Phosphorylation of TIP60 Suppresses 53BP1 Localization at DNA Damage Sites

Abstract

A proper balance between the repair of DNA double-strand breaks (DSBs) by homologous recombination and nonhomologous end joining is critical for maintaining genome integrity and preventing tumorigenesis. This balance is regulated and fine-tuned by a variety of factors, including cell cycle and the chromatin environment. The histone acetyltransferase TIP60 was previously shown to suppress pathological end joining and promote homologous recombination. However, it is unknown how regulatory posttranslational modifications impact TIP60 acetyltransferase activity to influence the outcome of DSB responses. In this study, we report that phosphorylation of TIP60 on serines 90 and 86 is important for limiting the accumulation of the pro-end joining factor 53BP1 at DSBs in S and G₂ cell cycle phases. Mutation of these sites disrupts histone acetylation changes in response to DNA damage, BRCA1 localization to DSBs, and poly(ADP-ribose) polymerase (PARP) inhibitor resistance. These findings reveal that phosphorylation directs TIP60-dependent acetylation to promote homologous recombination and maintain genome stability.

Keywords: 53BP1; BRCA1; DNA repair; TIP60; acetylation; homologous recombination.

FIG 1

Loss of TIP60 increases 53BP1 localization at DSBs and end joining. (A) Schematic of TIP60 domains and loxP sites on the Tip60 fl allele. For the targeting scheme, see reference . Functional domains are represented as color-filled rectangles. Numbers above the diagram correspond to amino acid positions. CD, chromodomain; Zn, zinc finger; CoA, acetyl coenzyme A-binding domain. (B) Western blot of TIP60 in Tip60 f/f, ERT2-Cre MEFs with no treatment (No tx) versus after 2 to 3 days of 4-hydroxytamoxifen (4′OHT) treatment. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is used as the loading control. (C) Plating efficiency of indicated MEFs in the absence of damage as measured by clonogenic assay. cKO, TIP60 conditional knockout by OHT treatment. n = 15. (D) Immunofluorescence images of 53BP1 and BRCA1 protein localization at 5 h following 10-Gy ionizing radiation (IR) in MEFs. Bar, 10 μm. (E) Quantification of percentage of cells with more than 5 foci at different time points post-IR. n = 3. (F) Survival of MEFs treated with different doses of the PARP inhibitor olaparib as measured by clonogenic assay. n = 4. ****, P < 0.0001. (G) Photomicrographs illustrating chromosomal breaks and fusions on MEF metaphase spreads. (H) Quantification of chromosomal abnormalities on metaphase spreads after olaparib treatment. n = 5. For panels C, E, F, and H, error bars show SEM.

FIG 2

Serines 90 and 86 on TIP60 regulate DNA repair. (A) Representative cell cycle FLOW profiles of Tip60 f/f, ERT2-Cre MEFs at 2 to 3 days following no treatment versus OHT treatment. Green fill-in, G₁; yellow-green, S; blue, G₂. (B) Quantification of percentage of cells in each phase of cell cycle. n = 8. (C) Western blots of whole-cell lysate from Tip60 f/f, ERT2-Cre MEFs stably expressing FLAG-HA (FH)-tagged empty vector, wild-type TIP60, or mutant human TIP60. EV, empty vector; WT, wild type. Endogenous TIP60 (endo) and FLAG-HA-tagged exogenous TIP60 (tagged) are indicated. (D) Representative MEF cell cycle FLOW profiles at day 2 post-OHT addition (top panels) and day 5 post-OHT addition (bottom panels). Green fill-in, G₁; yellow-green, S; blue, G₂; uncolored, aneuploid. (E) Quantification of cell cycle distributions at day 2 post-OHT addition (top panel) and day 5 post-OHT addition (bottom panel). n = 3. (F) Survival of MEFs after different doses of IR as measured by clonogenic assay. DNA-PK inhibitor-treated cells are used as a negative control. n = 3. For panels B, E, and F, error bars show SEM.

FIG 3

Phosphorylation of TIP60 S90 and S86 promotes HR repair. (A) Western blots of Tip60-cKO MEFs complemented with FLAG-HA-tagged TIP60 variants 5 h after treatment with damaging agent etoposide. HD, HAT-deficient Q377E/G380E mutant. Phosphorylated KAP1 is used as a marker of ATM activity. (B) Quantification of 53BP1 focus formation 5 h after IR. n = 3. (C) Quantification of BRCA1 focus formation 5 h after IR. n = 3. (D) Survival of MEFs treated with different doses of olaparib as measured by clonogenic assay. The same set of data is broken up into two different graphs for presentation clarity. n = 3. (E) Quantification of abnormal fusion events on MEF metaphase spreads. For panels B to E, error bars show SEM.

FIG 4

TIP60 S90 regulates 53BP1 localization during S-G₂. (A) Western blots of pS90 and pS86 on the FLAG-HA-tagged WT TIP60 after 5 h of damage induction with etoposide. p-KAP1 is used as a marker of damage. (B) Western blots of pS90 and pS86 on FLAG-HA-tagged WT TIP60 in MEFs treated with 10 μM CDK1 inhibitor RO-3306 for various lengths of time. (C) Western blots of pS90 and pS86 on FLAG-HA-tagged WT TIP60 in MEFs treated with 2 μM CDK9 inhibitor SNS-032 for various lengths of time. p-Pol II CTD is used as a readout of CDK9 inhibition. (D) FLOW plots of cell cycle distributions of EdU⁻ and EdU⁺ MEFs to illustrate enrichment in G₂ of our scheme entailing EdU labeling and then wash-off. On the y axis, FL4-H indicates the intensity of EdU label (after conjugation with Alexa Fluor 647); on the x axis, FL2-H indicates propidium iodide (PI) stain intensity. Left panel, untreated cells; middle panel, cells have been labeled with EdU for 1 h with no wash-off; right panel, cells were EdU labeled for 1 h, washed, and then collected 3 h after wash-off. The labeling scheme corresponding to the right panel (highlighted by a red box) is the one that we employed for the experiments in panels E to G. Density is indicated by colors: red (highest density) > yellow > green > blue (lowest density). (E) Immunofluorescence images of 53BP1 ionizing radiation-induced focus (IRIF) formation in EdU⁺ (late S and G₂) versus EdU⁻ MEFs 5 h after IR and 3 h after EdU wash-off. Arrowheads point to EdU⁺ cells that have many bright 53BP1 foci. Bar, 10 μm. (F) ImageJ particle analysis quantification of 53BP1 foci per cell and relative mean focal intensity (RMFI) in EdU⁺ versus EdU⁻ cells. The dotted line is set to the mean in EdU⁺ control cells. n = 3. (G) ImageJ particle analysis quantification of BRCA1 foci per cell and relative mean focal intensity (RMFI) in EdU⁺ cells versus EdU⁻ cells. The dashed lines are set to the means in EdU⁺ control cells. n = 3. For panels F and G, error bars show SEM. *, P < 0.05; **, P < 0.01.

FIG 5

TIP60 S90 and S86 are dispensable for viability, complex formation, and recruitment to damage sites. (A) Plating efficiency of undamaged Tip60-cKO MEFs complemented with empty vector, wild-type TIP60, or mutant TIP60 as measured by clonogenic assay. n = 3. (B) Western blot of proteins immunoprecipitated with the indicated FLAG-HA-tagged constructs from HeLa S3 cells. (C) Table of analyses of mass spectrometry-identified canonical TIP60-p400 complex members copurified with FLAG-HA-tagged WT versus S90A TIP60 from HeLa S3 cells. Negative average log₂ (S90A⁺/WT⁺) intensity means that the S90A mutant exhibits decreased association with the indicated complex partner compared to the WT. n = 3. (D) Schematic of mCherry-LacI-FokI damage induction by addition of Shield1 and 4′OHT (8). A LacO-transgene array is stably integrated into the U2OS genome. mCherry-LacI-FokI is constantly degraded at baseline, stabilized by the Shield1 ligand, and translocates to the nucleus after 4′OHT treatment. (E) Western blots of stably expressed FLAG-HA-tagged TIP60 variants in LacI-FokI reporter cells. TIP60 (exo), exogenous tagged TIP60. (F) Schematic of TIP60-53BP1 proximity ligation assay in the mCherry-LacI-FokI reporter cells. Colocalization between TIP60 and 53BP1 is visualized by green fluorescence. (G) Representative PLA images of mCherry-LacI-FokI reporter cells complemented with FLAG-HA-tagged TIP60 variants or empty vector (EV). mCherry marks where LacI-FokI is localized, and green fluorescence marks sites of PLA positivity, where there is colocalization between 53BP1 and the tagged TIP60 (HA tag). Arrowheads indicate damage sites (mCherry foci) where both TIP60 and 53BP1 are recruited (PLA⁺ green foci). (H) Quantification of the percentage of damage sites (mCherry foci) where both TIP60 and 53BP1 are recruited, as indicated by PLA⁺ green foci colocalizing with mCherry foci. n = 3. For panels A and H, error bars show SEM.

FIG 6

S90 facilitates TIP60-dependent acetylation of histone H4 after damage. (A) Western blots of histones isolated from indicated MEFs at different time points after 10-Gy IR. (B and C) ImageJ quantification of Western blot band intensities of H4K16ac normalized to total H4 (B) and H2AZ K4/K7ac normalized to total H2AZ (C). n = 3. (D and E) Relative abundance of bulk unmodified and modified histone H4 peptide (amino acids [aa] 4 to 17) (D) and histone H2A.Z peptide (aa 1 to 19) (E) in etoposide-damaged MEFs as quantified by mass spectrometry. n = 10. (F) Schematic of ChIP-qPCR in the doxycycline (dox)-inducible LacI-FokI cell lines, in which pretreatment with doxycycline turns on transcription of the transgene and stimulates histone acetylation at the locus. (G) Normalized ratios of H4K16ac ChIP to total H4 ChIP and of H2AZ K4/K7ac ChIP to total H2AZ ChIP, with the control cell line expressing no exogenous TIP60 set to a normalized ratio of 1. n = 3. For panels B, C, D, E, and G, error bars show SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.