Expression analysis of DNMT3A mutant-transduced hematopoetic stem/progenitors

Despite the impact of DNMT3A mutation in acute myeloid leukemia has been emphasized, the precise molecular mechanisms in leukemogenesis are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant hematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are down-regulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells. DNMT3A mutant also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), leading to transcriptional silencing of PU.1. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. Taken together, our results highlight the functional role of DNMT3A mutation, forming the basis for leukemia development. GFP-labeled empty vector, DNMT3A wild-type (WT), R882H-transduced LSK cells derived from transplanted mice were utilized for compared the expression profiles (3 sorted empty vector-transduced LSK cells, 3 sorted DNMT3A WT-transduced LSK cells, and 3 sorted DNMT3A R882H-transduced LSK cells. Total RNA was extracted by TaKaRa NucleoSpin RNA XS according to the manufacturer’s protocol. Amplification and biotin labeling of fragmented cDNA was carried out from 3.67 ng of total RNA by using NuGen Ovation Pico WTA System V2 (NuGEN) and SureTag Complete DNA Labeling Kit (Agilent). Each 2 μg of cDNA were hybridized to the Agilent SurePrint G3 Mouse Gene Expression 8x60K (Agilent) using Gene Expression Hybridization Kit (Agilent). After scanning, the signal intensity for each feature was measured by Agilent Feature Extraction (Agilent).