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

Abstract

The KDM4 family of lysine demethylases consists of five members, KDM4A, -B and -C that demethylate H3K9me2/3 and H3K36me2/3 marks, while KDM4D and -E demethylate only H3K9me2/3. Recent studies implicated KDM4 proteins in regulating genomic instability and carcinogenesis. Here, we describe a previously unrecognized pathway by which hyperactivity of KDM4 demethylases promotes genomic instability. We show that overexpression of KDM4A-C, but not KDM4D, disrupts MSH6 foci formation during S phase by demethylating its binding site, H3K36me3. Consequently, we demonstrate that cells overexpressing KDM4 members are defective in DNA mismatch repair (MMR), as evident by the instability of four microsatellite markers and the remarkable increase in the spontaneous mutations frequency at the HPRT locus. 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.

Keywords: Chromosomal instability; DNA damage; KDM4 proteins; Lysine demethylation; Mismatch repair.

Fig. 1.. 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).

Fig. 2.. 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.

Fig. 3.. 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.

Fig. 4.. 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.