BMI1 reprogrammes histone acetylation and enhances c-fos pathway via directly binding to Zmym3 in malignant myeloid progression

Abstract

The polycomb group BMI1 is proved to be crucial in malignant myeloid progression. However, the underlying mechanism of the action of BMI1 in myeloid malignant progression was not well characterized. In this study, we found that the patients of both myelodysplastic syndromes and chronic myeloid leukaemia with BMI1 overexpression had a higher risk in malignant myeloid progression. In vitro gene transfection studies showed that BMI1 inhibited cell myeloid and erythroid differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and histone deacetylase inhibitor sodium butyrate respectively. BMI1 also resisted apoptosis induced by arsenic trioxide. Moreover, the transcript levels of Runx1 and Pten were down-regulated in Bmi1-transfected cells in company with histone deacetylation modification. By using chromatin immunoprecipitation (ChIP) collaborated with secondary generation sequencing and verified by ChIP-PCR, we found that BMI1 directly bound to the promoter region of Zmym3, which encodes a component of histone deacetylase-containing complexes. In addition, as one of the downstream target genes of this complex, c-fos was activated with increasing histone acetylation when ZMYM3 was suppressed in the Bmi1-transfected cells. These results suggested that BMI1 may reprogramme the histone acetylation profile in multiple genes through either indirect or direct binding effects which probably contributes to the malignant progression of myeloid progenitor cells.

Keywords: BMI1; ZMYM3; acetylation; chronic myeloid leukaemia; myelodysplastic syndrome.

Figure 1

Bmi1 transcript expression in myeloid progression (A–E). The Bmi1 transcript expression levels were all performed by quantitative PCR analysis (Q-PCR). GPADH was as control. Relative expression was calculated by the comparative Ct method. (A) The relative Bmi1 transcript expression in myelodysplastic syndromes (MDS) and non-MDS cytopaenia bone marrow mononuclear cells (BMMCs). “NR MDS” means MDS not in remission. “R MDS” means MDS in remission. (B) The relative BMI1 expression in two MDS groups separated by the ratio of blast cells in MDS bone marrow smear. (C) The transcription of Bmi1 in non-MDS cytopaenia, MDS, MDS transformed acute myeloid leukaemia [MDS-acute myeloid leukaemia (AML)] and de novo AML (dAML) CD34+ cells. (D) The correlation of BMI1 transcription in MDS CD34+ cells and international prognostic scoring system score was evaluated by linear regression. The patient with one arrow progressed to refractory anaemia (RA) with excess blasts (RAEB) from RA and the patients with double arrows transformed to AML from RAEB. (E) The relative Bmi1 transcription in chronic myeloid leukaemia in chronic phase (CML-CP), CML in blast phase (CML-BP) and matched dAML BMMCs. (A, B, E) Quartile figure is used to present the data, and bar represents the median of the data. “*” indicates statistically significant P < 0.05; and “**” means without statistically significant.

Figure 2

BMI1 enhances the malignancy of leukaemic cells (A) Western blotting of BMI1 in subclones of transfected U937 and K562 cells. Numbers are the folds compared to the first line control. SC1 and SC2 represent two subclones of Bmi1 transfected U937 and K562 cells. (B) The viability of cell was examined by vi-cell XR cell viability analyser after cells were cultured without foetal bovine serum for 72 hrs, n = 3. (C) Colony forming assay of K562 and Bmi1-transfected K562. Cells were cultured in MethoCult®H4230 with 5*104cells/mL for 7 days and then replated two cycles. Numbers are colonies with diameter larger than 0.3 mm, n = 3. The colony number of Bmi1-transfected K562 was greater than control with statistical significance, P < 0.05. The colony number means the total number of colonies/number of cell input after two cycles replating. (D) The apoptosis ratio of K562 after 48 hrs of 5 μM arsenic trioxide treatment tested by annexin V and propidium iodide in flow cytometry.

Figure 3

BMI1 inhibits 12-O-tetradecanoyl phorbol-13-acetate (TPA) and histone deacetylase inhibitor sodium butyrate (NaB) induced differentiation in K562 (A) Nitroblue tetrazolium (NBT) assay for cell differentiation induced by 20 nM TPA for 48 hrs. M ± D is the percentage of NBT-positive blue cells, n = 3. Bar stands for 10 μm. (B) The differentiation curve of TPA induced differentiation. (C) The mean fluorescence intensity and percentage of CD15 +  cells detected by flow cytometry (FCM) after 20 nM TPA treatment for 72 hrs. Isotype primary conjugated antibodies were served as a negative control. “P” means percentage. (D) Benzidine assay of erythroid differentiation in K562 induced by 0.5 mM NaB for 72 hrs. M ± D is the percentage of benzidine-positive cells, n = 3. Bar stands for 10 μm. (E) The differentiation curve of NaB induced differentiation. (F) The dot plot of the FCM analysis for CD71 and GPA staining after cells induced by 0.5 mM NaB for 72 hrs.

Figure 4

BMI1 indirectly inhibits Runx1 and Pten expression with histone deacetylation. (A) The heat map of poor diagnosis genes in six MDS CD34+ cells of two refractory anaemia (RA), two RA with excess blasts I (RAEB-1) and two RAEB-2 compared to the normal control on the cDNA microarray. (B) The relative transcription of Runx1 and Pten tested by Q-PCR in K562. The transcript level in K562 was set to 1. (C and D) The histone H3 acetylation (H3ac) and histone H4 acetylation (H4ac) at Runx1 and Pten promoter regions tested by chromatin immunoprecipitation (ChIP) and Q-PCR assay in K562, n = 3. The histone modification in 10% input DNA was set to 1. IgG was as negative control.

Figure 5

BMI1 directly suppresses Zmym3 and activates c-fos pathway. (A) Electrophoresis strips of Zmym3 promoter region in BMI1 and H2A with ubiquitination ChIP-PCR products. (B) The relative transcript level of Zmym3 in Bmi1-transfected K562 cells. The expression level in K562 was set to 1. (C) The relative transcript expression of c-fos in Bmi1-transfected K562. The expression level in K562 was set to 1. (D) The relative histone H3K27 acetylation (H3K27ac) and H3 acetylation (H3ac) level of c-fos promoter region, n = 3. The histone modification in 10% input DNA was set to 1. IgG was as negative control.