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Updated: Feb 11, 2026
Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells
Published on: July 29, 2010
CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell
Lissania Guerra-Calderas1, Rodrigo González-Barrios1, Carlos César Patiño1
1Cancer Biomedical Research Unit, Instituto Nacional de Cancerología (INCan), Mexico City, Mexico.
Abstract:
Histone demethylase KDM4A is involved in H3K9me3 and H3K36me3 demethylation, which are epigenetic modifications associated with gene silencing and RNA Polymerase II elongation, respectively. KDM4A is abnormally expressed in cancer, affecting the expression of multiple targets, such as the CHD5 gene. This enzyme localizes at the first intron of CHD5, and the dissociation of KDM4A increases gene expression. In vitro assays showed that KDM4A-mediated demethylation is enhanced in the presence of CTCF, suggesting that CTCF could increase its enzymatic activity in vivo, however the specific mechanism by which CTCF and KDM4A might be involved in the CHD5 gene repression is poorly understood. Here, we show that CTCF and KDM4A form a protein complex, which is recruited into the first intron of CHD5. This is related to a decrease in H3K36me3/2 histone marks and is associated with its transcriptional downregulation. Depletion of CTCF or KDM4A by siRNA, triggered the reactivation of CHD5 expression, suggesting that both proteins are involved in the negative regulation of this gene. Furthermore, the knockout of KDM4A restored the CHD5 expression and H3K36me3 and H3K36me2 histone marks. Such mechanism acts independently of CHD5 promoter DNA methylation. Our findings support a novel mechanism of epigenetic repression at the gene body that does not involve promoter silencing.
Insights
The histone demethylase KDM4A and CTCF form a complex that represses the CHD5 gene by reducing H3K36me3/2 marks in its first intron, independent of promoter methylation.
Area of Science:
- Epigenetics
- Molecular Biology
- Cancer Biology
Background:
- Histone demethylase KDM4A targets H3K9me3 and H3K36me3 epigenetic marks.
- KDM4A is aberrantly expressed in cancer and influences target gene expression, including CHD5.
- CTCF enhances KDM4A's demethylase activity, but the repression mechanism of CHD5 by CTCF and KDM4A is unclear.
Purpose of the Study:
- To elucidate the mechanism of CHD5 gene repression mediated by CTCF and KDM4A.
- To investigate the role of KDM4A and CTCF in regulating H3K36me3/2 marks at the CHD5 gene locus.
Main Methods:
- Protein complex formation analysis between CTCF and KDM4A.
- Chromatin immunoprecipitation to assess histone modifications (H3K36me3/2) at the CHD5 gene.
- siRNA-mediated depletion of CTCF and KDM4A.
- CRISPR-Cas9 knockout of KDM4A.
- Analysis of CHD5 gene expression and promoter DNA methylation.
Main Results:
- CTCF and KDM4A form a complex recruited to the first intron of CHD5.
- This complex reduces H3K36me3/2 histone marks, leading to CHD5 transcriptional downregulation.
- Depletion of CTCF or KDM4A reactivates CHD5 expression.
- KDM4A knockout restores CHD5 expression and H3K36me3/2 marks.
- The repression mechanism is independent of CHD5 promoter DNA methylation.
Conclusions:
- CTCF and KDM4A collaborate to epigenetically repress the CHD5 gene via gene body histone modification.
- This study reveals a novel mechanism of gene repression acting at the gene body, distinct from promoter silencing.
- The findings highlight a new pathway for epigenetic regulation relevant to cancer biology.
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