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Silencing of the mouse H-rev107 gene encoding a class II tumor suppressor by CpG methylation
Karim Roder1, Maria-Jesus Latasa, Hei Sook Sul
1Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720, USA.
Abstract:
H-rev107 is a tumor suppressor originally isolated in revertants of H-ras-transformed cell lines. The gene is ubiquitously expressed in normal tissues but down-regulated in primary carcinomas or in many cell lines derived from tumors, including WEHI 7.1 lymphoma cells. Here, we show that unlike in H-rev107-expressing cells or tissues the 5'-end of H-rev107 containing a CpG-rich region of 421 bp is highly methylated in WEHI 7.1 lymphoma cells, correlating with silencing of this gene. Repression of H-rev107 transcription in these cells could be relieved by chemically induced hypomethylation with 5-aza-dC. In addition, upon in vitro methylation, expression of the luciferase reporter gene driven by the H-rev107 promoter decreased by 80% in WEHI 7.1 and 293 cells. Furthermore, co-transfection of the methyl-CpG binding proteins, MeCP2 and MBD2, inhibited H-rev107 promoter activity up to 70% in SL2 cells when the promoter was methylated. By chromatin immunoprecipitation assays, we observed in vivo binding of MeCP2 and MBD2 to the 5'-end of H-rev107 in WEHI 7.1 cells, which was reduced to undetectable levels upon 5-aza-dC treatment, concluding that MeCP2 and MBD2 might be involved in silencing the methylated H-rev107 gene in lymphoma cells and probably certain tumors.
Insights
The tumor suppressor gene H-rev107 is silenced in lymphoma cells due to DNA methylation. This silencing can be reversed by hypomethylation, suggesting a therapeutic target for certain cancers.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- H-rev107 is a known tumor suppressor gene.
- It is expressed in normal tissues but downregulated in various cancers, including lymphoma.
- Its downregulation in WEHI 7.1 lymphoma cells was investigated.
Purpose of the Study:
- To investigate the mechanism of H-rev107 gene silencing in WEHI 7.1 lymphoma cells.
- To explore the role of DNA methylation and methyl-CpG binding proteins in this silencing.
- To assess the potential for reversing the gene silencing.
Main Methods:
- DNA methylation analysis of the H-rev107 5'-CpG-rich region.
- Gene expression analysis after chemical hypomethylation (5-aza-dC).
- Reporter gene assays to assess promoter activity upon methylation.
- Chromatin immunoprecipitation (ChIP) to detect protein binding.
Main Results:
- The H-rev107 5'-end was highly methylated in WEHI 7.1 cells, correlating with gene silencing.
- Hypomethylation with 5-aza-dC reversed H-rev107 repression.
- In vitro methylation reduced promoter activity, and methyl-CpG binding proteins (MeCP2, MBD2) inhibited it.
- MeCP2 and MBD2 were found to bind the H-rev107 promoter in vivo in WEHI 7.1 cells.
Conclusions:
- DNA methylation at the 5'-end of H-rev107 silences the gene in WEHI 7.1 lymphoma cells.
- Methyl-CpG binding proteins MeCP2 and MBD2 are involved in this silencing mechanism.
- Reversing DNA methylation may be a viable strategy to restore H-rev107 tumor suppressor function in certain cancers.