c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis
Shenghao Jin, … , Anna Kalota, Alan M. Gewirtz
Shenghao Jin, … , Anna Kalota, Alan M. Gewirtz
Published January 19, 2010
Citation Information: J Clin Invest. 2010;120(2):593-606. https://doi.org/10.1172/JCI38030.
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Research Article Hematology

c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis

Abstract

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.

Authors

Shenghao Jin, Huiwu Zhao, Yan Yi, Yuji Nakata, Anna Kalota, Alan M. Gewirtz

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Figure 6

Decrease in trimethylation of H3K4 and HOXA9 gene expression is specific for MYB silencing but not secondary to the antiproliferation effect of MYB silencing.

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Decrease in trimethylation of H3K4 and HOXA9 gene expression is specific...
(A and B) KCL-22 cells were nucleofected with control siRNA or B-MYB siRNA. Whole-cell extracts were prepared 3 days after the initial nucleofection according to the cell number counts and probed by immunoblotting with α–c-Myb, α–B-Myb, and α–β-actin (A), α-H3K4(Me)1-3, and α-H3 (B) antibodies. (C) Cell proliferation assays were carried out with KCL-22 cells nucleofected with mock, control, MYB, and B-MYB siRNAs at indicated time points. Silencing MYB or B-MYB inhibited cell proliferation. (D–G) KCL-22 cells were nucleofected with control, MYB, or B-MYB siRNA. RNA samples, prepared 2 days after the initial nucleofection, were reverse transcribed and used for qRT-PCR analysis for MYB (D), B-MYB (E), HOXA9 (F), and MEIS1 (G) gene expression determined in triplicate as described in Figure 5B. (H and I) KCL-22 cells were nucleofected with control, MYB, or HOXA9 siRNA. RNA samples, prepared 2 days after the initial nucleofection, were reverse transcribed and used for qRT-PCR analysis for MYB (H) and HOXA9 (I) gene expression determined in triplicate as described in Figure 5B. Error bars represent the standard deviation of assays performed in triplicate (C–I).