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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, proteins impairs MSH6 foci formation during S-phase.(A) Western blot analysis shows the levels of EGFP-KDM4A-C fusions in U2OS-TetON cell lines in comparison to the levels of the endogenous KDM4A-C proteins in MCF7 cell line. Protein extracts were prepared from MCF7 cell line and from doxycycline-treated U2OS-TetON cells expressing EGFP-KDM4A-C fusions and immunoblotted using the indicated antibodies. β-actin is used as a loading control. EGFP-KDM4A-C fusions and the endogenous KDM4A-C proteins are indicated by arrowheads and stars, respectively. (B–E) Shows that overexpression of EGFP-KDM4A-C, but not EGFP-KDM4D, catalyzes the removal of H3K36me3 methylation and impairs MSH6 foci formation during S phase. Cells were fixed and subjected to immunofluorescence analysis using antibodies against MSH6 (red) and H3k36me3 (gray). DNA is stained with DAPI (blue) and EGFP-KDM4A-D fusions are in green. Results shown in (B–E) are typical of two independent experiments and represent at least 30 different cells each. (F) Graph shows the number of MSH6 foci in untransfected U2OS cells and in U2OS cells expressing EGFP-KDM4A-D fusions (n = 30 cells). Foci were counted by eye. Error bars represent SD from two independent experiments. Scale bars = 10 µm (B,C,E); 5 µm (D).
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f01: Overexpression of KDM4A-C, but not KDM4D, proteins impairs MSH6 foci formation during S-phase.(A) Western blot analysis shows the levels of EGFP-KDM4A-C fusions in U2OS-TetON cell lines in comparison to the levels of the endogenous KDM4A-C proteins in MCF7 cell line. Protein extracts were prepared from MCF7 cell line and from doxycycline-treated U2OS-TetON cells expressing EGFP-KDM4A-C fusions and immunoblotted using the indicated antibodies. β-actin is used as a loading control. EGFP-KDM4A-C fusions and the endogenous KDM4A-C proteins are indicated by arrowheads and stars, respectively. (B–E) Shows that overexpression of EGFP-KDM4A-C, but not EGFP-KDM4D, catalyzes the removal of H3K36me3 methylation and impairs MSH6 foci formation during S phase. Cells were fixed and subjected to immunofluorescence analysis using antibodies against MSH6 (red) and H3k36me3 (gray). DNA is stained with DAPI (blue) and EGFP-KDM4A-D fusions are in green. Results shown in (B–E) are typical of two independent experiments and represent at least 30 different cells each. (F) Graph shows the number of MSH6 foci in untransfected U2OS cells and in U2OS cells expressing EGFP-KDM4A-D fusions (n = 30 cells). Foci were counted by eye. Error bars represent SD from two independent experiments. Scale bars = 10 µm (B,C,E); 5 µm (D).

Mentions: KDM4A-C proteins, but not KDM4D, demethylate H3K36me3 mark as we and others have shown (Couture et al., 2007; Hillringhaus et al., 2011; Klose et al., 2006b; Kupershmit et al., 2014; Shin and Janknecht, 2007b; Whetstine et al., 2006). H3K36me3 is involved in DNA MMR as it provides a binding site for the MMR protein MSH6 and enables MSH6 foci formation during S phase (Li et al., 2013). Therefore, we sought to assess whether overexpression of KDM4A-C proteins affects MSH6 foci during S phase. Toward this end, we used U2OS-TetON cell lines that conditionally express functional EGFP-KDM4A-C fusions upon the addition of doxycycline (Ipenberg et al., 2013; Kupershmit et al., 2014). Importantly, the expression levels of EGFP-KDM4A-C fusions are comparable to the levels of the endogenous KDM4A-C proteins found in human breast adenocarcinoma cell line, MCF7, known to have elevated levels of KDM4 proteins (Berry and Janknecht, 2013; Berry et al., 2012) (Fig. 1A). The cells were synchronized at G1/S border using double-thymidine block; samples were collected at 3 hr after the removal of thymidine and subjected to both fluorescence-activated cell sorter (FACS) and immunofluorescence (IF). Results show that 3 hr after thymidine removal the majority of the cells (83%) were at S phase (supplementary material Fig. S1). IF analysis shows that overexpression of EGFP-KDM4A-C fusions (green) diminished the intensity of H3K36me3 signal (gray) and impaired MSH6 foci during S phase (red) (Fig. 1B–D). On the other hand, U2OS-TetON cells expressing EGFP-KDM4D fusion (Khoury-Haddad et al., 2014), which does not demethylate H3K36me3, show no detectable effect on H3K36me3 levels and MSH6 foci (Fig. 1E). Quantitative measurements of the MSH6 foci reveal that KDM4A-C overexpression leads to 6–9 fold decrease comparing to control U2OS cell line or cells overexpressing KDM4D (Fig. 1F). We concluded therefore that EGFP-KDM4A-C overexpression leads to a dramatic reduction in H3K36me3 and impairs MSH6 foci during the S phase of the cell cycle. Our results are consistent with a recent report showing that MSH6 foci during S phase is impaired following the inhibition of H3K36me3 methylation by knocking down SETD2 methyltransferase (Li et al., 2013).


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, proteins impairs MSH6 foci formation during S-phase.(A) Western blot analysis shows the levels of EGFP-KDM4A-C fusions in U2OS-TetON cell lines in comparison to the levels of the endogenous KDM4A-C proteins in MCF7 cell line. Protein extracts were prepared from MCF7 cell line and from doxycycline-treated U2OS-TetON cells expressing EGFP-KDM4A-C fusions and immunoblotted using the indicated antibodies. β-actin is used as a loading control. EGFP-KDM4A-C fusions and the endogenous KDM4A-C proteins are indicated by arrowheads and stars, respectively. (B–E) Shows that overexpression of EGFP-KDM4A-C, but not EGFP-KDM4D, catalyzes the removal of H3K36me3 methylation and impairs MSH6 foci formation during S phase. Cells were fixed and subjected to immunofluorescence analysis using antibodies against MSH6 (red) and H3k36me3 (gray). DNA is stained with DAPI (blue) and EGFP-KDM4A-D fusions are in green. Results shown in (B–E) are typical of two independent experiments and represent at least 30 different cells each. (F) Graph shows the number of MSH6 foci in untransfected U2OS cells and in U2OS cells expressing EGFP-KDM4A-D fusions (n = 30 cells). Foci were counted by eye. Error bars represent SD from two independent experiments. Scale bars = 10 µm (B,C,E); 5 µm (D).
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f01: Overexpression of KDM4A-C, but not KDM4D, proteins impairs MSH6 foci formation during S-phase.(A) Western blot analysis shows the levels of EGFP-KDM4A-C fusions in U2OS-TetON cell lines in comparison to the levels of the endogenous KDM4A-C proteins in MCF7 cell line. Protein extracts were prepared from MCF7 cell line and from doxycycline-treated U2OS-TetON cells expressing EGFP-KDM4A-C fusions and immunoblotted using the indicated antibodies. β-actin is used as a loading control. EGFP-KDM4A-C fusions and the endogenous KDM4A-C proteins are indicated by arrowheads and stars, respectively. (B–E) Shows that overexpression of EGFP-KDM4A-C, but not EGFP-KDM4D, catalyzes the removal of H3K36me3 methylation and impairs MSH6 foci formation during S phase. Cells were fixed and subjected to immunofluorescence analysis using antibodies against MSH6 (red) and H3k36me3 (gray). DNA is stained with DAPI (blue) and EGFP-KDM4A-D fusions are in green. Results shown in (B–E) are typical of two independent experiments and represent at least 30 different cells each. (F) Graph shows the number of MSH6 foci in untransfected U2OS cells and in U2OS cells expressing EGFP-KDM4A-D fusions (n = 30 cells). Foci were counted by eye. Error bars represent SD from two independent experiments. Scale bars = 10 µm (B,C,E); 5 µm (D).
Mentions: KDM4A-C proteins, but not KDM4D, demethylate H3K36me3 mark as we and others have shown (Couture et al., 2007; Hillringhaus et al., 2011; Klose et al., 2006b; Kupershmit et al., 2014; Shin and Janknecht, 2007b; Whetstine et al., 2006). H3K36me3 is involved in DNA MMR as it provides a binding site for the MMR protein MSH6 and enables MSH6 foci formation during S phase (Li et al., 2013). Therefore, we sought to assess whether overexpression of KDM4A-C proteins affects MSH6 foci during S phase. Toward this end, we used U2OS-TetON cell lines that conditionally express functional EGFP-KDM4A-C fusions upon the addition of doxycycline (Ipenberg et al., 2013; Kupershmit et al., 2014). Importantly, the expression levels of EGFP-KDM4A-C fusions are comparable to the levels of the endogenous KDM4A-C proteins found in human breast adenocarcinoma cell line, MCF7, known to have elevated levels of KDM4 proteins (Berry and Janknecht, 2013; Berry et al., 2012) (Fig. 1A). The cells were synchronized at G1/S border using double-thymidine block; samples were collected at 3 hr after the removal of thymidine and subjected to both fluorescence-activated cell sorter (FACS) and immunofluorescence (IF). Results show that 3 hr after thymidine removal the majority of the cells (83%) were at S phase (supplementary material Fig. S1). IF analysis shows that overexpression of EGFP-KDM4A-C fusions (green) diminished the intensity of H3K36me3 signal (gray) and impaired MSH6 foci during S phase (red) (Fig. 1B–D). On the other hand, U2OS-TetON cells expressing EGFP-KDM4D fusion (Khoury-Haddad et al., 2014), which does not demethylate H3K36me3, show no detectable effect on H3K36me3 levels and MSH6 foci (Fig. 1E). Quantitative measurements of the MSH6 foci reveal that KDM4A-C overexpression leads to 6–9 fold decrease comparing to control U2OS cell line or cells overexpressing KDM4D (Fig. 1F). We concluded therefore that EGFP-KDM4A-C overexpression leads to a dramatic reduction in H3K36me3 and impairs MSH6 foci during the S phase of the cell cycle. Our results are consistent with a recent report showing that MSH6 foci during S phase is impaired following the inhibition of H3K36me3 methylation by knocking down SETD2 methyltransferase (Li et al., 2013).

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