Analysis of de novo methylation in mouse embryonic stem cells mediated by Dnmt3a2 PWWP domain variants

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulating this process is known to cause aberrant development and oncogenesis, yet how DNMT3A function is shaped holistically by its three domains remains challenging to study. Through in situ mutational scanning, we found mutations in the PWWP domain, a histone reader, that modulate enzyme activity by affecting DNA binding, despite preserving histone recognition and protein stability. To investigate the consequences of these mutations on DNMT3A function in cells, we expressed ectopic Dnmt3a2 variants in embryonic stem cells and used reduced representation bisulfite sequencing and histone modification ChIP-seq to study the resulting patterns of de novo DNA methylation. Our data show that PWWP domain DNA affinity is required for full activity in cells and constitutes a noncanonical function of the PWWP domain. Overall design: Reduced representation bisulfite sequencing was performed to characterize DNA methylation gain in Dnmt1/Dnmt3a/Dnmt3b triple-knockout (TKO) mouse embryonic stem cells (ESCs) reconstituted with ectopic Dnmt3a2 in biological duplicate (separate transfections). As a negative control, we included the E752K variant, which is catalytically dead. Additionally, ChIP-seq was performed for the H3K4me3, H3K27me3, and H3K36me2 histone modifications in the parent TKO ESC line, each in singlicate (including an Input control).