Loss of KMT2C reprograms the epigenomic landscape in hPSCs resulting in NODAL overexpression and a failure of hemogenic endothelium specification

Abstract

Germline or somatic variation in the family of KMT2 lysine methyltransferases have been associated with a variety of congenital disorders and cancers. Notably, KMT2A-fusions are prevalent in 70% of infant leukaemias but fail to phenocopy short latency leukaemogenesis in mammalian models, suggesting additional factors are necessary for transformation. Given the lack of additional somatic mutation, the role of epigenetic regulation in cell specification, and our prior results of germline KMT2C variation in infant leukaemia patients, we hypothesized that germline dysfunction of KMT2C altered haematopoietic specification. In isogenic KMT2C KO hPSCs, we found genome-wide differences in histone modifications at active and poised enhancers, leading to gene expression profiles akin to mesendoderm rather than mesoderm highlighted by a significant increase in NODAL expression and WNT inhibition, ultimately resulting in a lack of in vitro hemogenic endothelium specification. These unbiased multi-omic results provide new evidence for germline mechanisms increasing risk of early leukaemogenesis.

Keywords: Histone methyltransferases; chromatin remodelling; development; gene expression; hemogenic endothelium; mesoderm; pluripotency.

Figure 1.

KMT2CKO pluripotent cells fail to specify hemogenic endothelium. hiPSCs (A panels) and H1 hESCs (B panels) with and without KMT2C were directed through haematopoietic differentiation according to the protocol by Sturgeon et al. (Sturgeon CM, Nat Biotech 2014). In all four cell lines, comparable amounts of CD34+/CD43- cells were specified (A1, A2, B1, B2). To differentiate between arterial, venous and hemogenic endothelium, the CD34+ CD43- cells were further subsorted via CD73 and CD184. HE is CD73-CD184- (boxes). In both KMT2CKO pluripotent lines, there is a failure to specify hemogenic endothelium at levels equivalent to WT. Chi-square analyses found the decrease in hemogenic endothelium to be significant with p-values ≤0.01 for both human iPSCs and ESCs as listed in Experimental Procedures under ‘Directed hematopoietic differentiation.’

Figure 2.

RNA-seq reveals 319 differentially expressed genes in KMT2CKO hiPSCs. (a) Volcano plot for fold change in expression (Y-axis) against the log of the fold change (X-axis). (b) Triplicates of RNA-seq from each cell line show consistent differences in gene expression across 319 genes. (c) PCA analysis reveals that KMT2CKO cells more closely resemble mesendoderm than any of the three germ layers.

Figure 3.

Epigenome tracks showing histone modifications, ATAC-seq peaks and relative RNA expression for NODAL, its regulator CER1 and two of its ligands, BMP4 and WNT3 in WT and KMT2CKO hiPSCs. Each gene demonstrates higher expression in the KMT2CKO compared to WT (boxes in the RNA tracks) while NODAL, BMP4 and WNT3 show the expected decrease in H3K4me3 in the KMT2CKO (boxes in the H3K4me3 tracks).

Figure 4.

Schematic overview of how the lack of KMT2C-mediated histone modifications in hPSCs alters cell fate specification in vitro.