Sp17 gene expression in myeloma cells is regulated by promoter methylation

Abstract

The mechanisms underlying sperm protein 17 (Sp17) gene expression in myeloma cells remained unclear. Using reverse transcription-polymerase chain reaction (RT-PCR), Sp17 transcripts were detected in ARK-B, ARP-1, RPMI-8226 and KMS-11 but not in H929, IM-9, MM1-R and U266 cells. Using a panel of primer pairs in methylation-sensitive PCR to amplify overlapping gene segments, our screening studies showed that the HpaII sites at -359 and -350 are involved in the regulation of Sp17 gene expression. To confirm the differences in methylation status between Sp17-positive and Sp17-negative cell lines, KMS-11 cells (Sp17-positive) and IM-9 cells (Sp17-negative) were subjected to the more accurate method of bisulphite conversion. KMS-11 cells were more hypomethylated at these HpaII sites of exon 1 compared to IM-9 cells, indicating the association of hypomethylated promoter with Sp17 gene expression. In addition, the level of methylation at other CpG sites within the promoter sequence was also higher in IM-9 than KMS-11. Exon 1 was cloned into a reporter vector, pCAT*3 Enhancer. Chloramphenicol acetyl transferase (CAT) activity was restored in cells transfected with the recombinant plasmid, indicating the promoter function of exon 1. Exposure of Sp17-negative cell lines to the hypomethylating agent, 5-azacytidine, resulted in the upregulation of Sp17 gene expression. Our results therefore provide evidence for the regulation of Sp17 gene expression by promoter methylation.

Figures

Figure 1

Reverse transcription–polymerase chain reaction analysis using a pair of sequence-specific primers for Sp17 produced a positive signal of approximately 500 bp (lane 1=ARK-B; lane 2=ARP-1; lane 3=H929; lane 4=IM-9; lane 5=KMS-11; lane 6=MM1-R; lane 7=U266; and lane 8=RPMI 8226) (M=molecular marker; lane a=PCR without prior RT; lane b=RT–PCR for Sp17; lane c=control amplification for a β-actin gene segment).

Figure 2

Polymerase chain reaction analysis for Sp17 promoter sequence using six primer pairs to amplify across eight HpaII sites. Therefore, PCR products were only obtained if the target sequence are methylated at the HpaII sites (A=PCR without prior HpaII digest; B=PCR after HpaII digest) (lane 1=ARK-B; lane 2=ARP-1; lane 3=H929; lane 4=IM-9; lane 5=KMS-11; lane 6=MM1-R; lane 7=U266; and lane 8=RPMI 8226) (M=molecular marker).

Figure 3

Comparison of the levels of methylation at all 46 CpG sites within exon 1 between KMS-11 and IM-9 cells, showing that there was increased hypomethylation throughout exon 1 in KMS-11 cells that express Sp17 gene.

Figure 4

Analysis of 293 cells for CAT activities after plasmid transfection to confirm the role of Sp17 promoter methylation (lane 1=wild-type 293 cells; 293 cells transfected with pCAT*3 Enhancer-Sp17 promoter; lane 3=293 cells transfected with the positive control plasmid pCAT*3 Enhancer-SV40 promoter; lane 4=293 cells transfected with a control plasmid, pcDNA3.1; lane 5=293 cells transfected with pCAT*3 Enhancer plasmid that did not contain any promoter; lane 6=293 cells transfected with pCAT*3 Enhancer-Sp17 promoter/met; lane 7=product control; lane 8=substrate control).

Figure 5

Northern blot analysis to compare the levels of Sp17 transcripts before (a) and after (b) exposure to 5-azacytidine (lane 1=ARK-B; lane 2=ARP-1; lane 3=H929; lane 4=IM-9; lane 5=KMS-11; lane 6=MM1-R; lane 7=U266; and lane 8=RPMI 8226).