Preliminary Studies On The Methylation Of DAZAP2 Promoter In Multiple Myeloma

Multiple myeloma (MM) develops through several sequential and interrelated steps such as the progressive dysregulation of genes. In order to find new MM associated genes, Shi et al analyzed gene expression profiles between mRNA from bone marrow mononuclear cells of MM patients and normal controls through microarray assay and then identified that DAZAP2 (deleted in azoospermia associated protein 2) was a profoundly down-regulated gene. The down-expression of DAZAP2 was further validated in the bone marrow mononuclear cells of MM patients and normal controls by means of semi-quantitative RT-PCR and Western blotting. All these results indicated that the down-regulated DAZAP2 may be involved in critical steps of MM pathogenesis.To illuminate the epigenetic mechanism of down-regulated DAZAP2 in MM, we determinated the methylation status and obtained the detailed loci of the methylation in DAZAP2 promoter by bisulphite genomic-sequencing (BGS) method in KM3.We analyzed -632～+58 of DAZAP2 gene by means of bioinformatics, and found two CpG islands in this region. We amplified the two CpG islands of DAZAP2 by using Bisulfite-Sequencing PCR (BSP) primers, respectively. The two regions revealed densely methylated CpGs in comparison with DAZAP2 sequences through bisulfite sequencing of 5 individual clones of PCR products. The ration of methylated CpGs in the two CpG islands was 46.25%. Then, we analyzed DAZAP2 promoter by TFSEARCH software, and found some binding sequences of transcription factors such as p300, ADRI, CREB and AP-2. We also found some CpGs just locating at binding sequences of transcription factors in the DAZAP2 promoter. We presumed that methylation patterns of CpGs probably lead to transcriptional suppression of DAZAP2 through interfering with transcription factors binding to the promoter. However, further experiments such as EMSA should be taken to validate the hypothesis.Meanwhile, we investigated if the methylation patterns of CpG could repress gene expression in vitro, and tried to find the crucial nucleotide sequences in the DAZAP2 promoter. We amplified the two CpG islands locating in DAZAP2 promoter by PCR, respectively. We respectively inserted the three nucleotide sequences into luciferase reporter gene vectors (pGL2-Basic vector), and also ligated the three nucleotide sequences which were methylated by M.Sss I methylase into pGL2-Basic vector. Then we transfected the recombinant plasmids into COS-7 cells and detected the expressional activity of luciferase. The results showed that Sequence 1 presented the weak transcriptional activity, and both of Sequence 2 and 3 showed a remarkable increase in activity, whereas the activity of the methylated sequences were suppressed significantly. It is indicated that Sequence 2 containing the second CpG island may be a crucial sequence in DAZAP2 promoter. We also validated that methylation patterns of CpGs could repress gene expression.In conclusion, all of results could be demonstrated that methylated CpGs in the promoter may play a critical role in down-regulation of DAZAP2 in MM, and epigenetic silencing by the methylation mechanism of the DAZAP2 promoter may be a factor in the pathogenesis of MM.