Cross-talk between the H3K36me3 and H4K16ac histone epigenetic marks in DNA double-strand break repair

Abstract

Post-translational modifications of histone proteins regulate numerous cellular processes. Among these modifications, trimethylation of lysine 36 in histone H3 (H3K36me3) and acetylation of lysine 16 in histone H4 (H4K16ac) have important roles in transcriptional regulation and DNA damage response signaling. However, whether these two epigenetic histone marks are mechanistically linked remains unclear. Here we discovered a new pathway through which H3K36me3 stimulates H4K16ac upon DNA double-strand break (DSB) induction in human cells. In particular, we examined, using Western blot analysis, the levels of H3K36me3 and H4K16ac in cells after exposure to various DSB-inducing agents, including neocarzinostatin, γ rays, and etoposide, and found that H3K36me3 and H4K16ac were both elevated in cells upon these treatments. We also observed that DSB-induced H4K16 acetylation was abolished in cells upon depletion of the histone methyltransferase gene SET-domain containing 2 (SETD2) and the ensuing loss of H3K36me3. Furthermore, the H3K36me3-mediated increase in H4K16ac necessitated lens epithelium-derived growth factor p75 splicing variant (LEDGF), which is a reader protein of H3K36me3, and the KAT5 (TIP60) histone acetyltransferase. Mechanistically, the chromatin-bound LEDGF, through its interaction with KAT5, promoted chromatin localization of KAT5, thereby stimulating H4K16 acetylation. In this study, we unveiled cross-talk between two important histone epigenetic marks and defined the function of this cross-talk in DNA DSB repair.

Keywords: DNA repair; epigenetics; histone acetylation; histone methylation; posttranslational modification (PTM).

Figure 1.

Exposure to DNA DSB–inducing agents led to elevated levels of H3K36me3 and H4K16ac, and SETD2^−/− cells exhibited a decreased level of H4K16ac. A, the levels of H3K36me3 and H4K16ac were decreased in SETD2^−/− cells, where histones H3 and H4 were used as references, respectively. B, quantification of the Western blot results showed diminished H3K36me3 and H4K16ac levels in SETD2^−/− cells. **, p < 0.01. The p values were calculated using two-tailed, unpaired Student's t test. C–F, time course experiments showed a transient elevation in the levels of γH2AX, H3K36me3, and H4K16ac in HEK293T (293T) cells after treatment with 100 ng/ml NCS, where histones H4, H3, and H4 were used as references, respectively. The NCS-induced increase in H4K16ac was abolished in SETD2^−/− cells. The quantification results displayed in D–F represent the mean and S.E. of results obtained from three independent biological replicates conducted on 3 separate days for all experiments, except that the time course experiment for H3K36me3 was conducted in five independent biological replicates performed on 5 separate days (see supplemental Fig. S2 for Western blot images obtained from other biological replicates). G and H, the chromatin localization of KAT5, but not hMOF, was increased in HEK293T cells after NCS treatment, and this increase in chromatin binding of KAT5 was lost in SETD2^−/− cells. Actin and histone H3 were employed as loading controls for the soluble fraction (SF) and chromatin fraction (CF) fractions, respectively. The relative values below the lanes were calculated from the ratios of band intensities of CF over SF, which were normalized against histone H3 and actin, respectively.

Figure 2.

Temporal responses of H3K36me3 and H4K16ac levels in different cell lines following DNA DSB induction. A–C, time course experiments revealed the dynamic changes in H3K36me3 and H4K16ac levels following NCS treatment. Shown are the Western blot results (A) and the quantification data for the relative levels of H3K36me3 and H4K16ac (B and C). The relative levels of these two histone modifications were calculated using histones H3 and H4 as references, respectively. The quantification data represent the mean and S.E. of results obtained from three independent biological replicates for all experiments, with the exception that the time course experiment for H3K36me3 was conducted in five independent biological replicates; each biological replicate was conducted on a separate day. Western blot images for data obtained from other biological replicates for LEDGF^−/− and KAT5^−/− cells are shown in supplemental Fig. S5.

Figure 3.

LEDGF facilitates the recruitment of KAT5 to chromatin following DNA DSB induction. A, FLAG-tagged LEDGF, but not LEDGF-Y18AW21A, could pull down histone H3 and H3K36me3. IP, immunoprecipitation. B, chromatin localization of LEDGF protein in HEK293T and SETD2^−/− cells. Knockout of SETD2 confers diminished chromatin localization of LEDGF. C and D, the chromatin localization of C-terminally FLAG-tagged KAT5 (KAT5-FLAG), but not hMOF, was increased in HEK293T cells upon NCS treatment, and this increase in chromatin binding of KAT5-FLAG was abolished in LEDGF^−/− cells. Actin and histone H3 were employed as loading controls for the SF and chromatin CF, respectively. The values below the lanes were calculated from the band intensities normalized against histone H3. E and F, reciprocal pulldown experiments revealed the interaction between LEDGF and KAT5. KAT5-HA and LEDGF-FLAG were co-transfected into cells, and pulldown experiments were conducted using anti-HA beads or anti-FLAG beads. Results from another biological replicate is shown in supplemental Fig. S8, F and G. G and H, reciprocal pulldown experiments revealed the interaction between endogenous LEDGF and ectopically expressed HA-KAT5. Results from another biological replicate are shown in supplemental Fig. S8, H and I. The nonspecific band in H was labeled with an asterisk. LEDGF Ab, LEDGF antibody used for immunoprecipitation.

Figure 4.

Changes in H3K36me3 and H4K16ac levels around a site-specifically generated DNA DSB site, and the modulations of the levels of these two histone modifications by SETD2 or LEDGF. A, schematic showing the locations of two sets of target fragments used in the ChIP assay. The numbers below indicate the numbers of base pairs between the target fragments and the I-SceI cleavage site. B and C, the relative occupancies (enrichment) of H3K36me3 (B) and H4K16ac (C) as revealed by ChIP assay. U2OS cells were transfected with or without siRNA targeting SETD2 or LEDGF gene for 48 h. The I-SceI plasmid was subsequently transfected into cells, and the cells were harvested 4 h following transfection. Cells without I-SceI transfection were used as negative control. For ChIP experiments, the relative enrichment was calculated by comparing the antibody binding results with the amounts of input DNA, and each value was normalized against the negative control without I-SceI transfection. The data represent the mean and S.E. of results from three biological replicates. D, a model showing the H3K36me3-H4K16ac cross-talk in response to DNA DSB induction and the involvement of LEDGF and KAT5 in this cross-talk.