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Overexpression of KDM4 lysine demethylases disrupts the integrity of the DNA mismatch repair pathway.

Awwad SW, Ayoub N - Biol Open (2015)

Bottom Line: We show that overexpression of KDM4A-C, but not KDM4D, disrupts MSH6 foci formation during S phase by demethylating its binding site, H3K36me3.Furthermore, we show that the defective MMR in cells overexpressing KDM4C is mainly due to the increase in its demethylase activity and can be mended by KDM4C downregulation.Altogether, our data suggest that cells overexpressing KDM4A-C are defective in DNA MMR and this may contribute to genomic instability and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technion - Israel Institute of Technology, Haifa 3200003, Israel.

No MeSH data available.


Related in: MedlinePlus

Overexpression of KDM4A-C, but not KDM4D, displays MSI phenotype.(A–E) Microsatellite instability (MSI) assay showing the analysis of PCR product patterns of four microsatellite markers in subclones derived from U2OS-TetON expressing EGFP fused to KDM4A (A), KDM4B (B), KDM4C (C), KDM4D (D) and U2OS-TetON control cells (E). To determine the microsatellite stability, genomic DNA was extracted from 20 single clones derived from different single cells of each cell line. The indicated microsatellite markers were then amplified using specific primers pairs, resolved by polyacrylamide-urea electrophoresis and visualized by SYBR-Gold staining. Δ and * show clones exhibiting complete deletion of the tested microsatellite markers or new repeat species, respectively.
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f02: Overexpression of KDM4A-C, but not KDM4D, displays MSI phenotype.(A–E) Microsatellite instability (MSI) assay showing the analysis of PCR product patterns of four microsatellite markers in subclones derived from U2OS-TetON expressing EGFP fused to KDM4A (A), KDM4B (B), KDM4C (C), KDM4D (D) and U2OS-TetON control cells (E). To determine the microsatellite stability, genomic DNA was extracted from 20 single clones derived from different single cells of each cell line. The indicated microsatellite markers were then amplified using specific primers pairs, resolved by polyacrylamide-urea electrophoresis and visualized by SYBR-Gold staining. Δ and * show clones exhibiting complete deletion of the tested microsatellite markers or new repeat species, respectively.

Mentions: It was shown that H3K36me3-MSH6 interaction is essential for intact DNA MMR (Li et al., 2013). We predicted therefore that the removal of H3K36me3 mark following KDM4A-C overexpression should disrupt the integrity of DNA MMR. Given that microsatellite instability (MSI) is a common hallmark of MMR-defective cells (Boland et al., 1998; Bocker et al., 1997; Ellegren, 2004; Hsieh and Yamane, 2008), we sought to test the stability of the mononucleotide (BAT25, BAT26) and the dinucleotide (D2S123, D5S346) microsatellite markers in U2OS-TetON cell lines overexpressing EGFP-KDM4A-D proteins. MSI assay was performed on genomic DNA, which was extracted from 20 clones derived from different single cells of each cell line. Results show that cells overexpressing KDM4A-C, but not KDM4D, exhibit MSI as evidence by the appearance of new repeat species (marked by *) and complete deletions of the tested markers (marked by Δ). As shown in Fig. 2A, 40% (8/20 clones), 55% (11/20) and 30% (6/20) of the clones expressing KDM4A, B and C respectively, show either deletion or novel microsatellite mark. On the other hand, no detectable alterations in the length of the four tested microsatellite markers were obtained in clones overexpressing KDM4D (Fig. 2D) and only one clone shows deletion in the control U2OS-TetON cells (Fig. 2E).


Overexpression of KDM4 lysine demethylases disrupts the integrity of the DNA mismatch repair pathway.

Awwad SW, Ayoub N - Biol Open (2015)

Overexpression of KDM4A-C, but not KDM4D, displays MSI phenotype.(A–E) Microsatellite instability (MSI) assay showing the analysis of PCR product patterns of four microsatellite markers in subclones derived from U2OS-TetON expressing EGFP fused to KDM4A (A), KDM4B (B), KDM4C (C), KDM4D (D) and U2OS-TetON control cells (E). To determine the microsatellite stability, genomic DNA was extracted from 20 single clones derived from different single cells of each cell line. The indicated microsatellite markers were then amplified using specific primers pairs, resolved by polyacrylamide-urea electrophoresis and visualized by SYBR-Gold staining. Δ and * show clones exhibiting complete deletion of the tested microsatellite markers or new repeat species, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400592&req=5

f02: Overexpression of KDM4A-C, but not KDM4D, displays MSI phenotype.(A–E) Microsatellite instability (MSI) assay showing the analysis of PCR product patterns of four microsatellite markers in subclones derived from U2OS-TetON expressing EGFP fused to KDM4A (A), KDM4B (B), KDM4C (C), KDM4D (D) and U2OS-TetON control cells (E). To determine the microsatellite stability, genomic DNA was extracted from 20 single clones derived from different single cells of each cell line. The indicated microsatellite markers were then amplified using specific primers pairs, resolved by polyacrylamide-urea electrophoresis and visualized by SYBR-Gold staining. Δ and * show clones exhibiting complete deletion of the tested microsatellite markers or new repeat species, respectively.
Mentions: It was shown that H3K36me3-MSH6 interaction is essential for intact DNA MMR (Li et al., 2013). We predicted therefore that the removal of H3K36me3 mark following KDM4A-C overexpression should disrupt the integrity of DNA MMR. Given that microsatellite instability (MSI) is a common hallmark of MMR-defective cells (Boland et al., 1998; Bocker et al., 1997; Ellegren, 2004; Hsieh and Yamane, 2008), we sought to test the stability of the mononucleotide (BAT25, BAT26) and the dinucleotide (D2S123, D5S346) microsatellite markers in U2OS-TetON cell lines overexpressing EGFP-KDM4A-D proteins. MSI assay was performed on genomic DNA, which was extracted from 20 clones derived from different single cells of each cell line. Results show that cells overexpressing KDM4A-C, but not KDM4D, exhibit MSI as evidence by the appearance of new repeat species (marked by *) and complete deletions of the tested markers (marked by Δ). As shown in Fig. 2A, 40% (8/20 clones), 55% (11/20) and 30% (6/20) of the clones expressing KDM4A, B and C respectively, show either deletion or novel microsatellite mark. On the other hand, no detectable alterations in the length of the four tested microsatellite markers were obtained in clones overexpressing KDM4D (Fig. 2D) and only one clone shows deletion in the control U2OS-TetON cells (Fig. 2E).

Bottom Line: We show that overexpression of KDM4A-C, but not KDM4D, disrupts MSH6 foci formation during S phase by demethylating its binding site, H3K36me3.Furthermore, we show that the defective MMR in cells overexpressing KDM4C is mainly due to the increase in its demethylase activity and can be mended by KDM4C downregulation.Altogether, our data suggest that cells overexpressing KDM4A-C are defective in DNA MMR and this may contribute to genomic instability and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Technion - Israel Institute of Technology, Haifa 3200003, Israel.

No MeSH data available.


Related in: MedlinePlus