actively transcribed gene promoters, while MLL2 targets transcriptionally inactive regions based on underlying CpG composition (upper panel). When SETD1B is ablated, H3K4me3 is lost at a subset of active promoters, resulting in downregulation of transcription (lower panel). Loss of SETD1B alters the activity of MLL2, permitting MLL2 to deposit H3K4me3 at CpG-rich regions, many of which should otherwise be DNA methylated. Thus, it is evident that MLL2 and de novo DNMTs compete for genomic occupancy late in oogenesis, and loss of SETD1B disrupts the balance of these mechanisms.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In the revised manuscript we have added new temporal analyses which help provide the following insights: 1) To improve clarity on the timing and dependencies of promoter H3K4me3 and the links to gene expression, H3K4me3 ChIP-seq datasets from stages across oogenesis, from Mll2 cKO oocytes (Hanna et al. 2016 Nat. Struct. Mol. Biol) and from Cxxc1 cKO oocytes (Sha et al. 2021 Nucl. Acids Res.) were analysed. These analyses revealed that most promoters rely on both SETD1B/CXXC1 and MLL2 for establishment of H3K4me3. However, a subset acquires H3K4me3 early in oogenesis and relies solely on SETD1B/CXXC1: these are the subset that lose expression in the Setd1b and Cxxc1 cKOs. These findings are detailed in a new section of the Results (page 7-8), Figure 3 and Supplementary Figure 4. 2) To improve understanding of links between disrupted H3K4me3 and DNA methylation patterning in Setd1b cKO oocytes, DNA methylation datasets from Cxxc1 cKO oocytes (Sha et al. 2021 NAR), and from d12 and d15 oocytes (Dahl et al. 2016 Nature), and from H3K4me3 from Dnmt3-null oocytes (Hanna et al. 2016 NSMB) were analysed. This shows that the loss of DNA methylation in Setd1b cKO (and Cxxc1 cKO) oocytes occurs at regions that acquire DNA methylation late in oogenesis and the gains of H3K4me3 across these domains recapitulate the gains present in oocytes that lack DNA methylation. These findings support a model in which MLL2 and de novo DNMTs compete for targets in late oogenesis and disruption of the balance between these mechanisms (such as in the Setd1b, Cxxc1 or Dnmt3 cKOs) leads to impaired patterning of the oocyte epigenome. These findings are detailed in the Results, Figure 5, and Supplementary Figure 5.
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167987