c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis
Shenghao Jin, Huiwu Zhao, Yan Yi, Yuji Nakata, Anna Kalota, Alan M. Gewirtz
Shenghao Jin, Huiwu Zhao, Yan Yi, Yuji Nakata, Anna Kalota, Alan M. Gewirtz
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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 4

Loss of c-Myb affects global H3K4 methylation level in K562 cells but not in human SEM-K2 leukemic cells.

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Loss of c-Myb affects global H3K4 methylation level in K562 cells but no...
(A and B) K562 cells were nucleofected with control siRNA or 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-menin, α-WDR5, α-MLLC, α-RbBP5, α–β-actin (A), α-H3K4(Me)1-3, and α-H3 (B) antibodies. Mock indicates K562 cells nucleofected with the same amount of nuclease-free water without siRNA. (C) ChIP assay was performed to assess the occupancy of c-Myb on the HOXA9 gene locus of HL-60 cells. Chromatin was immunoprecipitated with α–c-Myb antibody. The presence of the HOXA9 locus DNA in the chromatin precipitates was assessed by standard PCR. Negative and positive controls consisted of IgG and α–histone H3 antibody, respectively. (D) ChIP analysis of HL-60 cells nucleofected with control siRNA or MYB siRNA was performed using antibodies specific for MLLC and menin. The presence of the HOXA9 locus DNA in the chromatin precipitates was assessed by standard PCR. ChIP using α–histone H3 antibody served as a positive control. (E) SEM-K2 cells were nucleofected with control siRNA or 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-H3K4(Me)1-3, α-H3, and α–β-actin antibodies.