Mammalian Alteration/Deficiency in Activation 3 (Ada3) Is Essential for Embryonic Development and Cell Cycle Progression*

Background: Ada3 is a core component of HAT containing coactivator complexes. Results: Germline deletion of Ada3 is embryonic lethal, and cell deletion leads to abnormal cell cycle progression. Conclusion: Ada3 is a critical protein at organismic and cellular level. Significance: This study describes a novel role of Ada3, a component of HAT complexes, as a critical regulator of cell survival. Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3FL/FL mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G1 to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G2/M to G1 transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex.

The eukaryotic cell cycle progression depends on proper coordination of DNA replication and duplication of chromo-somes to daughter cells (1), a process precisely regulated by modification of chromatin that allows the accessibility to factors involved in transcription (2). Thus, proteins involved in modulating the structure of chromatin play an important role in cell cycle progression. The post-translational modification of core histones (H2A, H2B, H3, and H4) is an essential process for altering chromatin structure (3,4). Histone acetyl transferases (HATs) 6 and histone deacetylases are required to maintain steady state levels of acetylation (5). Several HAT enzymes, such as general control nonderepressible 5 (Gcn5), p300/CBPassociated factor (PCAF), p300, and CREB-binding protein (CBP), have been identified over the years (6,7). Most of the HATs are part of large complexes such as the human TBP-free TAF complex (TFTC); the Spt3/Taf9/Gcn5 acetyltransferase complex (STAGA) (human homolog of yeast SAGA complex) and the Ada2a-containing (ATAC) complex that play a role in several important processes, such as cell cycle (8,9). Additionally, previous studies from our laboratory and that of others have demonstrated the presence of p300 HAT in Ada3-containing protein complexes (10,11). Given the combined presence of Ada3 with Gcn5 in a number of distinct HAT complexes, recent evidence for a role of Gcn5 in regulating DNA replication as well as mitosis (12)(13)(14) suggest that Ada3 may also play a role in cell cycle. Despite the range of established and potential cellular functions of Ada3 as part of multiple HAT complexes, the in vivo physiological role of mammalian Ada3 is not known.
We previously identified human Ada3 as a novel human papillomavirus 16 E6-binding protein (15). Human Ada3 is the homologue of the yeast Ada3, an essential component of the Ada transcriptional coactivator complex composed of Ada2, Ada3, and a HAT component Gcn5 (16). Genetic studies in yeast have demonstrated that Ada3 functions as a critical component of coactivator complexes that link transcriptional activators, bound to specific promoters, to histone acetylation and basal transcriptional machinery (17)(18)(19). We showed that Ada3 binds and stabilizes the tumor suppressor p53 protein and is required for p53 acetylation by p300 (20). Work from our laboratory has also shown that Ada3 is required for HAT recruitment to estrogen receptors and their transcription activation function (11). We and others have shown that Ada3 also associates with and regulates transcriptional activity of other nuclear hormone receptors, including retinoic acid receptor (21) and androgen receptor (22).
Here, we used conditional deletion of mouse Ada3 gene to explore the physiological importance of mammalian Ada3. We demonstrate that homozygous deletion of Ada3 is early embryonic lethal. Ada3 deletion in Ada3 Flox/Flox (Ada3 FL/FL ) MEFs showed that Ada3 is required for efficient cell cycle progression through G 1 to S transition as well as for proper mitosis. Detailed analyses in this system revealed an Ada3-c-Myc-Skp2-p27 axis that controls G 1 to S phase progression and partly contributes to cell cycle delay upon Ada3 deletion. Additionally, loss of Ada3 showed dramatic decrease in acetylation of core histones that are known to play an important role in cell cycle. Loss of Ada3 also resulted in several changes in gene expression as observed by microarray analyses. Notably, many of the genes affected were involved in mitosis. Taken together, we present evidence for an essential role of mammalian Ada3 in embryonic development and cell cycle progression.

EXPERIMENTAL PROCEDURES
Generation of Ada3 Gene-targeted Mice, Isolation of Mouse Embryos and PCR Genotyping-Details concerning generation of conditional Ada3 knock-out construct and Ada3 knock-out mouse as well as PCR genotyping strategies are described in the supplemental data.
Cell Culture Procedures and Viral Infections-Embryonic day 13.5 embryos were dissected from Ada3 FL/ϩ intercrossed females, and MEFs were isolated and immortalized following the 3T3 protocol (23). MEFs were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Adenoviruses expressing EGFP-Cre or enhanced green fluorescent protein (EGFP) alone were purchased from the University of Iowa (Gene Transfer Vector Core). An adenovirus dose of 50 -100 MOI diluted in 4 ml of serum-free medium was added to cells in 100-mm culture dishes (at about 30% confluence) and incubated for 1 h each at room temperature and at 37°C followed by the addition of 7 ml of complete medium. After overnight incubation at 37°C, medium was replaced with complete medium, and cells were carried further for various experiments. To generate retroviral FLAG-hAda3 vector, fulllength FLAG-hAda3 (15) was cloned into pMSCVpuro vector (Clontech). Retroviruses were generated by transiently transfecting this retroviral construct into the Phoenix ecotropic packaging cell line using the calcium phosphate co-precipitation method. The retroviruses were transduced into Ada3 FL/FL MEFs by three infections at 12-h intervals using supernatant from transfected Phoenix cells to generate Ada3 FL/FL MEFs expressing FLAG-hAda3. Scrambled shRNA (5Ј-GGTTAAA-ACCTTACGATGT-3Ј) or p27 shRNA (5Ј-GTGGAATTTCG-ACTTTCAG-3Ј) was introduced into Ada3 FL/FL MEFs by using three infections at 12-h intervals of the shRNA bearing pSU-PER.retro.puro (Oligoengine) retrovirus containing supernatants from Phoenix cells. Retroviral infections were carried out in the presence of 8 g/ml Polybrene (Sigma) and were followed by selection in 2 g/ml puromycin for 48 h until complete loss of uninfected cells.
Proliferation Assay, Colony Formation Efficiency Assay, and Cell Cycle Analysis-To perform proliferation assays, 1 day after adenovirus infection, cells were plated at different numbers in 6-well plates in triplicates (5 ϫ 10 4 (for counting on day 3), 2.5 ϫ 10 4 (for counting on day 5), 1.25 ϫ 10 4 (for counting on day 7), and 0.625 ϫ 10 4 (for counting on day 9) and counted at the indicated time points. For colony formation assay, cells 3 days after adenovirus-infection were trypsinized and plated at 1000 cells per 100-mm culture dishes in triplicates and carried for 15 more days with medium change as required. At the end of incubation, colonies in dishes were fixed and stained with crystal violet solution (0.25% crystal violet in 25% methanol) and photographed. For cell cycle analysis, 2 days after plating and adenoviral infection of 2 ϫ 10 5 cells in 100-mm culture dishes, cells were synchronized by replacing the complete medium with DMEM ϩ 0.1% FCS and incubating for 72 h. Synchronized cells were stimulated with complete medium (DMEM ϩ 10% FCS) for various time points and harvested and stained with propidium iodide (PI) for FACS analysis. For synchronization of cells at G 2 /M phase, 48 h after adenovirus infection, cells were switched to complete medium containing 125 ng/ml nocodazole for 18 h. Following synchronization, cells were washed three times with PBS and stimulated with complete medium for various time points and analyzed by FACS after PI staining.
Generation of Ada3 Monoclonal Antibody and Immunoblotting-Antibodies used in this study can be found in the supplemental data.
Analysis of the p27 Protein Turnover-Ada3 FL/FL MEFs were plated in 100-mm dishes and infected with control or Cre adenoviruses. For analyzing p27 protein half-life in exponentially growing cells, 2 days after adenovirus infection, cells were treated with 50 g/ml cycloheximide (Sigma) and harvested at the indicated time points. For analyzing p27 protein half-life in serum-starved cells, 2 days after adenovirus infection, cells were starved for 72 h in 0.1% serum-containing medium. Subsequently, 50 g/ml cycloheximide was added to the medium, and cells were harvested at the indicated time points. Total cell extracts were prepared, and equivalent amounts were run on SDS-PAGE and analyzed by Western blotting. Densitometry analysis was carried out on scanned images using ImageJ software.
RNA Extraction and Quantitative Real-time PCR-TRIzol reagent (Invitrogen) was used to isolate total RNA from MEFs infected with control virus or Cre adenovirus. 2 g of total RNA was used for reverse transcriptase reaction using Super-Script TM II reverse transcriptase (Invitrogen). Real-time PCR quantification was performed in triplicates using SYBR Green PCR master mix (Applied Biosystems) and the primers listed in supplemental Table S3. Expression levels were normalized against ␤-actin mRNA levels, and the results were calculated by the ⌬⌬C t method.
Generation of Recombinant Baculoviruses and Ada3-His Expression Using Bac-to-Bac Expression System-Ada3 baculoviral construct information and recombinant protein purification are detailed in the supplemental data.
HAT Assay-Protocol used for in vitro HAT assay can be found in the supplemental data.
Microarray Analyses-Protocol for microarray analyses is described in the supplemental data. The microarray data from this publication have been submitted to the GEO database and have been assigned the following Series record: GSE37542.

Deletion of Ada3 Leads to Early Embryonic Lethality in Mice-
The targeting construct generated using the recombineering technique (supplemental Fig. S1A; see supplemental Materials and Methods) was electroporated into an ES cell line derived from the 129/Ola strain of mice. Screening of resultant neomycin-resistant colonies yielded three correctly targeted clones (supplemental Fig. S1B). One positive clone was microinjected into blastocysts. The resulting chimeras transmitted the targeted allele to their progeny as verified by PCR. The neomycin cassette flanked by Frt recombination sites was removed by crossing the Ada3-targeted mice to FlpE recombinase transgenic mice (B6.Cg-Tg (ACTFLPe) 9205Dym/J; stock number 005703). Homozygous Ada3 FL/FL mice were viable and fertile and exhibited no gross abnormalities when compared with Ada3 FL/ϩ or Ada3 ϩ/ϩ controls. To achieve Ada3 deletion, heterozygous Ada3-targeted mice (Ada3 FL/ϩ mice) were bred with transgenic mice expressing the adenovirus EIIa promoterdriven Cre (B6.FVB-Tg (EIIa-Cre) C5379Lmgd/J). EIIa directs Cre expression in a wide range of tissues including germ cells. Heterozygous Ada3-targeted, Cre transgene-positive mice were crossed to C57BL/6J (wild-type) mice to generate heterozygous Ada3-deleted, Cre transgene-negative (Ada3 ϩ/Ϫ ) mice. Heterozygous Ada3 ϩ/Ϫ mice of a mixed 129/Sv ϫ C57BL/6 background were viable and fertile, and their median life span of more than 18 months was comparable with that of their control littermates (data not shown). Heterozygous Ada3 ϩ/Ϫ mice were intercrossed to obtain homozygous Ada3null mice. No Ada3 Ϫ/Ϫ mice were observed among 224 live born pups screened ( Table 1). The ratio of wild type to heterozygous offspring was 1:2, indicating that the loss of one Ada3 allele does not lead to haploinsufficiency in mice.
To assess the specific period of developmental failure in the Ada3 knock-out mice, embryos derived from Ada3 ϩ/Ϫ intercrosses were genotyped at different stages of gestation using a duplex PCR method (supplemental Fig. S1, C and D). Because no homozygous mutant embryos were recovered beyond embryonic day 8.5 (E8.5; Table 1), blastocysts were isolated at 3.5 days postcoitum and genotyped directly by PCR (supplemental Fig. S1E). When compared with blastocysts of Ada3 ϩ/ϩ and Ada3 ϩ/Ϫ genotypes, Ada3 Ϫ/Ϫ blastocysts that attached to culture dishes showed severe growth retardation of the trophoblast layer, and the inner cell mass was absent (supplemental Fig. S1F). PCR analysis revealed that ϳ25% of blastocysts analyzed were null for Ada3 (Table 1). These results demonstrate that Ada3 plays a critical role in early embryogenesis in mice. The failure of Ada3 Ϫ/Ϫ embryos to remain viable beyond E3.5 suggests a potential role of Ada3 in cell proliferation because extensive cellular proliferation occurs during this early stage of embryogenesis (see later sections).

Ada3 Is Ubiquitously Expressed in Adult Mouse
Tissues-Embryonic lethality of Ada3 Ϫ/Ϫ mice suggested a potential role of Ada3 in growth and development of many tissues. To examine whether Ada3 is expressed in adult tissues, we analyzed the relative levels of Ada3 protein expression in a range of adult mouse tissues. For this purpose, lysates from various tissues of 8-week-old wild-type mice were subjected to immunoblotting using an anti-Ada3 monoclonal antibody generated in our laboratory (see supplemental Materials and Methods). As seen in supplemental Fig. S2, Ada3 is ubiquitously expressed in all the tissues with higher levels seen in the mammary gland, lung, and thymus. These results suggest potentially ubiquitous functional roles of Ada3 and are consistent with embryonic lethal phenotype of its germline deletion.
Conditional Ada3 Deletion in MEFs Leads to Proliferation Arrest-Given the embryonic lethality as a result of Ada3 deletion, we resorted to a cellular model of conditional Ada3 deletion to investigate its roles at the cellular level. For this purpose, we generated Ada3 FL/FL mice by interbreeding Ada3 FL/ϩ mice and established MEFs from these mice. Conditional Ada3 deletion was obtained by infecting Ada3 FL/FL MEFs with an adenovirus expressing the Cre recombinase (adeno-Cre), with adeno-GFP serving as a control. To assess the effects of Ada3 on cell proliferation, equal numbers of control-and adeno-Cre-infected MEFs were plated a day after adenoviral infection, and cells were counted at the indicated time points up to 9 days. Notably, Ada3-deleted MEFs exhibited a significantly slower rate of proliferation when compared with control MEFs (Fig. 1A, left). To confirm that the defect in cell proliferation was specifically due to depletion of Ada3, we generated Ada3 FL/FL/hAda3 MEFs by retrovirally introducing human Ada3 (hAda3) with an N-terminal FLAG tag into Ada3 FL/FL MEFs. These transfectants were verified to be expressing the exogenous FLAG-tagged Ada3 protein (Fig. 1B). Similar to Ada3 FL/FL MEFs, adeno-Cre infection of these cells led to deletion of endogenous Ada3 and loss of its protein product (Fig. 1B). Notably, however, Cre-mediated deletion of Ada3 in Ada3 FL/FL/hAda3 MEFs had a minimal effect on the proliferation of MEFs, whereas similar treatment of Ada3 FL/FL MEFs led to reduction in the rate of proliferation; thus, the proliferative defect induced by deletion of mouse Ada3 in MEFs was rescued by exogenous hAda3 (Fig. 1A, right). Colony formation efficiency assay, as an independent method to measure the extent of cell proliferation, further confirmed the proliferative defect of Ada3-deleted MEFs that could be rescued by reconstitution with exogenous hAda3 (Fig. 1, C and D).
Ada3 Is Required for Cell Cycle Progression through G 1 to S Phase-We reasoned that the proliferation defect upon Ada3 deletion in MEFs could reflect a role of Ada3 in cell cycle progression. To directly examine whether Ada3 plays a role in cell cycle progression, Ada3 FL/FL MEFs were infected with control and Cre adenoviruses, arrested in G 0 /G 1 by serum deprivation for 72 h, and then synchronously released into cell cycle by serum stimulation. FACS-based cell cycle analysis of propidium iodide-stained cells showed significant delay in G 1 to S progression in Ada3-deleted MEFs when compared with control MEFs ( Fig. 2A). Of note, the relative distribution of S phase in Ada3-null MEFs after 20 h of serum stimulation was about half (31.6 Ϯ 2.33 S.E. %) of the control virus-infected MEFs (56.05 Ϯ 4.71 S.E. %) (Fig. 2B). These results demonstrate that conditional deletion of Ada3 leads to delay in G 1 to S progression in MEFs, indicating an essential role of Ada3 in efficient G 1 /S progression.  AUGUST 24, 2012 • VOLUME 287 • NUMBER 35

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Elevated p27 Kip1 Levels and Impaired Rb Phosphorylation upon Conditional Ada3 Deletion-Given the delay in G 1 /S progression imposed by induced Ada3 deficiency, we examined the status of key proteins known to control the G 1 /S transition. A well established and critical event during G 1 to S progression is the phosphorylation of Rb by Cdk complexes (particularly complexes containing Cyclins D, E, or A), such as Cdk4/6 and Cdk2 (24,25); phosphorylation of Rb leads to its release from Rb/E2F complexes, relieves E2Fs from repression, and facilitates the expression of E2F-responsive genes important for S phase progression (24,25). Furthermore, degradation of Cdk inhibitors, such as p27, is required for progression of cells from G 1 to S phase (26,27). Therefore, we carried out Western blotting of cell lysates obtained from control versus conditional Ada3-deleted MEFs released into synchronous cell cycle progression to assess the levels of proteins relevant to the G 1 to S phase transition. Notably, although minimal to no changes were observed in the levels of Cdk2, Cdk4, Cdk6, p16, p21, cyclin E, and cyclin D, a significant increase in p27 levels, a delay in the cell cycleassociated increase in cyclin A levels, and a lower level of Rb phosphorylation were observed in MEFs upon Ada3 deletion when compared with control cells (Fig. 3A).
In view of increased levels of p27 without a significant change in the levels of Cdk proteins in cells with Ada3 deletion, we assessed the level of Cdk2 kinase activity using an in vitro kinase assay on immunoprecipitates from cells. Although the Cdk4/6 kinase activity was comparable between control-and adeno-Cre-infected MEFs (Fig. 3B), the level of Cdk2 kinase activity was substantially reduced in Cre-infected MEFs when compared with control MEFs (Fig. 3B). These results suggest the potential reduction of Cdk2 kinase activity in the Ada3-deleted cells as a result of an increase in the levels of p27, accounting for defective Rb phosphorylation.
Accumulation of p27 upon Ada3 Deletion Is due to Increased Stability of p27-As accumulation of p27 levels upon Ada3 deletion appeared to be functionally important, we examined whether this accumulation was at the transcriptional or posttranscriptional level. Real-time PCR analysis showed that serum stimulation resulted in a marked reduction in the levels of Cdkn1b mRNA in both the control-infected and the Creinfected cells (Fig. 4A); furthermore, the levels of Cdkn1b mRNA at various time points after serum addition remained comparable between the two cell populations, reinforcing the idea that the increase in p27 protein levels in Ada3-deleted cells was likely to be at a post-transcriptional level. As alterations in protein stability are a prominent mechanism to control Cdk inhibitor levels (28), we compared the half-life of p27 protein in WT versus Ada3-deleted MEFs using two distinct experimental formats; the first one utilized exponentially growing cultures, whereas the second one utilized cells first arrested in G 1 by serum deprivation for 72 h followed by synchronous release into cell cycle by serum addition. In each case, Ada3 FL/FL MEFs infected with control or Cre adenoviruses were treated with cycloheximide to block new protein synthesis, and p27 levels in cell lysates following cycloheximide treatment were quantified using immunoblotting at various time points. Previous work has shown that p27 half-life in exponentially growing MEFs is about 3 h and increases to about 8 h in serum-starved cells (29). We found the p27 half-life in cells infected with control adenovirus was consistent with published results, i.e. approximately 2 h and 40 min in exponentially growing MEFs, whereas in growth-arrested cells, half-life was approximately 3 h and 30 min (Fig. 4, B-E). Notably, in both experimental formats, we observed a substantial increase in p27 protein half-life upon Cre-dependent Ada3 deletion, with approximate half-lives of 4 h and 10 min and 6 h in exponentially growing versus synchronous culture formats, respectively. These results strongly support our conclusion that accumulation of p27 protein upon Ada3 deletion is due to its increased stability.
Depletion of p27 from Conditionally Deleted Ada3 MEFs Causes a Partial Rescue of G 1 /S Progression Defects-Reduced activity of the p27 target Cdk2 in Ada3-deleted MEFs strongly suggested a role for p27 in defective cell cycle progression in these cells. To directly establish whether this is the case, we generated stable p27 knockdown Ada3 FL/FL MEFs (Ada3 FL/FL/p27shRNA ) by infecting Ada3 FL/FL MEFs with a retro- virus expressing a p27-specific shRNA followed by selection in puromycin, which resulted in a significant knockdown of p27 expression in these cells (Fig. 5A). Next, we infected the Ada3 FL/FL/p27shRNA MEFs with control or Cre adenovirus and analyzed these for cell cycle progression using serum deprivation followed by serum stimulation, as above (Fig. 5B). Notably, a partial but clear rescue of the G 1 /S delay was observed in p27 shRNA-expressing cells, as seen by a much larger percentage of cells entering the S phase ( 2B). Importantly, the levels of cyclin A, which is known to be expressed during G 1 /S transition and to peak in the S phase, as well as hyperphosphorylation of Rb, were essentially fully rescued by p27 shRNA knockdown (Fig. 5D; compare with Fig.  3A). Taken together, these results clearly demonstrate an important role of Ada3-dependent control of p27 levels in promoting cell cycle progression.
Deletion of Ada3 Leads to Reduced Protein and mRNA Levels of Skp2 and c-Myc-Given the causal link established above between p27 accumulation and G 1 /S cell cycle delay upon Ada3 deletion, we wished to examine the molecular mechanism by which loss of Ada3 promotes p27 stability. Published studies have established a major role of Skp2-containing E3 ubiquitin ligases in regulating p27 protein turnover during cell cycle progression (30). As Skp2 is a transcriptional target of c-Myc (31) and Ada3-containing STAGA complex has been shown to increase myc mRNA transcription (32,33), the possibility of an Ada3-c-Myc-Skp2-p27 regulatory pathway appeared to be a plausible mechanism for our findings. To explore this hypothesis, we first examined the effects of Ada3 deletion on the levels of Skp2 mRNA (real-time PCR) and protein (immunoblotting). For this purpose, Ada3 FL/FL cells infected with control or Cre adenovirus were serumdeprived and released into synchronous cell cycle progression by adding serum followed by analyses of Skp2 mRNA and protein at various time points. Notably, Skp2 mRNA and protein levels were substantially lower at each comparable time point in adeno-Cre-infected versus control MEFs (Fig.  6, A and B). These results indicate that Ada3 deletion indeed leads to reduction in Skp2 levels and that this effect is likely due to reduced Skp2 gene transcription.
Next, we asked whether Ada3 deletion alters c-Myc mRNA levels and whether Ada3 directly binds to c-myc promoter. Indeed, analysis of control versus Ada3-deleted MEFs stimulated with serum to undergo cell cycle progression demonstrated that c-Myc mRNA as well as protein levels were significantly lower at each time point examined upon deletion of Ada3 from cells (Fig. 6, C  and D). Consistent with this, we observed lower occupancy of mouse Skp2 promoter by c-Myc upon deletion of Ada3, which supports our results (supplemental Fig. S3). Finally, to establish that Ada3 indeed participates in the enhancement of myc gene transcription, we carried out ChIP analysis to assess whether Ada3 is recruited to c-myc enhancer during cell cycle progression. Indeed, a rapid recruitment of Ada3, as well as RNA polymerase II (used as positive control), to c-myc enhancer at Ϫ1.4 kb relative to transcription start site (but not to a distal site at Ϫ5 kb) was seen upon serum stimulation of MEFs (Fig. 6E). As expected, we did not detect any signals after immunoprecipitation with anti-Ada3 antibody in cells infected with adeno-Cre. These results therefore sup- port the existence of a novel cell cycle-associated, Ada3-regulated signaling pathway that promotes G 1 /S cell cycle progression by regulating p27 stability through Myc-dependent control of Skp2 expression.
Ada3 Deletion Leads to Decreased Histone Acetylation-As we observed a partial rescue of G 1 /S transition in Ada3-deleted MEFs after knockdown of p27, we speculated that Ada3 deletion-induced cell cycle arrest may involve other pathways as well. Given the known literature on Ada3 as part of HAT complexes (8,9), we examined whether Ada3 is involved in controlling global histone acetylation. Therefore, we assessed the effect of Ada3 deletion on lysine acetylation of various core histones. We expressed Cre recombinase in Ada3 FL/FL MEFs and harvested protein samples from asynchronous cultures after 3 days of infection. Western blotting using antibodies against important acetylated lysine residues of all four core histones (H2A-K5, H2B-K5, H3-K9, H3-K56, and H4-K8) showed a significant reduction in acetylation at all these sites in Ada3-deficient MEFs when compared with control MEFs (Fig. 7A), indicating that Ada3 is essential in maintaining global histone acetylation.
We further examined the effect of Ada3 deletion on acetylation of core histones after synchronizing cells in G 1 phase and subsequent release. There was a dramatic down-regulation of H3-K9 acetylation and a slight decrease in acetylation of H2B-K5 in Ada3-deleted MEFs when compared with control-MEFs, whereas this defect was rescued in Ada3 FL/FL MEFs reconstituted with exogenous human FLAG-Ada3 (Fig. 7B), suggesting that the defect in histone acetylation seen in Ada3deleted MEFs was a consequence of Ada3 deletion. Histone acetylation has been shown to be important for deposition of histones during replication-coupled nucleosome assembly as well as for chromatin maturation following DNA replication (34,35). Thus, the partial rescue in G 1 to S transition observed upon knockdown of p27 in Ada3-deficient cells could be attributed to massive histone acetylation defects, which would create difficulties for cells to undergo DNA replication and thus delay transition through S phase. B, 48 h after adenovirus infection, MEFs were treated with 50 g/ml cycloheximide and harvested at the indicated time points, and p27 and ␤-actin protein levels were analyzed by immunoblotting. C, the intensity of p27 bands was quantified by densitometry, normalized to ␤-actin using ImageJ software, and plotted against the time of cycloheximide treatment. Each decrease of 1 unit of log 2 is equivalent to one half-life. The lines were generated by linear regression formula. D, after 48 h of adenovirus infection, MEFs were starved using 0.1% serum-containing medium for 72 h and subsequently treated with 50 g/ml cycloheximide and harvested at the indicated time points. Cell lysates were analyzed by Western blotting using antibodies against p27 and ␤-actin. E, graph made from experiment in D by using the same procedure as in C.

Recombinant Ada3 Stabilizes HAT Enzymes and Enhances
Their Activity-Ada3 protein has been identified as an important component of protein complexes containing HAT enzymes. Therefore, we subjected samples harvested after 3 days of Ada3 deletion to immunoblotting with two important HATs such as p300 and PCAF. Indeed, deletion of Ada3 caused drastic down-regulation of p300 and PCAF in MEFs (Fig. 7C). Notably, Ada3 deletion had no effect on the mRNA levels of p300 and PCAF (data not shown). Thus, the defects in histone acetylation seen in Ada3-null MEFs could be attributed to the effect of Ada3 deletion on stability of important HATs in cells.
In addition to the role of Ada3 in stability of HAT enzymes, we explored whether Ada3 catalyzes the activity of HAT enzymes. Although Ada3 is shown to be important in maintaining stability of HAT complexes, it has not been demonstrated whether Ada3 directly modulates the activity of known HAT enzymes such as p300. Thus, we expressed and purified baculoviral hAda3 and used it in an in vitro assay in which HAT activity of p300 histone acetyl transferase enzyme on histone substrates was measured. As seen in Fig. 7D, increasing amounts of Ada3 resulted in increased acetylation of histone H1 and histone H3 by p300, suggesting that Ada3 plays an important role in enhancing the HAT activity of p300. To further explore the role of Ada3 in histone acetylation, we used only histone H3 as a substrate and observed an Ada3 dose-dependent increase in acetylation of histone H3 by p300 (Fig. 7E). Thus, Ada3 manifests its effect on histone acetylation by main-taining the integrity of various HAT complexes and by enhancing the catalytic activity of HATs.
Deletion of Ada3 Leads to Global Gene Expression Changes-Given the links between Ada3 and transcriptional activation, we used control and Ada3-deleted cells to perform microarray analyses. As expected, the expression of multiple genes was altered; 539 genes were down-regulated and 928 genes were up-regulated Ն 1.5-fold upon Ada3 deletion (supplemental Table S1). Validation of some of the deregulated genes from microarray by real-time PCR showed good co-relation with the microarray data (supplemental Fig. S4). Ingenuity pathway analyses showed that most of the genes affected were involved in controlling cell growth, proliferation, and cell death (supplemental Table S2, top biological functions affected; cell growth and proliferation (386 genes) and cell death (359 genes)). The top network affected was the RNA posttranscriptional modification and cellular assembly and organization network, whereas the cell cycle, endocrine system development and function, and cancer network was the third most affected network (supplemental Fig. S5). Notably, c-myc and Skp2 genes that we described above were down-regulated 1.4-and 1.43fold, respectively. This is lower than what we observed by real-time PCR and could be attributed to the fact that microarray data were performed on asynchronous populations, whereas the real-time PCR data were performed on synchronous cells (Fig. 6, A and C). Interestingly, many of the genes present in cell growth and proliferation set were those involved in controlling cell division as well as some involved in DNA replication ( Table 2).  AUGUST 24, 2012 • VOLUME 287 • NUMBER 35

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Ada3 Deletion Leads to Defects in Cell Division and Accumulation of Abnormal Nuclei-Based on our microarray analyses where several mitotic genes were affected upon deletion of Ada3 and a recent study showing the role of Ada3 in mitosis upon shRNA deletion (14), we examined the effect of Ada3 deletion on mitotic phase of cell cycle. These analyses showed that Cre-mediated Ada3 deletion led to increased accumulation of cells with abnormal nuclei when compared with control MEFs. Ada3-deficient MEFs showed various nuclear abnormalities such as fragmentation, lobulation, and multinucleation (Fig. 8A). When compared with 13.08 Ϯ 2.39 S.E. % control MEFs, 83.41 Ϯ 3.45 S.E. % of Ada3-deficient MEFs showed abnormal nuclei (Fig. 8B). Live imaging of cells for 24 h showed that the majority of Ada3-deleted cells failed to divide normally. Some of the cells snapped back while attempting to undergo cytokinesis, leading to the formation of binucleated cells, whereas other cells that had normal nucleus before mitosis showed fragmented nuclei afterward and were unable to divide. In other cases, cell division resulted in the formation of anucleated daughter cells (Representative images shown in supplemental Fig. S6). Taken together, these results demonstrate an indispensable role of Ada3 in normal cell cycle progression. The cell division defect results reported here corroborate with an earlier published study showing similar defects upon shRNA knockdown of Ada3 (14). Mitotic defects observed in their study were attributed to acetylation of a non-histone substrate cyclin A, and no changes in histone acetylation upon knockdown of Ada3 were reported. In contrast, we observed a dramatic change in global histone acetylation and expression of various genes involved in mitosis. Although at present we cannot explain this discrepancy, the differences in the results may be partly attributable to the use of different cellular systems and differences in approaches followed such as shRNA or Cre-mediated to delete Ada3.
Deletion of Ada3 Leads to Delay in G 2 /M to G 1 Progression-As deletion of Ada3 in MEFs led to defects in cell division, we reasoned that the disruption of Ada3 should exert an effect on G 2 /M to G 1 transition. To examine this effect, we synchronized control-and Cre-adenovirus-infected Ada3 FL/FL MEFs at G 2 /M checkpoint by treating them with nocodazole and released them from synchrony followed by cell cycle analysis using flow cytometry (Fig. 8C). Nocodazole-synchronized Ada3-deleted MEFs showed a lower percentage of cells in G 2 /M phase (61%) at the 0-h time point when compared with control MEFs (80%) (Fig. 5C). On the contrary, we observed a higher percentage (20%) of Ada3-deleted MEFs in G 1 phase when compared with control MEFs (7%) after synchronization. We speculate that Ada3-deficient MEFs that are exhibiting a delay in G 1 to S transition were unable to get completely synchronized at G 2 /M checkpoint as these cells are potentially moving slowly through the G 1 to S transition and require a prolonged treatment with nocodazole to show a complete synchronization as seen in control MEFs. When we compared the percentage of cells moving into G 1 phase on release from nocodazole treatment in both Ada3-deficient and control MEFs, a significant impairment in G 2 /M to G 1 transition in Ada3-deleted MEFs was observed (Fig. 8D). Taken together, these results demonstrate a critical role of Ada3 in both G 1 to S transition as well as G 2 /M to G 1 transition in MEFs, indicating that the cell proliferation defect observed in Ada3-deficient MEFs is due to a combined defect in G 1 to S as well as G 2 /M to G 1 transition.

DISCUSSION
Regulated cell cycle entry and progression are essential for precise developmental programs as well as to maintain organ homeostasis in adult animals. Although the basic components of cell cycle have been largely defined, regulatory control mechanisms that ensure orderly proliferative responses to physiological cues and whose aberrations underlie the vast instances of altered proliferation in cancer continue to be elucidated. We previously identified the ADA complex component Ada3 as a human papillomavirus E6 oncoprotein partner as well as a coactivator of cell cycle checkpoint regulator and tumor suppressor p53 (15,20). Several in vitro studies have shown that Ada3 is an essentially universal component of a multitude of HAT-based transcriptional regulatory complexes (8,9), and it has become essential to define its physiological roles using in vivo animal models.
Here, we demonstrate that Ada3 is essential for embryonic development in mice and that Ada3-null embryos undergo very early lethality. As an essential component of the transcriptional coactivator complexes that include HATs and promote histone acetylation of key gene targets, Ada3 is known to be essential for growth in yeast (16) as well as in model metazoan organisms such as Drosophila where Ada3 deficiency is associated with arrest in early development (36). However, this study is the first direct demonstration of an essential role of Ada3 in mammalian embryonic development. Notably, the embryonic developmental block imposed by Ada3 deletion occurs very early, resulting in arrest of development at the blastocyst stage, the stage of embryonic development at which extensive cell proliferation occurs (37). Notably, studies that employed gene knockouts of subunits of several chromatin-modifying complexes, including Gcn5, Trrap, Ep300, CBP, Hdac3, or Atac2, also lead to early embryonic lethality (34, 38 -42), consistent with an essential role of chromatin modification machinery in mammalian growth and development. However, except for Trrap knockout, which produces lethality at the blastocyst stage (42), knockouts of other genes produce embryonic developmental arrest at much later stages: for example, Gcn5 (E9.5-E11.5), Ep300 (E9.5-E10.5), and Atac2 (E11.5) in comparison with E3.5 block observed in Ada3-null mice. The relatively early developmental arrest of Ada3-null mice when compared with other regulators could reflect the role of Ada3 as a component of multiple chromatin-remodeling complexes (see Introduction and below). The distinct times of arrest seen with Gcn5-null and Ada3-null embryos are somewhat surprising and suggest the possibility that Ada3 may mediate early developmental roles through complexes in which Gcn5 is not a critical component or is functionally redundant with other HATs. Consistent with this hypothesis, we observed that Ada3-deleted cells exhibit defects in multiple histone acetylations and show decrease in the levels of PCAF and p300 proteins.
We used the conditional deletion feature of the mouse model to assess the critical functional roles of Ada3 by utilizing Credependent gene deletion in MEFs from Ada3 FL/FL mice. This system provided a clear evidence that Ada3 plays an essential

List of deregulated genes involved in cell division and DNA replication
Genes down-regulated at least 1.5-fold upon loss of Ada3 as obtained from microarray analyses. The genes were classified based upon gene ontology biological processes. Origin recognition complex, subunit 1-like (S. cerevisiae) 1.6

Rpa1
Replication protein A1 1.6 Cdt1 Chromatin licensing and DNA replication factor 1 1.6 Gins2 GINS complex subunit 2 (Psf2 homolog) 1.5 Rbbp4 Retinoblastoma-binding protein 4 1.5 Chaf1b Chromatin assembly factor 1, subunit B (p60) 1.5 Tk1 Thymidine kinase 1 1.5 role in cell proliferation by promoting G 1 to S as well as G 2 /M to G 1 cell cycle progression. Furthermore, the proliferative arrest imposed by conditional deletion of Ada3 was reversed by ectopic expression of human Ada3, indicating that the loss of Ada3 itself, rather than alteration of any neighboring gene product, was responsible for the observed cell cycle phenotype. Cell cycle progression is a tightly regulated process and is dependent on sequential and stringently controlled, concerted activation of Cdks and their inhibition by Cdk inhibitors. The novel cell cycle regulatory pathway downstream of Ada3 was suggested by our initial analyses of alterations in the levels of core components of mammalian cell cycle machinery. These analyses revealed a dramatic reduction in the key propeller of G 1 /S phase transition, hypophosphorylated Rb when Ada3 was deleted. Association of this defect with reduced Cdk2 activity without a reduction in Cdk2 levels suggested the role of elevated p27, which we established directly by demonstrating that shRNA knockdown of p27 substantially alleviated the G 1 /S block imposed by Ada3 deficiency. Further biochemical connections were suggested by recent findings that STAGA complex, which includes Ada3 as a component, enhances c-myc transcription (32,33). Because c-Myc is shown to regulate the transcription of Skp2, an essential component of the SCF(Skp2) cell cycle-associated E3 ligase that regulates p27 levels, we sought and established evidence that cell cycle-associated Myc transcription is Ada3-dependent and that Ada3 is required for Skp2 transcription (which is a downstream target of Myc) and p27 stability (regulated by SCF(Skp2)). We provided direct evidence for key elements of this model, including ChIP analyses that demonstrated the cell cycle-associated early recruitment of Ada3 to c-myc enhancer elements. This result is consistent with independent findings from two groups that STAGA complex is recruited to c-Myc enhancer and regulates c-myc transcription (32,33). In addition to control of c-myc gene transcription by Ada3-containing STAGA complex, studies have shown that STAGA associates with c-Myc on c-Myc target gene promoters and is required for efficient transcription activation by c-Myc (43,44). This provides an additional mechanism by which Ada3 could control c-Myc-driven target genes that regulate cell proliferation. Thus, Ada3 might be involved in controlling both c-myc transcription as well as c-Myc function. Consistent with our observations, it is noteworthy that c-myc knock-out mice are embryonic lethal (45). Defective regulation of c-Myc transcription by Ada3-containing (STAGA or other) complexes might contribute to the early embryonic lethality seen in Ada3-null mice; further analyses of Myc-dependent pathways upon germline or conditional deletion of Ada3 during embryogenesis should help establish whether this is the case.
Although regulation of p27 protein stability by Ada3 through control of c-myc transcription forms an important basis for G 1 /S transition defects, we were not able to fully rescue the defect in cell cycle by using p27 shRNA, suggesting the involvement of other cellular pathways. To this end, examining global histone acetylations in Ada3-deficient cells revealed dramatic defects in histone acetylation. Because Ada3 forms a core structural component of various different HAT complexes in the cell, the presence of Ada3 is highly essential for structural maintenance and proper functioning of these complexes in cells. Additionally, loss of Ada3 led to substantial depletion of important HATs, p300, and PCAF proteins but not mRNA, which further explains the profound defects in histone acetylation seen upon loss of Ada3. This is consistent with the fact that PCAF and p300 are present in Ada3-containing protein complexes (8 -11). These defects in histone acetylation could explain the partial rescue upon knockdown of p27 as histone acetylation has been shown to have an important role in the process of DNA replication (34,35).
Given the role of Ada3 in regulating global histone acetylation and that histone acetylation is important in transcriptional activation of genes, we performed microarray analysis and showed that several genes were deregulated upon Ada3 deletion. Analysis of these genes by ingenuity pathway analysis revealed the RNA post-transcriptional modification and cellular assembly and organization network as the top affected network, with the cell cycle, endocrine system development and function, and cancer network as the third most affected. The top network affected in the microarray data is consistent with an earlier study, which showed that Ada3-containing STAGA complex interacts with pre-mRNA splicing machinery, components suggesting a role for this complex in mRNA splicing (46). Importantly, the top biological functions affected upon deletion of Ada3 included those involved in cell growth and proliferation with 386 deregulated genes involved in this process. Thus, our microarray data confirmed a role of Ada3 in cell cycle progression. Additionally, some of the top physiological functions affected upon deletion of Ada3 were those involving tissue development and organismal survival (supplemental Table S2), which could be linked to the early embryonic lethality observed upon knock-out of Ada3 in mouse.
Notably, many of the genes that were involved in regulating cell growth and proliferation were those involved in mitosis and some that were involved in DNA replication. This led us to examine cell division upon deletion of Ada3. Consistent with the microarray data, we observed massive nuclear abnormalities, cell division defects, and delay in G 2 /M to G 1 phase progression upon deletion of Ada3. Our observed phenomenon of cell division defects upon deletion of Ada3 is consistent with a recently published study (14). The authors showed that ATAC HAT complex is specifically involved in regulating mitosis and that shRNA-mediated knockdown of Ada3 or Ada2a led to defects in cell division, which were attributed to stabilization of cyclin A upon disruption of ATAC complex. Although we did not observe an increase in cyclin A levels (in fact the converse) in our system, we did observe a similar effect on nuclear abnormalities and a clear defect in mitosis. Furthermore, the authors did not observe any changes in histone acetylation defects upon depletion of Ada3, which is not consistent with our results. Of note, Ada2a is a component of only ATAC complex; however, Ada3 has been shown to be a core component of a number of HAT complexes. The authors used depletion of Ada3 as an indication of disruption of only ATAC complex; however, deletion of Ada3 would affect several HAT complexes and not just ATAC complex. Thus, deletion of Ada3 would cause disruption of several HAT complexes that function in different phases of the cell cycle leading to defects in various phases of the cell cycle. Based on these findings, we propose the following working model of Ada3 regulation of cell cycle progression. As part of a chromatin-remodeling complex, likely the STAGA complex, Ada3 is recruited to and modifies the c-myc transcriptional regulatory elements to enhance Skp2 transcription. This leads to destabilization of p27 by the SCF(Skp2) E3 ligase, resulting in increased Cdk2 activity and Rb phosphorylation to promote G 1 /S progression. Additionally, Ada3, by regulating the number of genes involved in mitosis, regulates cell division. Lastly, Ada3 as part of ATAC and STAGA complex regulates transcription of various genes by recruiting HATs and acetylating histones. Combination of these functions led to severe cell cycle defect and embryonic lethality upon Ada3 deletion (Fig. 9). Finally, although our studies here have focused on the role of Ada3 in cell cycle progression, future studies using cell type-or stage-specific condi- As a core structural component of various HAT complexes, Ada3 maintains the integrity of HAT complexes and thus regulates global histone acetylation. Ada3 regulates G 1 to S transition by controlling transcription of c-myc gene, which in turn controls Skp2 gene expression by binding to its promoter. Skp2 as an E3 ubiquitin ligase causes timely degradation of p27 protein so that cells can enter into S phase by increasing Cdk2 kinase activity, thus inducing hyperphosphorylation of Rb and cell progression from G 1 to S phase of cell cycle. Additionally, through controlling global histone acetylation, Ada3 controls transcription of various genes involved in cell division and is required for cells to undergo normal mitosis and G 2 /M to G 1 progression. tional deletion of Ada3 in mouse to assess its role in functions other than transcriptional activation, including optimal transcription elongation, mRNA export, and nucleotide excision repair, need to be explored (8,46,47).
In conclusion, we demonstrate that the evolutionarily conserved Ada3 protein as an essential component of HAT complex plays an important role in embryogenesis and cell division. Thus, our studies identify Ada3 as a novel component of the physiological regulation of mammalian cell cycle progression and set the stage for future studies to assess the role of Ada3 in cell cycle progression during in vivo physiological and pathological settings. Use of Ada3 FL/FL mice should facilitate these analyses to functionally dissect the in vivo roles of Ada3.