Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

Abstract

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2'-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5' region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5' CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

Figure 1

Expression of BNIP3 in haematopoietic tumour cell lines. (A) A panel of haematopoietic tumours cell lines was analysed for BNIP3 expression by RT–PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for the integrity of the cDNA. Corresponding negative controls (amplification without RT) are shown as RT-negative. Cell lines and tissues used are shown on the top. (B) Effect of hypoxia on expression of BNIP3. Real-time PCR was carried out using cDNA from cells subjected to normoxic (20% O₂, hypoxia−) or hypoxic (1% O₂, hypoxia+) conditions for 24 h. Signals normalised to GAPDH are shown on the Y-axis. Cell lines examined are shown below the columns. (C) Western blot analysis of HIF-1α and BNIP3. Cells were incubated for 24 h under normoxic (20% O₂, hypoxia-) or hypoxic (1% O₂, hypoxia+) conditions, after which the proteins were separated by SDS–PAGE, and HIF-1α, BNIP3 and actin proteins were detected using appropriate antibodies.

Figure 2

Analysis of BNIP3 methylation in a panel of haematopoietic tumour cell lines. (A) CpG island of BNIP3; CpG sites are shown by vertical bars. The region analysed by COBRA is shown by a solid bar. Exon 1 is shown by a solid box on a solid line. The transcription start site is shown by an arrow. (B) Aberrant methylation of BNIP3 in haematopoietic tumour cell lines. Methylation of BNIP3 was examined using COBRA. Percentages of methylated alleles are shown below the gels. M: methylated allele. (C) Analysis of BNIP3 expression before and after treatment with 5-aza-dC. Cell lines were treated for 96 h with 2.0 μM 5-aza-dC and then harvested, after which RNA was extracted.

Figure 3

Bisulphite-sequencing of BNIP3. (A) Representative results of bisulphite-sequencing. Amplified PCR products were purified from gels and then directly sequenced. The cell lines examined are shown below the column; U, unmethylated CpG sites; M, methylated CpG sites. (B) Summary of bisulphite-sequencing. Each circle represents a CpG dinucleotide. Methylation status: open circles, unmethylated; black circles, methylated. At least seven clones were sequenced for each case. The CpG sites in the region analysed are indicated by vertical bar (top).

Figure 4

Restoration of hypoxia-mediated apoptosis by treatment with 5-aza-dC. TALL1 cells were treated with either mock or 1 μM 5-aza-dC for 72 h followed by incubation under normoxic or hypoxic conditions for 96 h. Percentages of apoptotic cells were determined by flow cytometry. The unmethylated Jurkat cell line served as a control.

Figure 5

The role of histone deacetylation in silencing BNIP3 gene expression. (A) Effects of DNA methyltransferase and/or histone deacetylase inhibitor on the expression of BNIP3. SupT1 cells, which show BNIP3 methylation, were treated with 0.2 μM 5-aza-dC, 300 nM TSA or both. (B) Histone acetylation examined by ChIP followed by PCR. Chromatin immunoprecipitation analysis was carried out using DNA precipitated with antiacetylated histone H3 antibody; the bars show the levels of histone acetylation determined by real-time-PCR normalised to the GAPDH signal. The cell lines examined are indicated below.

Figure 6

Analysis of BNIP3 methylation in a panel of primary haematopoietic tumours. (A) Methylation of BNIP3 examined using COBRA. Tumour type is shown above the gels. Percentages of methylated alleles were calculated by densitometry and are shown below the gels. (B) Summary of bisulphite-sequencing. Each circle represents a CpG dinucleotide. Methylation status: open circles, unmethylated; black circles, methylated. The cases examined are shown on the right.