Dnmt3b alters the expression profile of LSCs and leads to downregulation of stem cell genes. (A) Genome-wide mRNA expression profiles of c-Kit⁺MLL-AF9 LSCs and c-Kit⁻MLL-AF9 leukemic bulk cells were generated using Affymetrix Mouse Gene ST 2.0 arrays. Numbers of up- and downregulated genes in c-Kit⁺ and c-Kit⁻ bone marrow cells from primary recipients of wild-type and Dnmt3b-knock-in MLL-AF9 leukemic cells were compared. (B) Gene set enrichment analysis of expression profiles of c-Kit⁺ wild-type and Dnmt3b-knock-in MLL-AF9 LSCs. Stemness genes and genes expressed in LICs were found in wild-type LSCs (left), whereas the expression profile of Dnmt3b-knock-in LSCs was enriched for genes expressed in mature hematopoietic cells or genes that are downregulated in LSCs and LICs (right). (C) Differentially expressed genes were filtered for those with reduced expression levels with DNA hypermethylation in both MLL-AF9 and Myc-Bcl2 leukemias at the same time. Relative expression levels of representative candidate genes were measured by real-time PCR analysis. Mean ± SD values are from 3 replicates normalized to expression levels of GAPDH. (D-E) Gene expression profiles of representative candidate genes in HSCs, granulocyte-macrophage progenitors, and mature granulocytes. Microarray data sets were analyzed using the Leukemia Gene Atlas (http://www. leukemia-gene-atlas.org). ***P < .001