The trithorax protein partner menin acts in tandem with EZH2 to suppress C/EBPα and differentiation in MLL-AF9 leukemia

Abstract

Trithorax and polycomb group proteins antagonistically regulate the transcription of many genes, and cancer can result from the disruption of this regulation. Deregulation of trithorax function occurs through chromosomal translocations involving the trithorax gene MLL, leading to the expression of MLL fusion proteins and acute leukemia. It is poorly understood how MLL fusion proteins block differentiation, a hallmark of leukemogenesis. We analyzed the effect of acute depletion of menin, a close partner of MLL that is critical for MLL and MLL-AF9 recruitment to target genes, on MLL-AF9 leukemia cell differentiation using an in vivo model. We performed cDNA microarray analysis of menin-regulated genes from primary leukemia cells to determine menin-regulated pathways involved in suppressing MLL-AF9 leukemia cell differentiation. We found that menin binds the promoter of the polycomb gene Ezh2, and promotes its expression. EZH2 interacts with the differentiation-promoting transcription factor C/EBPα and represses C/EBPα target genes. Menin depletion reduces MLL binding to the Ezh2 locus, EZH2 expression, and EZH2 binding and repressive H3K27 methylation at C/EBPα target genes, thereby inducing the expression of pro-differentiation C/EBPα targets. In conclusion, our results show that in contrast to its classical role antagonizing trithorax function, the polycomb group protein EZH2 collaborates with trithorax-associated menin to block MLL-AF9 leukemia cell differentiation, uncovering a novel mechanism for suppression of C/EBPα and leukemia cell differentiation, through menin-mediated upregulation of EZH2.

Figure 1.

Acute menin depletion leads to MA9 cell differentiation in culture. (A) Genotype for Men1 excision in AT-1 cells 2 days after 4-OHT treatment. (B) Western blot for menin in control and Men1-excised AT-1 cells 4 days after 4-OHT treatment. (C) Flow cytometry for Gr-1 cell surface expression in control and Men1-excised AT-1 cells. (D) Wright-Giemsa staining of control and Men1-excised AT-1 cells 6 days after 4-OHT treatment. (E) Real-time PCR examining Hoxa9 transcript levels in control and Men1-excised AT-1 cells. (F) Flow cytometry for Gr-1 cell surface expression in control and Men1-excised AT-1 cells with or without Hoxa9/Meis1 overexpression day 6 after 4-OHT treatment. (G) Real-time PCR examining transcript levels of genes associated with differentiation in control and Men1-excised AT-1 cells.

Figure 2.

Menin depletion results in MA9 cell differentiation in vivo. (A) A schematic for examining the acute effect of menin depletion on MA9 cells in vivo. Men1^f/f; Cre-ER bone marrow was transduced with MA9-ires-GFP and transplanted into lethally irradiated recipients. Recipient mice were treated with corn oil (CO) as a control or with CO plus tamoxifen (TAM) to excise the floxed Men1 allele. GFP⁺ cells were analyzed for MA9 cell immunophenotype. (B) FACS plot showing increased Gr-1^high percentage in Men1-excised MA9 primary cells 7 days after-initial TAM treatment. (C) Hematoxylin and eosin staining of spleen sections from control (left) and TAM-treated (right) Men1^f/f; Cre-ER MA9 primary recipients 7 days after-initial TAM treatment. (D–E) A summary of the Gr-1^high (D) and c-kit^high (E) MA9 cell population in response to menin depletion 4 and 7 days after-initial TAM treatment.

Figure 3.

Men1 excision leads to C/EBPα target gene upregulation but does not affect C/EBPα expression. (A) Gene set enrichment analysis analysis comparing Men1^f/f; Cre-ER MA9 primary cells treated with tamoxifen in vivo to the C/EBP target data set. (B) Flow cytometry analysis of Gr-1 cell surface expression in vector or C/EBPα-ER transduced AT-1 cells 2 days after 4-OHT treatment. (C) Western blot for C/EBPα expression in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (D) ChIP for C/EBPα enrichment at the Mcsfr promoter in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment.

Figure 4.

Menin promotes EZH2 expression in MA9 cells. (A) Real-time PCR analysis of Mecom and Ezh2 transcript levels in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (B) Western blot for EZH2 in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (C–D) ChIP assays for menin, AF9c (C), and MLL-C (D) binding at the Ezh2 promoter in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (E) ChIP assays for H3K4m3 and H3K79m2 at the Ezh2 promoter 6 days after 4-OHT treatment.

Figure 5.

EZH2 interacts with C/EBPα in MA9 cells and represses C/EBPα target genes. (A) Luciferase assay in HEK 293T cells with a C/EBPα binding site-containing promoter-driven luciferase plasmid, C/EBPα and increasing amounts of EZH2. (B) Luciferase assay in HEK 293T cells with a Gli-1 binding site-containing promoter-driven luciferase plasmid, Gli-1 and increasing amounts of EZH2. (C) Luciferase assay in RAW264.7 cells with a C/EBPα binding site-containing promoter-driven luciferase plasmid, C/EBPα and increasing amounts of EZH2. (D) Immunoprecipitation (IP) for C/EBPα followed by western blotting for EZH2 (top) or C/EBPα (bottom). (E) IP for EZH2 followed by western blotting for C/EBPα (top) and EZH2 (bottom) in THP-1 cells.

Figure 6.

EZH2 knockdown induces MA9 cell differentiation. (A) ChIP assay for EZH2 enrichment at the Mcsfr, Id2, and Pparg loci in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (B) ChIP assay for H3K27m3 enrichment at the Mcsfr promoter in control or Men1-excised AT-1 cells 6 days after 4-OHT treatment. (C) Western blot for EZH2 expression in Scr control and EZH2 knockdown THP-1 cells. (D) Real-time PCR analysis of Scr control and EZH2 KD THP-1 cells for C/EBPα target genes. (E) Flow cytometry analysis of CD11b cell surface expression in Scr and EZH2 knockdown THP-1 cells. (F) A model for the role of EZH2 in MA9 leukemia.