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Updated: Apr 29, 2026
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Inhibition of intestinal tumor formation by deletion of the DNA methyltransferase 3a
B Weis1, J Schmidt1, H Maamar2
1Division of Epigenetics (A130), German Cancer Research Center (DKFZ), Heidelberg, Germany.
Abstract:
Aberrant de novo methylation of DNA is considered an important mediator of tumorigenesis. To investigate the role of de novo DNA methyltransferase 3a (Dnmt3a) in intestinal tumor development, we analyzed the expression of Dnmt3a in murine colon crypts, murine colon adenomas and human colorectal cancer using RNA fluorescence in situ hybridization (FISH), quantitative PCR and immunostaining. Following conditional deletion of Dnmt3a in the colon of APC((Min/+)) mice, we analyzed tumor numbers, genotype of macroadenomas and laser dissected microadenomas, global and regional DNA methylation and gene expression. Our results showed increased Dnmt3a expression in colon adenomas of APC((Min/+)) mice and human colorectal cancer samples when compared with control tissue. Interestingly, in tumor tissue, RNA FISH analysis showed highest Dnmt3a expression in Lgr5-positive stem/progenitor cells. Deletion of Dnmt3a in APC((Min/+)) mice reduced colon tumor numbers by ~40%. Remaining adenomas and microadenomas almost exclusively contained the non-recombined Dnmt3a allele; no tumors composed of the inactivated Dnmt3a allele were detected. DNA methylation was reduced at the Oct4, Nanog, Tff2 and Cdkn1c promoters and expression of the tumor-suppressor genes Tff2 and Cdkn1c was increased. In conclusion, our results show that Dnmt3a is predominantly expressed in the stem/progenitor cell compartment of tumors and that deletion of Dnmt3a inhibits the earliest stages of intestinal tumor development.
Insights
DNA methyltransferase 3a (Dnmt3a) drives intestinal tumor growth by increasing DNA methylation in stem cells. Deleting Dnmt3a reduces tumor development and reactivates tumor-suppressor genes.
Area of Science:
- Oncology
- Epigenetics
- Molecular Biology
Background:
- Aberrant DNA methylation is a key factor in cancer development.
- DNA methyltransferase 3a (Dnmt3a) plays a role in de novo methylation.
- Understanding Dnmt3a's role in intestinal tumorigenesis is crucial.
Purpose of the Study:
- To investigate the role of Dnmt3a in intestinal tumor development.
- To analyze Dnmt3a expression in normal and cancerous colon tissues.
- To determine the effect of Dnmt3a deletion on tumor formation and progression.
Main Methods:
- Analyzed Dnmt3a expression using RNA FISH, qPCR, and immunostaining in mouse and human tissues.
- Generated conditional Dnmt3a knockout mice (APC((Min/+))) to study tumor development.
- Assessed tumor number, genotype, DNA methylation, and gene expression after Dnmt3a deletion.
Main Results:
- Dnmt3a expression was elevated in colon adenomas and colorectal cancer.
- Highest Dnmt3a expression was observed in Lgr5-positive stem/progenitor cells within tumors.
- Dnmt3a deletion in APC((Min/+)) mice reduced tumor numbers by approximately 40%.
- Remaining tumors predominantly carried the active Dnmt3a allele.
- DNA methylation decreased at specific gene promoters (Oct4, Nanog, Tff2, Cdkn1c), and tumor-suppressor gene expression (Tff2, Cdkn1c) increased.
Conclusions:
- Dnmt3a is primarily expressed in the stem/progenitor cell compartment of intestinal tumors.
- Dnmt3a deletion inhibits early stages of intestinal tumor development.
- Dnmt3a contributes to tumorigenesis by promoting DNA methylation and suppressing tumor-suppressor genes.
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