TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.

We have recently uncovered a novel function for TPX2 in DNA damage response [15]. DNA damage response activates so-called 'checkpoint pathways' that halt the cell cycle in order to repair the insulted DNA. Alternatively, DNA damage response induces cell death or senescence if the damage is too severe [16][17][18][19][20]. Together, this ensures genomic stability whereas inability to mount a proper response to DNA damage can promote development of cancer [21,22]. A central step in the cellular response to DNA double strand breaks is the formation of c-H2AX, i.e. phosphorylation of histone H2AX (on serine139 in human) in chromatin adjacent to DNA ruptures [16,[23][24][25]. Our previous study demonstrated that the levels of TPX2 inversely correlate with the levels of c-H2AX generated by the ATM kinase [15]. Particularly during G1-phase of the cell cycle, a dramatic increase in H2AX phosphorylation was observed in the absence of TPX2 upon treatment with clastogenic ionizing radiation [15]. However, the number of ionizing radiation-induced DNA double strand breaks was unperturbed in TPX2-depleted cells [15]. Instead, these cells exhibited an increased intensity of c-H2AX ionizing radiationinduced foci (i.e. the microscopic visualization of c-H2AX at DNA breaks), indicating hyper-phosphorylation of H2AX at DNA lesions [15]. TPX2's active function in DNA damage response is further highlighted by its accumulation on chromatin that surrounds chromosomal breaks [15].
Importantly, both types of enzymes have been implicated in DNA damage response [37][38][39][40][41][42]. For example, the HATs MOF and TIP60 induce acetylation (ac) of histone H4 on lysine16 (H4K16) during DNA damage response and are important for DNA repair and cellular survival [37,[42][43][44]. On the other hand, the SIRT1 HDAC antagonizes these actions by deacetylating H4K16ac and promoting the proteasomal degradation of MOF [40,45,46]. Although a transient delay (up to 15 min upon genomic insult) in H2AX phosphorylation was observed in cells with decreased H4K16ac levels [43], it remains unknown whether this histone modification further impacts c-H2AX signaling. Downstream of H2AX phosphorylation and dependent on the acetylation status of H4K16, 53BP1 accumulates on chromatin flanking DNA breaks. This matures the DNA damage response and promotes DNA repair and cellular survival [16,42,47].
In the present study, we report that overexpression of TPX2 alters the DAPI staining pattern of interphase cells. Furthermore, depletion of TPX2 decreases H4K16ac levels. These phenotypes are observed in the absence of exogenously induced DNA damage. Upon ionizing irradiation, the constitutive TPX2 depletiondependent decrease in H4K16ac levels correlates with increased levels of c-H2AX. Altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2, also changes both H4K16ac and c-H2AX levels in an inverse manner. Together, these data indicate a balance between H4K16ac and c-H2AX levels that can be modified by TPX2 and SIRT1. Finally, TPX2 depletion also interferes with 53BP1 ionizing radiation-induced foci formation, an event that depends on H4K16ac and c-H2AX levels. Our study indicates that TPX2 influences the chromatin environment with potential implications for DNA damage response.

Cell cycle synchronization and flow cytometry
HeLa EM2-11-TPX2 cells were plated 24 h before being subjected to the following synchronization procedure (i.e. double thymidine block): Treatment with thymidine (2 mM) for 20 h was followed by a passage into thymidine-free media for 15 h and subsequent treatment with thymidine (2 mM) for an additional 14 h. To induce expression of the TPX2 targeting miRNA, doxycycline was added to the media in parallel with the second thymidine treatment as indicated. After the double thymidine block, cells were released into thymidine-free media to allow synchronous cell cycle progression. Cells were then treated with 10 Gy of ionizing radiation (or left untreated) 11 h, 12 h, and 13 h after release from the double thymidine block (i.e. while progressing through G1-phase). Subsequently, cells were incubated for 1 h of recovery, harvested by trypsinization, and analyzed by Western blot. An aliquot (20%) of non-irradiated control cells was fixed with ethanol, stained with propidium iodide, and used for flow cytometry-based cell cycle profiling to ensure equal cell cycle synchronicity in all individual sample populations.

Chromatin fractionation
Cells were rinsed twice with PBS (37uC) and harvested in ice cold NETN buffer [150 mM NaCl, 1 mM EDTA, 50 mM Tris-Cl pH 7.4, 1% NP40, and 1x protease inhibitor cocktail complete Mini-EDTA free (Roche)]. The obtained cell extracts were sonicated once for 5 seconds with a Sonic Dismembrator Model 100 at Level 4. The insoluble chromatin fraction was pelleted for 20 minutes at 4uC at maximum speed in a table top centrifuge. The soluble NETN fraction was analyzed and nuclear lamins but no histones were found to be contained in this NETN fraction. The insoluble chromatin fraction (containing the histones but no nuclear lamins) was washed twice in 1 ml NETN buffer. Solubilization of the chromatin fraction was achieved by addition of 1% SDS in PBS followed by one freeze and thaw cycle at 2 80uC, incubation at 95uC for 15 minutes, and sonication for 15 seconds. Protein concentrations were measured with the Bio-Rad DC protein assay (chromatin fraction) or Bio-Rad Protein (Bradford) assay (NETN fraction).

Co-immunoprecipitations
Co-immunoprecipitation experiments have been described previously [15] and were carried out in the absence or presence of 50 mg/ml ethidium bromide (EtBr). Antibodies specific for TPX2 (a kind gift from Dr. Oliver J. Gruss-University of Heidelberg; [2]) or SIRT1 (Upstate) were used.

Immunofluorescence staining and microscopy analysis
Cells were fixed (paraformaldehyde), permeabilized (Triton X-100), blocked (BSA), and stained with antibodies against H4K16ac (Abcam), 53BP1 (Novus Biologicals), and the Xpress-tag encoded by His-TPX2 as per standard laboratory procedures. Nuclei were counterstained with DAPI. Images were acquired with a Nikon Eclipse TE2000-E confocal microscope.

Ionizing radiation
Treatment with ionizing radiation was performed using a source of Cs 137 from a MDS Nordion Gammacell 1000.

TPX2 associates with the chromatin and TPX2 overexpression alters the DAPI staining pattern
Since TPX2 partially co-localizes with DNA during interphase [2,15], we investigated a potential constitutive association of TPX2 with the chromatin. In the absence of exogenously induced DNA damage, TPX2 is readily found in chromatin fractions obtained from MCF7 cells and HeLa cells (Fig.1A). These fractions contain histone proteins but no nuclear lamins (Fig. 1A), indicating high stringency of the chromatin purification method. Expression of a doxycycline-inducible TPX2 targeting miRNA in HeLa cells [48] or transient transfection of MCF7 cells with a TPX2 targeting siRNA depleted the protein from these chromatin fractions (Figs.1A, 1D, 1F). Efficiencies and specificities of the two TPX2 targeting RNAi sequences have been determined previously [15,48]. The use of two independent RNAi approaches in two different cell lines provides strong evidence that the observed protein is indeed endogenous TPX2. It is noteworthy that the abundance of TPX2 in chromatin fractions increases after treatment with ionizing radiation (Fig.1B). This finding is in agreement with our published work documenting the recruitment of TPX2 to DNA double strand breaks [15].
Compatible with the presence of TPX2 in chromatin fractions, we found that overexpression of either His-TPX2 or GFP-TPX2 in non-irradiated MCF7 cells causes abnormal DAPI (49,6diamidino-2-phenylindole) staining patterns (Fig.1C). In these cells, the DAPI staining is more structured and compartmentalized than the uniformly distributed DAPI signal found in surrounding non-transfected control cells or cells expressing GFP (Fig.1C).

TPX2 depletion decreases the levels of H4K16ac
We previously reported that TPX2 regulates phosphorylation of H2AX upon ionizing irradiation [15]. In addition to H2AX, several other histones are also post-translationally modified during DNA damage response. Notably, the acetylation status of H3K9, H3K56, and H4K16 is changed upon breakage of chromosomes [37,42,47,50]. In light of results showing that ectopic expression of TPX2 alters DAPI staining patterns in non-irradiated cells (Fig.1C), we determined whether TPX2 also affects post-translational modification of histones in the absence of exogenously induced DNA damage. As previously shown, no substantial induction of c-H2AX was observed in TPX2-depleted cells before treatment with ionizing radiation ( [15] and Fig.2A). In nonirradiated MCF7 cells, the levels of H3K9ac and H3K56ac remained unchanged upon TPX2 depletion by siRNA (Fig.1D-E). Intriguingly, the levels of H4K16ac markedly decreased in these cells (D,76%; p(t test) = 0.003; 3 independent experiments; Fig.1D and Fig.2A-B for quantifications). To ensure specificity of this phenotype, we also examined H3K9ac, H3K56ac, and H4K16ac levels in HeLa cells depleted of TPX2 by miRNA. Consistently, we observed a substantial decrease in H4K16ac levels in these cells whereas H3K9ac and H3K56ac levels remained unchanged (Fig.1F). Thus, TPX2 impacts the levels of H4K16ac independently of DNA damage in two different cell types.
The TPX2 depletion-dependent decrease in H4K16ac is unaffected by ionizing irradiation but correlates with increased c-H2AX during DNA damage response Since acetylation of H4K16 is modulated upon genomic insult [37,47], we next sought to determine whether the constitutive TPX2 depletion-dependent decrease in H4K16ac levels ( Fig.1) is affected by ionizing irradiation. In agreement with recent findings [47], we found that H4K16ac levels in control MCF7 cells were slightly decreased after treatment with 10 Gy of ionizing radiation ( Fig.2A). This phenotype was consistent and statistically significant [ Fig.2B; control siRNA -IR (10.0+/21.0) vs. control siRNA + IR (6.1+/20.9); p(t test) = 0.044; group (mean of H4K16ac +/2SE, A.U.); n = 3 independent experiments; IR: ionizing radiation]. However, non-irradiated MCF7 (and HeLa; Fig.2C) cells depleted of TPX2 by siRNA (or miRNA; Fig.2C) already exhibited significantly lower H4K16ac levels than non-irradiated or irradiated control cells [ Fig.2A-B; control siRNA -IR (10.0+/2 1.0) vs. TPX2 siRNA -IR (2.4+/20.7); p(t test) = 0.003; group (mean of H4K16ac +/2SE, A.U.); n = 3 independent experiments]. Upon treatment with ionizing radiation, TPX2-depleted cells did not exhibit further decrease in H4K16ac levels [ Fig.2A Intriguingly, we found that the TPX2 depletion-triggered decrease in H4K16ac levels correlates with an increase in c-H2AX levels after treatment with ionizing radiation ( Fig.2A). Because TPX2's DNA damage response function is particularly evident during G1-phase (see [15]), we next determined the TPX2-dependent levels of H4K16ac at this cell cycle stage. To do so, we utilized the HeLa cell line expressing a doxycyclineinducible TPX2 miRNA and synchronized these cells with a double thymidine block [15,48]. Long-term depletion of TPX2 is known to impact cell cycle progression [2,8]. Therefore, we chose a minimal TPX2 knockdown time of less than 27h for these experiments. Our previously published data indicates that HeLa cell cultures are synchronized for S-phase, G2-phase, and M-phase at 2 h, 6 h, and 9 h after release from a double thymidine block. G1-phase occurs from 11 h-12 h after release [15]. In this study, we found that irradiated TPX2-depleted G1-phase-enriched cell cultures with 3.  Fig.2A-B), the TPX2 depletion-dependent decrease in H4K16ac levels observed in G1-phase HeLa cells appears to be independent of ionizing irradiation (Fig.2C).
Of note, 13 h after release from the double thymidine block the percentage of cells in G1-phase decreased (control: 71.4%; TPX2 miRNA: 76.4% G1-phase cells) and cells started to enter S-phase . This attenuation of the H4K16ac phenotype was accompanied by a diminished magnitude and reduced statistical significance of the TPX2 depletion-dependent c-H2AX increase (Fig.2C-E). The latter is in agreement with our previously published data documenting that TPX2 depletion has no effect on c-H2AX in cell cycle phases other than G1 and G0 [15].
In brief, our results indicate that TPX2 constitutively affects the levels of H4K16ac in G1-phase. During DNA damage response, the levels of H4K16ac and c-H2AX exhibit an inverse correlation. Therefore, the ionizing radiation-independent impact of TPX2 on H4K16ac levels (Figs.1D, 1F, 2A-C) may affect the phosphorylation of H2AX once DNA damage response is launched. Intriguingly, single cell analysis via confocal microscopy did not reveal a notable decrease in global acetylation of H4K16 upon TPX2 depletion (Fig.2F). This suggests that TPX2-dependent changes in H4K16ac levels may be restricted to certain genomic loci. Further studies are necessary to decipher where exactly in the genome TPX2 impacts the levels of H4K16ac (see Discussion).

SIRT1 impacts the levels of H4K16ac and c-H2AX and associates with TPX2
H4K16ac is a substrate of SIRT1 HDAC [40,45,46]. Interestingly, SIRT1 knockout mice display increased levels of H4K16ac that correlate with decreased levels of ionizing radiation-triggered c-H2AX [36]. The increase in H4K16ac levels in these animals is presumably due to loss of SIRT1 HDAC activity [36,46]. In  agreement with these data we observed that siRNA-mediated depletion of SIRT1 in HeLa cells increases H4K16ac levels and decreases c-H2AX levels compared to controls (Fig.3A). Conversely, overexpression of SIRT1 in MCF7 cells results in decreased H4K16ac levels that correlate with increased levels of ionizing radiation-triggered c-H2AX compared to controls (Fig.3B). Note that these MCF7 cells are caspase-3-deficient and do not undergo ionizing radiation-induced apoptosis [51,52]. The observed increase in c-H2AX levels upon SIRT1 overexpression is therefore not an epiphenomenon of apoptosis, known to also induce c-H2AX during apoptotic DNA fragmentation [53]. Thus, our data reveal an inverse correlation between the levels of H4K16ac and c-H2AX. Both TPX2 and SIRT1 can modulate the levels of these post-translationally modified histones (Figs. 2A-E and 3A-B).
In the absence of reports or sequence motifs suggesting an enzymatic activity intrinsic to TPX2, we hypothesized that TPX2 may be part of a regulatory complex that controls the levels of H4K16ac and c-H2AX. SIRT1 may be a member of this complex since it also modifies H4K16ac and c-H2AX levels (Fig.3A-B). In support of this hypothesis we found that TPX2 antibodies coimmunoprecipitated a subpopulation of SIRT1 (Fig.3C). Furthermore, SIRT1 antibodies also co-immunoprecipitated a subpopulation of TPX2 (Fig.3D). The yield of SIRT1 in the TPX2 coimmunoprecipitations was not affected by the presence of ethidium bromide, suggesting that the association between TPX2 and SIRT1 is not mediated by chromatin (Fig.3C). Finally, ionizing irradiation did not impact the association between TPX2 and SIRT1 in these co-immunoprecipitation experiments (Fig.3C-D). The significance of the TPX2/SIRT1 interaction is analyzed in the discussion.

TPX2 depletion causes defects in 53BP1 recruitment to exogenously induced chromosomal breaks
The recruitment of 53BP1 to DNA double strand breaks occurs downstream of c-H2AX signaling and is dependent on the acetylation status of H4K16 [16,20,42,47,54,55]. Since TPX2depleted cells exhibit altered levels of c-H2AX [15] and H4K16ac represent SE). (C) HeLa cell cultures enriched for G1-phase cells via release from a double thymidine block exhibit constitutively decreased levels of H4K16ac and increased ionizing radiation-dependent levels of c-H2AX upon depletion of TPX2 compared to controls (no TPX2 miRNA induction). Flow cytometry based cell cycle profiles (bottom histograms) derived from the non-irradiated cell cultures analyzed by western blots are shown. Note that the TPX2 depletion-dependent c-H2AX and H4K16ac phenotypes are particularly pronounced 11 h and 12 h after release. During G1/S transition (i.e. 13 h after release), c-H2AX and H4K16ac levels start to normalize in TPX2-depleted cells. See text for details. (D) Quantification of H4K16ac levels after irradiation throughout G1-phase from control (Ctrl) and TPX2 miRNA expressing HeLa cells (n = 3 independent experiments; Error bars represent SE). The relative increase of c-H2AX levels upon TPX2 depletion compared to controls is shown for each time point (light grey). (E) p-values (unpaired student's t test) describing differences in c-H2AX levels (n = 3 independent experiments) or H4K16ac levels (n = 3 independent experiments), respectively, between control (Ctrl) and TPX2 miRNA expressing HeLa cells at indicated time points after release from a double thymidine block. Note that the statistically significant (i.e. p,0.05) increase in c-H2AX levels and decrease in H4K16ac levels upon TPX2 depletion is attenuated at the G1/S transition (i.e. 13 h after release). Levels of H4 and H2AX were used to normalize for loading. NS: non significant, * p,0.05, ** p,0.01, *** p,0.001; IR: ionizing radiation. (F) No apparent global decrease in acetylated H4K16 in TPX2-depleted cells 12 h after release from a double thymidine block (i.e. G1-phase). Expression of the inducible TPX2 miRNA also triggers expression of a GFP reporter. doi:10.1371/journal.pone.0110994.g002   Fig.1-2), we hypothesized that 53BP1 ionizing radiation-induced foci formation is also disturbed in these cells.
We first analyzed the total amount of 53BP1 in TPX2-depleted cells and found no change in 53BP1 protein levels before and after treatment with ionizing radiation compared to controls (Fig.4A). We then performed a time-course analysis of 53BP1 ionizing radiation-induced foci formation in HeLa cells expressing the doxycycline-induced TPX2 targeting miRNA [48]. 53BP1 ionizing radiation-induced foci formation was significantly impaired in TPX2-depleted cells from 15 min to 2h after an irradiation dose of 2 Gy (Fig.4B). However, 53BP1 focus formation is not inhibited per se in the absence of TPX2. Infrequent endogenous DNA double strand breaks that arise at so called ''fragile sites'' in the absence of ionizing radiation treatment [56][57][58] still recruit 53BP1 upon TPX2 miRNA expression (Fig.4C).
Since depletion of TPX2 has been associated with mitotic arrest in HeLa cells [2], we determined whether the TPX2 depletiondependent 53BP1 phenotype is the result of increased numbers of mitotic cells, known to exclude 53BP1 from their repair foci [59,60]. Although we found a modest ,5% increase of the G2/M population upon TPX2 depletion via flow cytometry-based cell cycle profiling (Fig.4D), this slight increase can not account for the defect in 53BP1 ionizing radiation-induced foci formation exhibited by more than 55% of TPX2-depleted cells (Fig.4B).

Discussion
The nuclear functions of TPX2 are poorly understood. In the present study, we found that TPX2 associates constitutively with the chromatin and that TPX2 overexpression alters the DAPI staining pattern (Fig.1). Importantly, depletion of TPX2 decreases selectively the levels of H4K16ac (Figs.1-2). This phenotype is particularly evident in cell cultures synchronized at G1-phase ( Fig.2) and thus, it is not an artifact of changed cell cycle profiles. Taken together, our results indicate a role for TPX2 in chromatin biology.
Although the significance of TPX2-dependent H4K16ac levels in unperturbed cell cycles remains to be investigated, we propose that this histone modification may impact DNA damage response. Specifically, in TPX2-depleted G1-phase cells the constitutive decrease in H4K16ac levels is paralleled by an ionizing radiationdependent increase in c-H2AX levels. These correlating TPX2dependent H4K16ac and c-H2AX phenotypes are simultaneously attenuated at the end of G1-phase (Fig.2). Additional evidence for a link between H4K16ac and c-H2AX levels is provided by our observation that SIRT1 can impact both histone modifications (Fig.3). Thus, the acetylation status of H4K16 may influence the extent of c-H2AX formation. Upon TPX2 depletion, the decreased H4K16ac levels may prime the chromatin for excessive accumulation of c-H2AX. H4K16 may also be part of a broader TPX2 and/or SIRT1-dependent chromatin remodeling program that affects phosphorylation of H2AX via unidentified mechanisms.
It remains unclear where (Fig.2F) and how TPX2 affects acetylation of H4K16. Based on our co-immunprecipitations of SIRT1 and TPX2 (Fig.3), we hypothesize that these two proteins may collaborate to regulate H4K16ac levels. In this context, it will be interesting to identify the specific genomic loci occupied by TPX2 and to determine whether these loci also contain SIRT1, H4K16ac, and/or ionizing radiation-inducible c-H2AX. However, it remains unclear why TPX2 depletion does not impact other SIRT1 substrates, e.g. H3K9ac and H3K56ac [45,61,62]. Although TPX2/SIRT1 may specifically control loci that are enriched for H4K16ac but devoid of H3K9/56ac, TPX2 and SIRT1 could also act independently of each other. Further studies are required to define the mechanisms that regulate the TPX2dependent H4K16ac/c-H2AX levels.
Downstream of c-H2AX, 53BP1 accumulates on chromatin flanking DNA lesions. This accumulation of 53BP1 also requires deacetylated H4K16, since acetylation of this histone-site inhibits binding of 53BP1's TUDOR domains to constitutively expressed di-methylated H4K20 [42,47]. We found that depletion of TPX2 inhibits 53BP1 ionizing radiation-induced foci formation (Fig.4). Due to the increased c-H2AX and constitutively decreased H4K16ac levels exhibited by TPX2-depleted cells, one might actually expect the opposite, i.e an increase in 53BP1 ionizing radiation-induced foci. However, our data are supported by several observations. First, mimicking globally deacetylated H4K16 does not increase 53BP1 foci formation either [47]. Second, excessive DNA damage caused by more than 2 Gy also interferes with formation of 53BP1 ionizing radiation-induced foci [57]. Such excessive damage that inhibits accumulation of 53BP1 at chromosomal breaks could be simulated by the pronounced DNA damage signaling of TPX2-depleted cells. Specifically, the increased c-H2AX and decreased H4K16ac levels in TPX2depleted cells (Figs.1-2) could signal more DNA damage than actually present and thus, misguide the 53BP1 system; (note that modulation of H4K16ac levels and c-H2AX formation are both natural responses to chromosomal breakage; Fig.2A-B). This interpretation can be reconciled with the observation that a limited number of DNA lesions can still be efficiently decorated with 53BP1 upon depletion of TPX2 (Fig. 4C). In this context, one or two endogenous DNA double strand breaks with de-regulated H2AX phosphorylation and altered H4K16ac levels might not mimic beyond the threshold level of excessive DNA damage that exhausts the 53BP1 system [57].
Finally, since TPX2 has an active role during DNA damage response [15], we do not exclude that it contributes to the ionizing radiation-triggered modulation of H4K16ac levels [42,47]. The idea that TPX2 may exert analogous chromatin modifying functions during physiological and DNA damaged contexts is exciting, novel, but not unprecedented. The ATM kinase that generates the majority of c-H2AX during DNA damage response [16,32] is also implicated in phosphorylation of H2AX on undamaged mitotic chromatin. The latter has been demonstrated to be important for chromosomal separation [63]. Furthermore, BRCA1-mediated ubiquitination of H2A, known to maintain integrity of heterochromatin [14], might also be important during DNA damage response. Downstream of c-H2AX signaling, ubiquitination of DNA double strand break-flanking chromatin is essential for a functional DNA damage response [16,17]. Interestingly, in BRCA1-depleted cells this ubiquitination of damaged chromatin is diminished [64][65][66]. TPX2 may be an addition to this list of factors that mediate analogous chromatin modifications during physiological conditions and DNA damage response.