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Published on: September 7, 2017
Sp17 gene expression in myeloma cells is regulated by promoter methylation
1Division of Hematology and Oncology, Texas Tech University Health Sciences Center, Amarillo, TX, USA.
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.
Insights
Promoter methylation regulates sperm protein 17 (Sp17) gene expression in myeloma cells. Hypomethylation of Sp17 promoter sites correlated with Sp17 expression, and demethylation agents induced Sp17.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Biology
Background:
- Sperm protein 17 (Sp17) gene expression mechanisms in myeloma cells were previously unclear.
- Sp17 transcripts were detected in specific myeloma cell lines (ARK-B, ARP-1, RPMI-8226, KMS-11) but absent in others (H929, IM-9, MM1-R, U266).
Purpose of the Study:
- To investigate the role of promoter methylation in regulating Sp17 gene expression in myeloma cells.
- To identify specific CpG sites involved in Sp17 gene regulation.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to detect Sp17 transcripts.
- Methylation-sensitive PCR and bisulphite conversion followed by sequencing to analyze promoter methylation status.
- Reporter gene assay (chloramphenicol acetyl transferase - CAT) to confirm promoter function.
- Treatment with a hypomethylating agent (5-azacytidine) to assess its effect on Sp17 expression.
Main Results:
- Specific HpaII sites (-359 and -350) in the Sp17 promoter were identified as key regulatory regions.
- Sp17-positive cell lines (KMS-11) exhibited hypomethylation at these sites compared to Sp17-negative cell lines (IM-9).
- Exon 1 of the Sp17 gene demonstrated promoter activity.
- Treatment with 5-azacytidine induced Sp17 gene expression in previously negative cell lines.
Conclusions:
- Promoter methylation, particularly hypomethylation at specific sites in exon 1, is a critical regulator of Sp17 gene expression in myeloma.
- The findings provide a mechanistic link between epigenetic modifications and Sp17 expression in myeloma cells.
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