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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

KDM4C over-activity disrupts DNA MMR.(A) Western blot analysis showing that overexpression of EGFP-KDM4C-S198M has no detectable effect on the levels of H3K36me3. Protein extracts were prepared from U2OS-TetON cells expressing either EGFP-KDM4C-WT or EGFP-KDM4C-S198M and immunoblotted using the indicated antibodies. (B) MSI assay was performed as described in Fig. 2 except that genomic DNA was extracted from 20 single clones derived from U2OS-TetON cells expressing KDM4C-S198M. Results show that only one clone out of 20 clones shows instability in a single marker. Δ shows clones exhibiting complete deletion of the tested microsatellite markers.
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f03: KDM4C over-activity disrupts DNA MMR.(A) Western blot analysis showing that overexpression of EGFP-KDM4C-S198M has no detectable effect on the levels of H3K36me3. Protein extracts were prepared from U2OS-TetON cells expressing either EGFP-KDM4C-WT or EGFP-KDM4C-S198M and immunoblotted using the indicated antibodies. (B) MSI assay was performed as described in Fig. 2 except that genomic DNA was extracted from 20 single clones derived from U2OS-TetON cells expressing KDM4C-S198M. Results show that only one clone out of 20 clones shows instability in a single marker. Δ shows clones exhibiting complete deletion of the tested microsatellite markers.

Mentions: We show that the decrease in H3K36me3 levels following KDM4A-C overexpression impairs MSH6 foci formation and disrupts the integrity of DNA MMR (Figs 1, 2 and Table 1). These findings suggest that the levels of H3K36me3 have a critical role in regulating DNA MMR pathway. To further validate this, we monitored the integrity of MMR in cells overexpressing KDM4C demethylase-dead mutant. Previously, we have established a catalytically inert KDM4C-S198M mutant that does not demethylate H3K9me3 (Kupershmit et al., 2014). Here, we validated by western blot that KDM4C-S198M overexpression also has no effect on the levels of H3K36me3 (Fig. 3A). To assess the integrity of MMR, cells overexpressing KDM4C-S198M were subjected to MSI and HPRT mutability assays. The MSI results show no MSI-H clones and only one out of the 20 tested clones overexpressing KDM4C-S198M shows complete deletion of BAT25 marker (Fig. 3B). In addition, the HPRT mutability assay shows that the mutation frequency in cells overexpressing KDM4C-S198M is ∼7 fold less than in cells overexpressing KDM4C-WT (Table 1). Nonetheless, KDM4C-S198M overexpression shows significant increase in the mutation frequency (∼75 fold) at the HPRT locus comparing to U2OS-TetON cells. Altogether, our findings confirm that the defective DNA MMR in cells overexpressing KDM4 results mainly from the increase in the demethylase activity. However, overexpression of the catalytically inert mutant has also a relatively minor effect on the integrity of DNA MMR. This result suggests that one mechanism by which KDM4C-S198M interferes with MMR might be by competing with MSH6 on binding to H3K36me3.


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

Awwad SW, Ayoub N - Biol Open (2015)

KDM4C over-activity disrupts DNA MMR.(A) Western blot analysis showing that overexpression of EGFP-KDM4C-S198M has no detectable effect on the levels of H3K36me3. Protein extracts were prepared from U2OS-TetON cells expressing either EGFP-KDM4C-WT or EGFP-KDM4C-S198M and immunoblotted using the indicated antibodies. (B) MSI assay was performed as described in Fig. 2 except that genomic DNA was extracted from 20 single clones derived from U2OS-TetON cells expressing KDM4C-S198M. Results show that only one clone out of 20 clones shows instability in a single marker. Δ shows clones exhibiting complete deletion of the tested microsatellite markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400592&req=5

f03: KDM4C over-activity disrupts DNA MMR.(A) Western blot analysis showing that overexpression of EGFP-KDM4C-S198M has no detectable effect on the levels of H3K36me3. Protein extracts were prepared from U2OS-TetON cells expressing either EGFP-KDM4C-WT or EGFP-KDM4C-S198M and immunoblotted using the indicated antibodies. (B) MSI assay was performed as described in Fig. 2 except that genomic DNA was extracted from 20 single clones derived from U2OS-TetON cells expressing KDM4C-S198M. Results show that only one clone out of 20 clones shows instability in a single marker. Δ shows clones exhibiting complete deletion of the tested microsatellite markers.
Mentions: We show that the decrease in H3K36me3 levels following KDM4A-C overexpression impairs MSH6 foci formation and disrupts the integrity of DNA MMR (Figs 1, 2 and Table 1). These findings suggest that the levels of H3K36me3 have a critical role in regulating DNA MMR pathway. To further validate this, we monitored the integrity of MMR in cells overexpressing KDM4C demethylase-dead mutant. Previously, we have established a catalytically inert KDM4C-S198M mutant that does not demethylate H3K9me3 (Kupershmit et al., 2014). Here, we validated by western blot that KDM4C-S198M overexpression also has no effect on the levels of H3K36me3 (Fig. 3A). To assess the integrity of MMR, cells overexpressing KDM4C-S198M were subjected to MSI and HPRT mutability assays. The MSI results show no MSI-H clones and only one out of the 20 tested clones overexpressing KDM4C-S198M shows complete deletion of BAT25 marker (Fig. 3B). In addition, the HPRT mutability assay shows that the mutation frequency in cells overexpressing KDM4C-S198M is ∼7 fold less than in cells overexpressing KDM4C-WT (Table 1). Nonetheless, KDM4C-S198M overexpression shows significant increase in the mutation frequency (∼75 fold) at the HPRT locus comparing to U2OS-TetON cells. Altogether, our findings confirm that the defective DNA MMR in cells overexpressing KDM4 results mainly from the increase in the demethylase activity. However, overexpression of the catalytically inert mutant has also a relatively minor effect on the integrity of DNA MMR. This result suggests that one mechanism by which KDM4C-S198M interferes with MMR might be by competing with MSH6 on binding to H3K36me3.

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