The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation

Abstract

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.

Keywords: 53BP1; H2AK15; H4K20; MBTD1; NuA4; TIP60; acetylation; histone methylation; homologous recombination; ubiquitylation.

Figure 1. MBTD1 is a stable subunit of the human TIP60/NuA4 acetyltransferase complex

(A) Purification of native TIP60/NuA4 complex from K562 cells. The TIP60 complex was purified using a K562 cell line modified to stably express Tip60-3xFLAG (Fig. S1A). Anti-FLAG immunoprecipitation was performed on nuclear extract, bound material was then eluted with 3xFLAG peptide and analyzed on 4-12% SDS-PAGE followed by silver staining. A mock purification was performed on a control cell line expressing an empty 3xFLAG tag. Subunits were identified by western blotting and mass spectrometry (Tables S1-S2). (B-C) Purification of native TIP60 complexes from K562 cells expressing EPC1-3xFLAG or MBTD1-3xFLAG (as in (A)) (*non-specific band). (D) Purified complexes from (B) and (C) were analyzed by western blot with the indicated antibodies to confirm the equivalent presence of known subunits of the TIP60 complex. The doublet appearing with the anti-MBTD1 antibody in the MBTD1-FLAG purification corresponds to inefficient self-cleaving peptide to remove the puromycin resistance portion of the tagged protein (also identified in (A-C)). (E) Purification of the TIP60 complex from a cell line expressing a truncated EPC1(1-581)-3xFLAG protein and analyzed as in (A). (F) MBTD1 association to TIP60 depends on EPC1 C-terminus. Western blot analysis of EPC1(1-581)-3xFLAG and EPC1-3xFLAG purifications. (G) MBTD1 directly interacts with EPC1 C-terminus through its MBT domains. Western blot analysis of GST pulldowns using EPC1 C-terminus (aa582 to 836) incubated with the indicated MBTD1 fragments. (H) Schematic representation of EPC1 and MBTD1 functional domains and interactions.

Figure 2. MBTD1 binds the H4K20me1/2 histone mark and colocalizes with the TIP60 complex at gene regulatory elements in the genome

(A) GST-tagged MBT domains of MBTD1 analyzed by peptide pulldown with the indicated biotinylated H4 peptides (aa1 to 23) (left panel); as positive control, the GST-tagged tandem Tudor domain of 53BP1 (aa1484 to 1603) is shown (right panel). Pulldowns were revealed by Western blotting with anti-GST. (B) Overexpression of MBTD1 suppresses cell growth by clonogenic assay. A MBTD1 triple-mutant in the fourth MBT domain is also shown. U2OS cells were transfected in triplicates with WT HA-MBTD1 or F526-W529-Y533A mutant (3A mutant) or empty vector for 48h (expression verified in fig. S2B). Hygromycin-resistant colonies were stained with Giemsa. (C) MBTD1, Tip60/KAT5 and EPC1 co-localize near the transcriptional start site (TSS) of the actively transcribed RPSA gene. Genome wide MBTD1 localization was analyzed after ChIP-seq and compared to data obtained in parallel with EPC1, EPC1 C-terminal truncation (EPC1(1-581)) and Tip60/KAT5. Anti-FLAG chromatin immuno-precipitations were performed in tagged and mock K562 cell lines used in figure 1. The values correspond to reads per million of total reads (rpm). The H4K20me1 profile presented is from K562 cells in the Encode consortium dataset (direct reads). (D) MBTD1 is important for binding of the TIP60 complex on the promoter of the MAPK8IP2 gene. ChIP-seq data at the MAPK8IP2 gene is shown as an example where the EPC1(1-581) signal is lost compared to wild type EPC1, indicating a specific role of MBTD1 for the association of the TIP60 complex to this region. (E) MBTD1 impacts TIP60 complex localization at specific loci within the genome. Common genomic loci strongly bound by EPC1, Tip60/KAT5 and MBTD1 identified 368 high confidence targets of the TIP60 complex in K562 cells (Fig. S2C, Table S3). Comparison with the EPC1(1-581)-3xFLAG signals reveals that 160 out of these 368 bone fide TIP60 targets are affected by the absence of MBTD1 from the complex (log10pvalue ≥ 52.66)(Fig. S2D-H for additional analyses/loci).

Figure 3. MBTD1 is implicated with TIP60 in the repair of DNA double strand breaks by homologous recombination

(A) MBTD1 is recruited at DNA double strand breaks. K562 cells expressing FLAG-tagged MBTD1 or EPC1 from the endogenous genes (Dalvai et al., 2015) were transfected with a vector expressing a zinc-finger nuclease (ZFN) targeting the AAVS1 safe harbor followed 18h later by anti-FLAG ChIP. Primers for qPCR are located 0,5 and 3,5kb from the break as well as a negative control locus. Values represent % of input. Error bars are from two independently performed experiments (Fig. S3A for positive control). (B) Depletion of MBTD1 affects γ-H2AX foci dynamics after γ-radiations. 293 cells were transfected with the indicated siRNAs for 48h, treated with 2Gy γ-irradiations and processed for γ-H2AX immunofluorescence. Results are presented over time after irradiation as the percentage of cells with more than 4 γ-H2AX foci (mean ± s.e.m, n=3). 100 cells were analyzed for each time-point. Similar results were obtained with shRNAs against other TIP60 subunits and in U2OS cells (Fig. S3C-D). (C) Measurement of homology directed repair of an inducible I-Sce1 DSB in U2OS cells using an integrated the DRGFP reporter (schematic on the top). Cells were transfected with the indicated siRNAs for 36h, infected with I-Sce1 adenovirus to induce DSB and then assessed 48h later by FACS analysis for GFP expression. Results represent the percentage of GFP positive cells normalized to the control siRNA, from 2 independent and representative experiments. (D) Measurement of I-Sce1 DSB repair by non-homologous end joining in U2OS cells using an integrated PC222/GFP-RFP reporter (schematic on the top). Cells were treated as in (C) and assessed by FACS for RFP and GFP expression. Results represent the percentage of cells that are RFP positive but GFP negative, normalized to the control siRNA, from 2 independent experiments. (E) Complementation assay in the DR-GFP reporter system (from C). Cells were transfected with CTL or MBTD1 siRNAs for 16h, then with pCAG-I-Sce1, pmCherry and siRNA-resistant MBTD1-expressing (WT or 3A mutant F526-W529-Y533A) or empty vectors and finally assessed 48h later by FACS for mCherry and GFP co-expression. Results represent the percentage of mCherry and GFP positive cells normalized to the control siRNA, from 2 independent experiments.

Figure 4. MBTD1 can displace 53BP1 from the H4K20me2 histone mark and affects the dynamics of 53BP1 foci after DNA damage

(A) MBTD1 can remove 53BP1 from H4K20me2 peptides. In vitro competition experiment between GST-tagged 53BP1 tandem Tudor domain and MBP-tagged MBTD1 MBT-repeat domain using H4K20me2 peptides (aa1-23). A two-fold excess of 53BP1 tandem Tudor domain (WT or mutant D1521R) was incubated with biotinylated H4 peptide followed by the addition of increasing amounts of purified MBT-repeat domain of MBTD1 (or BSA). The level of binding by the tandem Tudor vs the MBT-repeat was measured by western blot on the peptide bound material using anti-GST or anti-MBP, respectively. (B) (C) Tip60/KAT5 affects the dynamics of 53BP1 foci after DNA damage. U2OS cells were transfected with control siRNA (B) or siTip60 (C) and irradiated (2Gy) 48h post-transfection. Cells were processed for γ-H2AX and 53BP1 immunofluorescence 0, 1 and 5h after irradiation. (D) MBTD1 knock down phenocopies Tip60 effect on the stability of 53BP1 foci. (E) Quantification of the dynamics of 53BP1 foci appearance/disappearance after irradiation in control conditions or when Tip60/MBTD1 are depleted. Data are presented as percentage of cells with more than five 53BP1 foci (mean ± s.d, n=2). Two independent siRNAs for MBTD1 were used (Fig. S3E and S4).

Figure 5. MBTD1 knockout cells show DNA repair deficiency and increased formation of 53BP1 foci after DNA damage

(A) Alkaline comet assay of MBTD1 WT or KO cells (U2OS), non-treated and 6h after etoposide treatment (50μM, 15min). One representative experiment on 2 different KO clones compared to WT (n>60 per condition, mean ± s.d., p=0.0006). Tail moments 30min after treatment are similar in WT and KO cells with a mean around 100. (B) (C) (D) Dynamics of IR-induced 53BP1 foci in MBTD1 WT and KO cells. U2OS control cells (B) or KO clones # 15 (C) and #25 (D) were irradiated (2Gy) and processed for γ-H2AX and 53BP1 immunofluorescence 0, 1 and 5h after irradiation (scale bar is 5μm). (E) Re-introduction of MBTD1 in KO cells restores 53BP1 foci dynamics. KO cells (#25 is shown) were transfected with empty vector or FLAG-MBTD1, irradiated (2Gy) 24h post-transfection and processed for FLAG and 53BP1 immunofluorescence 0, 1 and 5h after irradiation. (F) Quantification of (B) (C) (D) and rescue experiment (E). At each time point, the number of 53BP1 foci was counted in at least 50 cells, averaged and normalized against the average number of foci counted in the same cell line at time 0 (T0 = 1). (means ± s.e.m., n = 3; rescue: means ± s.e.m., n ≥ 2).

Figure 6. H2AK15 ubiquitylation by RNF168 versus acetylation by the TIP60 complex

(A) H2A ubiquitylation by RNF168 specifically inhibits TIP60-dependant acetylation of nucleosomes. Native mononucleosomes from HeLa cells were ubiquitylated in vitro with RNF168/E1/E2 (500ng; Fig. S6A) and used as substrate in HAT assays with purified TIP60 complex. Another histone H4-specific HAT complex, HBO1, was used as control. Values are normalized on the activity measured on control nucleosomes prepared without RNF168 (mean ± s.d, n=2). (B) H2AK15 is acetylated by the TIP60 complex on nucleosomes in vitro; validation of H2AK15ac antibody specificity and interrelation with H4K20me2. Recombinant mononucleosomes (NCP) were used as substrates with purified TIP60 complex (EPC1-3xFLAG, Fig. 1). Wild type and mutant NCPs were used as well as one carrying H4Kc20me2 (methyllysine analog). (C) H2AK15 is a substrate of TIP60 in vivo. Chromatin was extracted from 293T cells transfected with a nucleosomal HAT sub-complex of TIP60 (piccolo-TIP60/NuA4) containing EPC1, ING3, MEAF6, MBTD1 and wild type Tip60 or the enzymatically dead E403Q mutant (Fig. S6C). H2AK15, H2AK5ac, H4ac and H3 signals were analyzed by western blotting. (D) Tip60 knockdown affects H2AK15ac level in vivo. Acid extracted histones from U2OS cells transfected with the indicated siRNAs for 48h were analyzed by western blotting for different histone modifications. (E) Acetylation of H2AK15 is regulated during the cell cycle. H2AK15 acetylation was analyzed by western blotting in U2OS cells synchronized by double-thymidine block and release (lanes 4 & 5; 0 and 2h, respectively) or thymidine-nocodazole block and release (lanes 2 & 3; 0 and 30 min, respectively). Asynchronous cells are shown as control (lane 1, ASN) and H3S10p signal is shown as G2/M marker (Fig. S6F for FACS). (F) H2AK15ac is differentially regulated in response to DNA damage. Acid extracted histones from 293T cells transfected with MBTD1 or control vector and treated or not with etoposide (20μM, 1h) were analyzed by western blotting for different histone modifications. (G) Nuclear H2AK15ac increases after DNA damage. U2OS cells were irradiated with 5 Gy, processed after 0, 1 or 5h for H2AK15ac and γ-H2AX immunofluorescence and DAPI. (H) H2AK15ac increase upon DNA damage is dependent on Tip60. U2OS cells were irradiated with 5 Gy, 48h post transfection with the indicated siRNAs, and processed after 0, 1 or 5h for H2AK15ac immunofluorescence and DAPI. H2AK15ac signals were normalized on the DAPI signal in the same nuclei (n>80 per condition in duplicate, mean ± s.d, p<0.0001).

Figure 7. Acetylation of H2AK15 by TIP60 is linked to breaks preferentially repaired by HR during S/G2

(A) (B) H2AK15 is acetylated at pro-HR DSB in S/G2 cells. γ-H2AX and H2AK15ac ChIP experiments in AID-DIvA cells (Aymard et al., 2014) after synchronization by double thymidine block and treatment with 4-OHT. Results are normalized for nucleosome occupancy over H3 IP % and represent a duplicate of independent experiments (Fig. S7B for other DSB sites). (C) Schematic representation of TIP60 complex composition and its multiple histone mark reader subunits that regulate its interaction with chromatin. (D) Model for TIP60 regulation of 53BP1 during DSB repair pathway choice. TIP60 regulates DSB repair pathway selection by sharing common nucleosomal histone targets with 53BP1 on nucleosomes and by creating an ubiquitylation / acetylation switch at H2AK15. MBTD1 and 53BP1 both target H4K20me2 which promotes their binding to chromatin. RNF168 ubiquitylates H2AK15 in response to DNA damage to recruit 53BP1 on damaged chromatin. During NHEJ, H2AK15 ubiquitylation blocks TIP60 (EPC1) binding to the H2A tail and H4 acetylation, favoring 53BP1 retention at the break and inhibition of resection. On the other hand, TIP60 acetylates H2AK15 and therefore blocks ubiquitylation by RNF168 and 53BP1 binding. This can prevent promiscuous DNA damage-induced ubiquitylation of H2A throughout the genome, but also participates with H4K20me2 binding and H4 acetylation by TIP60/MBTD1 in disrupting 53BP1 occupancy on damaged chromatin to allow resection and repair by homologous recombination. The action of specific deubiquitinase (DUB) and deacetylases (KDAC) will be important to determine which interactions and modifications are favored on specific nucleosomes.