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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

Downregulation of KDM4C restores the integrity of DNA mismatch repair.(A) An outline describing the experimental flow to assess the effect of KDM4C downregulation on the rate of mutation frequency at the HPRT gene. Cells were cultured with doxycycline for 7 days to induce EGFP-KDM4C expression, then the doxycycline was removed to shutdown the expression of EGFP-KDM4C and subsequently the mutation frequency at the HPRT gene was determined. (B) Western blot showing that doxycycline removal suppresses the expression of EGFP-KDM4C fusion. Protein lysates from untreated and doxycycline-treated U2OS-TetON-EGFP-KDM4C cells were immunoblotted using the indicated antibodies. (C) A model showing that KDM4A-C overexpression diminishes H3K36me3 signal, impairs MSH6 foci and impairs the integrity of DNA MMR pathway.
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f04: Downregulation of KDM4C restores the integrity of DNA mismatch repair.(A) An outline describing the experimental flow to assess the effect of KDM4C downregulation on the rate of mutation frequency at the HPRT gene. Cells were cultured with doxycycline for 7 days to induce EGFP-KDM4C expression, then the doxycycline was removed to shutdown the expression of EGFP-KDM4C and subsequently the mutation frequency at the HPRT gene was determined. (B) Western blot showing that doxycycline removal suppresses the expression of EGFP-KDM4C fusion. Protein lysates from untreated and doxycycline-treated U2OS-TetON-EGFP-KDM4C cells were immunoblotted using the indicated antibodies. (C) A model showing that KDM4A-C overexpression diminishes H3K36me3 signal, impairs MSH6 foci and impairs the integrity of DNA MMR pathway.

Mentions: Here, we asked whether the defective MMR in cell overexpressing KDM4C could be repaired by KDM4C downregulation. To address this question, we treated cells with doxycycline for five days to trigger EGFP-KDM4C overexpression and disrupt DNA MMR. Next, EGFP-KDM4C expression was shutdown by doxycycline removal and cells were subjected to HPRT mutability assay (Fig. 4A). To validate the shutting down of EGFP-KDM4C expression, we performed western blot before and after doxycycline removal (Fig. 4B). The HPRT mutability showed that the removal of doxycycline leads to a decrease of ∼26 fold in the mutation frequency comparing to cells overexpressing KDM4C protein (Table 1). This result strongly suggests that KDM4C downregulation restores the integrity of DNA MMR. Given that Microsatellite-stable (MSS) and MSI tumors display distinct responses to certain antitumor agents (Merok et al., 2013; Ribic et al., 2003; Vilar et al., 2008; Vilar and Tabernero, 2013), we speculate that small molecule inhibitors of KDM4C may restore the integrity of DNA MMR and alter the sensitivity to anticancer drugs.


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

Awwad SW, Ayoub N - Biol Open (2015)

Downregulation of KDM4C restores the integrity of DNA mismatch repair.(A) An outline describing the experimental flow to assess the effect of KDM4C downregulation on the rate of mutation frequency at the HPRT gene. Cells were cultured with doxycycline for 7 days to induce EGFP-KDM4C expression, then the doxycycline was removed to shutdown the expression of EGFP-KDM4C and subsequently the mutation frequency at the HPRT gene was determined. (B) Western blot showing that doxycycline removal suppresses the expression of EGFP-KDM4C fusion. Protein lysates from untreated and doxycycline-treated U2OS-TetON-EGFP-KDM4C cells were immunoblotted using the indicated antibodies. (C) A model showing that KDM4A-C overexpression diminishes H3K36me3 signal, impairs MSH6 foci and impairs the integrity of DNA MMR pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f04: Downregulation of KDM4C restores the integrity of DNA mismatch repair.(A) An outline describing the experimental flow to assess the effect of KDM4C downregulation on the rate of mutation frequency at the HPRT gene. Cells were cultured with doxycycline for 7 days to induce EGFP-KDM4C expression, then the doxycycline was removed to shutdown the expression of EGFP-KDM4C and subsequently the mutation frequency at the HPRT gene was determined. (B) Western blot showing that doxycycline removal suppresses the expression of EGFP-KDM4C fusion. Protein lysates from untreated and doxycycline-treated U2OS-TetON-EGFP-KDM4C cells were immunoblotted using the indicated antibodies. (C) A model showing that KDM4A-C overexpression diminishes H3K36me3 signal, impairs MSH6 foci and impairs the integrity of DNA MMR pathway.
Mentions: Here, we asked whether the defective MMR in cell overexpressing KDM4C could be repaired by KDM4C downregulation. To address this question, we treated cells with doxycycline for five days to trigger EGFP-KDM4C overexpression and disrupt DNA MMR. Next, EGFP-KDM4C expression was shutdown by doxycycline removal and cells were subjected to HPRT mutability assay (Fig. 4A). To validate the shutting down of EGFP-KDM4C expression, we performed western blot before and after doxycycline removal (Fig. 4B). The HPRT mutability showed that the removal of doxycycline leads to a decrease of ∼26 fold in the mutation frequency comparing to cells overexpressing KDM4C protein (Table 1). This result strongly suggests that KDM4C downregulation restores the integrity of DNA MMR. Given that Microsatellite-stable (MSS) and MSI tumors display distinct responses to certain antitumor agents (Merok et al., 2013; Ribic et al., 2003; Vilar et al., 2008; Vilar and Tabernero, 2013), we speculate that small molecule inhibitors of KDM4C may restore the integrity of DNA MMR and alter the sensitivity to anticancer drugs.

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