Hematopoietic differentiation of human iPSC is hardly affected by knockouts in DNMT3A exons despite loss of de novo DNA methylation [DNA methylation]

DNA methyltransferase 3A (DNMT3A) is the most frequently mutated gene in clonal hematopoiesis, indicating that it may be essential for hematopoietic differentiation. We therefore addressed the functional relevance of DNMT3A for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) by knocking out either exon 2, 19, or 23. Directed differentiation towards mesenchymal stromal cells or hematopoietic progenitor cells (iHPCs) was only slightly reduced in exon 19-/- lines. Notably, exon 19-/- and exon 23-/- lines revealed absence of almost the entire de novo DNA methylation during differentiation. Despite the drastic effects on DNA methylation, there was no significant impact on gene expression of iHPCs. Notably, DNA methylation differences in acute myeloid leukemia with/without DNMT3A are related to iHPCs with/without DNMT3A knockout. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A, while it has little impact on early cell-fate decisions. DNA was isolated from iPSCs, from iPSC-derived mesenchymal stromal cells after 35 days of mesenchymal differentiation, and from iPSC-derived hematopoietic progenitor cells after 16 days of hematopoietic differentiation. DNA was bisulfite converted and hybridized to the Illumina Infinium MethylationEPIC BeadChip.