Yu-Jie Wu, 1 Li-xia Li, 2 Lu Liu, 1 Si-shu Zhao, 1 Hai-rong Qiu, 1 Hui Wang 1 1Department of Hematology, The First Affiliated Hospital of Nanjing Medical University (Jiangsu Provincial People Hospital), Nanjing 210029, Jiangsu Province, People’s Republic of China; 2Department of Laboratory Medicine Center, The Second Affiliated Hospital of Nanjing Medical University (Jiangsu Provincial People Hospital), Nanjing 210011, Jiangsu Province, People’s Republic of ChinaCorrespondence: Yu-Jie Wu Email wuyujie123456@163.comBackground: Mixed lineage leukemia (MLL) fusion protein alone exhibits poor histone lysine methyltransferase (HKMT) activity in catalyzing histone H3 Lys4 trimethylation (H3K4me3) in MLL-rearranged acute leukemia.Methods: To explore the HKMT effect of another regulatory protein within the complex of proteins associated with Set 1 (COMPASS), we analyzed the H3K4me3 modification of the HOXC8 promoter under the action of ASH2L regulation. Small interfering RNA of ASH2L, chromatin immunoprecipitation, real-time-PCR (RT-PCR), and Western blotting were used to detect the expression of specific regions of the HOXC8 promoter, RBBP5, WDR5, MLL, and BRTF in two MLL-rearranged acute leukemia cell lines (RS4:11 and THP-1 cells).Results: The gene and protein expression levels of HOXC8 were significantly downregulated upon treatment with ASH2L-siRNA (as analyzed by targeting specific regions of the HOXC8 promoter located 0 and 3 kb (– 3.0 kb) upstream of the transcriptional start site in RSH:11 cells; and – 3.0 and – 2.0 kb upstream of the transcriptional start site, and +1.4 kb downstream of the transcriptional start site in THP-1 cells). The expression levels of the BRTF, RBBP5, WDR5, and MLL genes were significantly downregulated from the different transcriptional start sites of the HOXC8 promoter in the RSH:11 cell line (P < 0.05). Furthermore, the BPTF and RBBP5 genes were downregulated from the HOXC8 promoter in the THP-1 cell line (P < 0.05).Conclusion: Based on these results, we suggest a new concept of histone modification of the ASH2L protein in MLL-rearranged acute leukemia, which cannot carry out methyltransferase activity independently. The protein–protein interactions of ASH2L with other COMPASS members, such as MLL, WDR5, RBBP5, and chromatin remodeling factor BRTF, appear to be essential for its role in the activation of HOXC8 gene transcription.Keywords: MLL-rearranged acute leukemia, HOXC8 gene, ASH2L, COMPASS complex, H3K4 trimethylation