SRP310422 PRJNA714079 GSE168811 DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells [csRNA-seq] DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. It is the most common driver gene of age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly individuals. Most acquired DNMT3A mutations are heterozygous and predicted to cause loss-of-function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, whether and how DNMT3A haploinsufficiency affects the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairs DNA demethylation at thousands of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with acquired DNMT3A mutations and clonal hematopoiesis. Overall design: Capped small RNA-seq (csRNA-seq) can map and quantify all transcription start sites (TSSs), including those from enhancer RNA and other rapidly processed or degraded transcripts. csRNA-seq was applied to macrophages differentiated from wild-type (DNMT3A+/+), DNMT3A-haploinsufficient (DNMT3A+/-), or DNMT3A-null (DNMT3A-/-) human embryonic stem cells to quantaify enhancer RNA and assess enhancer activity. GSE168811 pubmed 33970190 parent_bioproject PRJNA713766