The emerging role of lysine demethylases in DNA damage response: dissecting the recruitment mode of KDM4D/JMJD2D to DNA damage sites

Abstract

KDM4D is a lysine demethylase that removes tri- and di- methylated residues from H3K9 and is involved in transcriptional regulation and carcinogenesis. We recently showed that KDM4D is recruited to DNA damage sites in a PARP1-dependent manner and facilitates double-strand break repair in human cells. Moreover, we demonstrated that KDM4D is an RNA binding protein and mapped its RNA-binding motifs. Interestingly, KDM4D-RNA interaction is essential for its localization on chromatin and subsequently for efficient demethylation of its histone substrate H3K9me3. Here, we provide new data that shed mechanistic insights into KDM4D accumulation at DNA damage sites. We show for the first time that KDM4D binds poly(ADP-ribose) (PAR) in vitro via its C-terminal region. In addition, we demonstrate that KDM4D-RNA interaction is required for KDM4D accumulation at DNA breakage sites. Finally, we discuss the recruitment mode and the biological functions of additional lysine demethylases including KDM4B, KDM5B, JMJD1C, and LSD1 in DNA damage response.

Keywords: DNA damage response (DDR); KDM4D; PARP1; and cancer; chromosomal instability; double strand break (DSB); lysine demethylases (KDM); poly(ADP-ribose)ylation (PARylation).

Figure 1.

KDM4D region spanning 350–474 amino acids binds PAR in vitro. PAR-binding assay with 6xHis tagged KDM4D full-length (FL) protein (523aa) (A), deletion mutants: N-terminal (1–350aa) (B), GST-tagged C-terminal (350–523aa) (C), truncated C-terminal (1–474aa) and internal deletion 350–474aa (D), and 6xHis tagged KDM4D-4M mutant (contains 4 mutations: E357A, R450A, R451A and R455A) (E). 6xHis-Rpn8, GST-only and BSA are used as negative controls and H3 as a positive control. Right: schematic representation of KDM4D mutants. IB: Immunoblot. ³²P: radiolabelled PAR.

Figure 2.

KDM4D PAR-binding region is essential for its recruitment to laser-microirradiated sites. Representative cells showing the localization of EGFP-KDM4D-WT EGFP-KDM4D^1-474aa and EGFP-KDM4D^Δ350-474aa fusions before and 5 minutes after the induction of laser-microirradiation to a single region, marked with a white arrow. Each cell is representative of at least 20 different cells. The graph shows the increase in the relative fluorescence intensity of KDM4D fusions at laser-microirradiated sites. Error bars represent SD of 10 different cells.

Figure 3.

KDM4D-RNA interactions are essential for KDM4D accumulation at DNA damage sites. Representative cells showing the localization of EGFP-KDM4D-WT and EGFP-KDM4D-1H4R-HRK fusions before and 5 minutes after the induction of laser-microirradiation to a single region, marked by a white arrow. Each cell is representative of at least 20 cells. The graph shows the increase in the relative fluorescence intensity of EGFP-KDM4D-WT and EGFP-KDM4D-1H4K-HRK at laser-microirradiated sites. Error bars represent SD of 10 different cells.