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Updated: May 3, 2026
Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
Published on: June 16, 2017
Silencing of CHD5 gene by promoter methylation in leukemia
Rui Zhao1, Fanyi Meng2, Nisha Wang1
1Institute of Molecular Biology, Southern Medical University, Guangzhou, PR China.
Abstract:
Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter.
Insights
Chromodomain helicase DNA binding protein 5 (CHD5) is downregulated in leukemia due to promoter DNA methylation. Restoring CHD5 expression may offer a new therapeutic strategy for leukemia treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- Chromodomain helicase DNA binding protein 5 (CHD5) is a known tumor suppressor.
- CHD5 is downregulated in various cancers, including leukemia, contributing to tumor development.
- The mechanisms behind CHD5 downregulation in leukemia remain largely unelucidated.
Purpose of the Study:
- To investigate the molecular mechanisms responsible for Chromodomain helicase DNA binding protein 5 (CHD5) downregulation in human leukemia.
- To identify the role of epigenetic modifications, specifically DNA methylation, in regulating CHD5 expression in leukemia.
- To explore potential therapeutic strategies targeting CHD5 regulation in leukemia.
Main Methods:
- Quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and Western blotting to assess CHD5 expression levels.
- Luciferase reporter assays to analyze promoter activity and identify regulatory regions.
- Bisulfite DNA sequencing to evaluate DNA methylation status of the CHD5 promoter.
- Treatment with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor.
- Chromatin immunoprecipitation (ChIP) assays combined with PCR to identify transcription factor binding.
- In vitro transcription-factor activity studies.
Main Results:
- CHD5 expression was significantly downregulated in human leukemia cell lines and patient samples.
- The CHD5 promoter region, specifically between 500 and 200 bp upstream of the transcription start site, showed hypermethylation in leukemia cells.
- CHD5 expression levels were inversely correlated with promoter DNA methylation.
- Treatment with DAC reactivated CHD5 expression in leukemia cell lines.
- Methylation of the CHD5 promoter repressed its transcriptional activity.
- Activating protein 2 (AP2) was identified as a transcription factor involved in CHD5 regulation, with its recruitment to the promoter increased by DAC treatment.
- AP2 overexpression enhanced CHD5 promoter activity.
Conclusions:
- Repression of CHD5 gene expression in human leukemia is significantly mediated by DNA methylation of its promoter.
- Epigenetic silencing of the CHD5 gene is a key mechanism contributing to its downregulation in leukemia.
- Targeting DNA methylation could be a viable strategy to restore CHD5 expression and potentially treat leukemia.
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