Interplay Between P300 and HDAC1 Regulate Acetylation and Stability of Api5 to 2 Regulate Cell Proliferation

bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 Interplay between p300 and HDAC1 regulate acetylation and stability of Api5 to 2 regulate cell proliferation

3 Virender Kumar Sharma and Mayurika Lahiri*

4 Department of Biology, Indian Institute of Science Education and Research, Dr. Homi 5 Bhabha Road, Pune, Maharashtra 411008, India

6 *Author for correspondence ([email protected])

7 Orcid ID: M.L., 0000-0001-9456-4920, V.K.S- 0000-0003-1541-4006

8 Key words: Api5, p300 histone acetyl transferase, HDAC1, acetylation, cell cycle

10 Abstract

11 Api5, is a known anti-apoptotic and nuclear protein that is responsible for inhibiting cell 12 death in serum-starved conditions. The only known post-translational modification of Api5 is 13 acetylation at lysine 251 (K251). K251 acetylation of Api5 is responsible for maintaining its 14 stability while de-acetylated form of Api5 is unstable. This study aimed to find out the 15 enzymes regulating acetylation and deacetylation of Api5 and the effect of acetylation on its 16 function. Our studies suggest that acetylation of Api5 at lysine 251 is mediated by p300 17 histone acetyltransferase while de-acetylation is carried out by HDAC1. Inhibition of 18 acetylation by p300 leads to reduction in Api5 levels while inhibition of deacetylation by 19 HDAC1 results in increased levels of Api5. This dynamic switch between acetylation and 20 deacetylation regulate the localization of Api5 in the cell. This study also demonstrates that 21 the regulation of acetylation and deacetylation of Api5 is an essential factor for the 22 progression of the cell cycle.

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24 Introduction.

25 Apoptosis inhibitor 5 (Api5) is also known as anti-apoptotic clone 11, AAC-11. Api5 was 26 discovered as a nuclear protein, which was responsible for inhibition of apoptosis under 27 serum starvation conditions [1]. Api5 is a right handed superhelix which is composed of all

28 helical repeats. It has 19 α-helices and two 310 helices [2]. It has also been observed that all 29 these helices interact with neighbouring helices to form antiparallel helix pair. N-terminal of 30 Api5 comprises of α-helix from 1 to 11 which shares homology with the HEAT repeat 31 structures of other proteins such as Importin β. C- terminal of Api5 is made up of α-helices 32 from residues 12 - 19 and share homology with ARM-like repeat structures of other proteins, 33 for example, p120 catenin. The N-terminal of Api5 contains LxxLL motif which is an 34 amphipathic α-helix that forms nuclear receptor co-factor interaction regions. This motif is 35 present in the sixth α−helix and is required for maintaining stability of the protein by forming 36 hydrophobic interactions with neighbouring α-helices and is also the region that interacts 37 with FGF2 [3]. The C-terminal region of Api5 has a nuclear localization signal and a putative 38 Leucine Zipper domain (LZD). LZD is present in the α18 helix, between 371- 391 amino 39 acid residues. It interacts with Acinus, a protein which mediates fragmentation of chromatin 40 during apoptosis [4]. Global mass spectrometric analysis of Api5 suggests the presence of 41 one conserved acetylation site at lysine 251, which is present in the hinge region between 42 α13 and α14 [2] .

43 It has been proven that Api5 inhibits the dE2F1-mediated apoptosis in Drosophila cells [5]. 44 dE2F1 is a well-known pro-apoptotic gene responsible for apoptosis in the fruit flies. In the 45 Drosophila embryonic cells, SL2, RNAi-mediated depletion of Api5 was found to be 46 responsible for increased apoptotic cell death as compared to the dE2F1 over-expressed cells. 47 Similar result was also observed in the human osteosarcoma cell line, Saos-2, where ectopic 48 expression of Api5 decreased E2F1-mediated apoptosis in E2F1 over-expressing cells 49 without affecting its transcriptional activity [5].

50 According to the study by Rigou et al., AAC-11 binds to Acinus, a nuclear protein and plays 51 a significant role in chromatin condensation during apoptosis [4]. Binding of AAC-11 to 52 Acinus does not allow its cleavage by caspase-3, thus in turn inhibiting DNA fragmentation 53 and apoptosis [4].

54 Studies performed in melanoma cells, showed Api5 to modulate FGF2 and FGFR1 signalling 55 which activates ERK. This activated ERK phosphorylates Bim, a pro-apoptotic protein.

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56 Phosphorylated Bim is the target for proteosomal degradation. Thus ubiquitin-mediated 57 degradation of Bim is a means by which Api5 inhibits apoptosis in HeLa and 565mel cell 58 lines [6].

59 Mayank and group have reported Api5 to inhibit transcription of APAF1 gene. APAF1 is the 60 main component of the apoptosome complex. Thus Api5 prevents the formation of the 61 apoptosome, in turn inhibiting apoptosis [7]. A recent study suggests Api5 to physically 62 interact with caspase 2, and prevent its activation, thus inhibiting apoptosis [8].

63 Api5 has also been reported to be involved in the regulation of E2F1, a transcriptional 64 activator of G1/S cell cycle transition genes, by enhancing the binding affinity of E2F1 to its 65 target promoters. Knockdown of Api5 arrested H1299 cells at the G1 phase of the cell cycle, 66 thus proving that apart from regulating apoptosis, Api5 also plays a critical role in 67 maintaining normal cell cycle progression [9].

68 Navarro and co-workers have demonstrated that the levels of Api5 are regulated in a cyclic 69 manner [9]. It was observed that the levels of Api5 was higher in the G1 phase and was 70 stabilized during the G1/S transition. Interestingly, Api5 levels decreased as the cells 71 proceeded further in the cell cycle, from G2 to G2/M phase [9]. This suggests that Api5 72 undergoes degradation during cell cycle progression. Knockdown of Api5 arrests H1299 non- 73 small cell lung carcinoma cells at the G1 phase. This was further supported by Han and his 74 group where they showed acetylation of Api5 at lysine 251 to be associated with its stability 75 [2].

76 Api5 has been found to be overexpressed in different types of cancers like cervical, urinary 77 bladder, lung, ovarian and oesophageal cancers [6; 10; 11; 12; 13]. In cervical cancers, Api5 78 overexpression has been shown to promote invasion [12]. Api5 has also been shown to 79 promote the degradation of Bim, a pro-apoptotic protein [6]. In osteosarcomas, studies have 80 shown Api5 to inhibit E2F1 as well as Acinus-mediated apoptosis [4; 5]. Api5 has been 81 identified as a biomarker for cervical and ovarian cancers and a prognosis marker for non- 82 small cell lung carcinomas [11; 13]. High levels of Api5 provide cancer cells the ability to 83 evade immune response mediated cell death [6]. In breast cancers, Api5 interacts with the 84 estrogen receptor to promote proliferation [14]. It has also been reported that Api5 promotes 85 metastasis in breast cancers [14]. Higher levels of Api5 are associated with chemo-resistance 86 [15]. It has been shown that tamoxifen-resistant breast cancer cells show an upregulation of 87 Api5 [16], while cancer cells which are sensitive to anticancer agents like tocotreinol show

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88 reduced levels of Api5 [17]. Reduced and low levels of Api5 are associated with the increase 89 in cell death in various cancers. Knockdown of Api5 resulted in the reduction in in vivo 90 tumorogenicity in cancer cells [14].

91 Api5 acetylation at lysine 251 is conserved from protists to mammals [2]. De-acetylated form 92 of Api5 is not stable and therefore undergoes post translational degradation. However the 93 mechanism of degradation and the enzymes involved in the process of acetylation and de- 94 acetylation of Api5 is not yet known.

95 CBP/p300, GCN5/PCAF and TIP60/MYST1/2/3/4 are the major acetyltransferases involved 96 in acetylation of most of the cellular proteins. Among this, p300 acetylates proteins involved 97 in a number of diverse biological functions including proliferation, cell cycle regulation, 98 apoptosis, differentiation and DNA damage response [18; 19; 20; 21]. p300 histone acetyl 99 transferase was initially identified as a transcriptional activator that performs its function by 100 acetylating histones in eukaryotic cells . p300 is capable of acetylating all the four histones 101 [22; 23]. Later it was discovered that p300 also acetylates non-histone proteins like E2F1, 102 p53, p73, Rb, E2F, myb, myoD, HMG(I)Y, GATA1 and α-importin [24; 25; 26; 27; 28; 29; 103 30; 31; 32; 33]. The role of p300 histone acetyl transferase in the regulation of the cell cycle 104 is also known [34]. Activity of p300 is required for normal transition from G1 to S phase of 105 cell cycle [35]. Mutations or abnormality in function of p300 leads to multiple disorders and 106 cancer is most common amongst them [36; 37; 38; 39].

107 Histone de-acetylases (HDACs) are the major de-acetylases involved in the de-acetylation of 108 histones that regulate genetic expression of certain genes as well as non-histone proteins that 109 regulate the processes responsible for maintaining cellular homeostasis [40; 41; 42; 43]. 110 HDAC de-acetylates proteins involved in almost all the cellular events like cell cycle, 111 replication, proliferation and apoptosis. It has also been shown that most of the proteins that 112 get acetylated by p300 histone acetyltransferase also undergo de-acetylation by HDAC1, a 113 member of class1 HDACs, for example p53 and E2F1 [28; 42]. Han and group in their 114 studies have suggested the possibility of involvement of histone de-acetylases in the de- 115 acetylation of Api5 [2].

116 AMP-activated protein kinase (AMPK) is an energy sensor kinase that regulates almost all 117 cellular processes like cell division, DNA replication, transcription activation and apoptosis 118 [44; 45; 46] by sensing AMP to ATP ratio and thereby regulating cellular homeostasis. It has

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119 also been reported that AMPK regulates G1/S transition of cells [47]. Mutations in AMPK 120 can lead to multiple disorders including cancer [48].

121 Protein kinase B (Akt) has various functions in cells that include proliferation, migration and 122 transcription. Studies have shown PI3K/Akt pathway to also regulate cell cycle progression 123 by phosphorylating the inhibitors of cyclin-dependent kinases (CKI) p21 and p27 Cip/Kip 124 proteins [49; 50; 51]. Akt has also been shown to play a role in regulating different pro- 125 apoptotic molecules in order to enhance survival and proliferation of cancer cells [52].

126 In this study, we report two novel and key regulators of Api5: p300 and HDAC-1. We 127 observed p300 to be the enzyme that interacts with and regulate Api5 levels in cells. p300 128 mediated acetylation of Api5 at lysine 251 provided stability to Api5, while the HDAC1- 129 mediated deacetylation led to reduced levels of Api5 in the cells. Both these regulators also 130 regulated the subcellular localization of Api5 in cells. We observed that the de-acetylated 131 protein was transported to the cytoplasm while the acetylated protein was present in the 132 nucleus. The regulation of acetylation was also observed to regulate cell cycle progression. 133 We also demonstrated AMPK and Akt to be the players regulating p300 activity, thereby 134 leading to stability of Api5 in cells.

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135 Results

136 in silico analysis predicts p300 histone acetyltransferase to acetylate Api5 at lysine 251

137 It has been reported in previous studies that Api5 undergoes acetylation at lysine 251, 138 however, the enzyme that is required for the acetylation function has not yet been identified. 139 Therefore, to identify the enzyme involved in the acetylation of Api5 we performed in silico 140 analysis using the ASEB web server tool [53; 54] to predict the site as well as the enzyme 141 which may be responsible for acetylation of Api5 at lysine 251. It was observed that amongst 142 CBP/p300, GCN5/PCAF and TIP60/MYST1/2/3/4, CBP/ p300 had a lower p value 143 suggesting a higher possibility of acetylating Api5 at lysine 251 (Figure 1A). Active site 144 residues of p300 histone acetyltransferase were obtained from the literature [55]. Docking 145 was performed between p300 and Api5 using the HADDOCK webserver [56]. The output 146 was analyzed using Protein Interaction Z Score Assessment (PIZSA) [57] and the best 147 scoring model was chosen as shown in figure (1B-D). Interestingly the acetylating domain of 148 p300 showed interaction with three amino acid residues of Api5 including lysine 251 (Figure 149 1C-D).

150 p300 interacts with and regulates Api5

151 Acetylation of Api5 at K251 is responsible for maintaining its stability [2]. To prove whether 152 p300 histone acetyltransferase can regulate Api5 acetylation and thus stability, cells were 153 treated with different concentrations of a small molecule chemical inhibitor of p300 (p300i) 154 and levels of Api5 were analyzed. Api5 protein expression was reduced upon p300 inhibition 155 (Figure 2A-B) without affecting the transcript expression of Api5 (Figure 2C-D), thus 156 demonstrating that p300 might be regulating Api5 at the post-translational level. In order to 157 acetylate Api5, the interaction between p300 and Api5 is essential. Thus, to find out whether 158 p300 and Api5 physically interact or not, stable cells expressing mCherry-tagged Api5 were 159 immunoprecipitated using GFP- specific antibody (Figure 2E). The presence of p300 histone 160 acetyltransferase in the GFP pull down lysate confirmed the interaction between Api5 with 161 p300. This interaction was further corroborated by reverse immunoprecipitation using p300 162 antibody and the presence of Api5 was confirmed using GFP specific antibody (Figure 2F). 163 These results conclusively demonstrate p300 to interact with Api5 and thus regulate Api5 164 protein expression without affecting the transcript levels.

165 HDAC1 interacts with and regulates levels of Api5

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166 From the previous results, we discovered p300 to be the histone acetyltransferase to regulate 167 Api5 protein levels. Next, we were interested in identifying the de-acetylase enzyme which 168 might be involved in regulating the stability of Api5.

169 HDAC1 is one of the member of the large family of histone de-acetylases. De-acetylation 170 activity of HDAC1, a class 1 HDAC protein, is coupled with the acetylation activity of p300. 171 Interestingly, HDAC1 has also been reported to be a part of the Api5 interactome [58]. To 172 investigate whether HDAC1 regulates Api5, cells were treated with different concentrations 173 of romidepsin, a HDAC class 1 inhibitor (HDACi), and Api5 protein levels were analyzed. 174 Api5 protein levels increased upon treatment with HDACi suggesting that HDAC1 may be 175 involved in regulating the stability of Api5 (Figure 3A-B). The accumulation of Api5 upon 176 inhibition of HDAC1 could also be the result of increased transcription of Api5. To rule out 177 this possibility, Api5 transcript levels were analyzed upon HDAC1 inhibition. Api5 transcript 178 levels remain unaltered upon romidepsin treatment as shown in Figure 3C-D. These results 179 suggest HDAC1 to play a role in regulating the stability of Api5 at the post translational 180 level. In order to decipher whether HDAC1 interacted with Api5 to bring about the 181 regulation, immunoprecipitation studies were conducted. To demonstrate the interaction of 182 Api5 with HDAC1, whole cell lysates of mCherry-Api5 overexpressing stable cells were 183 immune-precipitated using GFP-specific antibody (Figure 3E). Presence of HDAC1 in the 184 GFP pull down lysates confirmed the interaction between HDAC1 and Api5. This interaction 185 was further corroborated by reverse immuno-precipitation and pull down was performed 186 using HDAC1 specific antibody. Api5 was found to be present in the HDAC1 pull down 187 lysates (Figure 3F). These studies thus validate that HDAC1 interacts with and regulate Api5 188 at the protein level.

189 Api5 is acetylated by p300 and de-acetylated by HDAC1 at lysine 251 residue

190 Previous experiments suggested p300 histone acetyltransferase and HDAC1 as the 191 acetylating and de-acetylating enzymes respectively that regulate the levels of Api5. It was 192 hypothesized that these two enzymes might also regulate the dynamics of acetylation and de- 193 acetylation of Api5. Therefore, the acetylation status of Api5 was investigated upon 194 inhibition of both p300 and HDAC1.

195 To investigate whether Api5 is acetylated by p300, mCherry-Api5 overexpressing stable cells 196 were treated with and without p300i and immunoprecipitation studies were performed. 197 Acetylated Api5 were detected using L-Acetyl lysine specific antibody. Api5 acetylation was

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198 observed to be reduced upon inhibition of p300 when compared to the untreated control as 199 shown in Figure 4A. To check for the effect of HDAC1 inhibition using romidepsin on the 200 acetylation status of Api5 similar immunoprecipitation experiments were performed with and 201 without romidepsin. Acetylated levels of Api5 increased upon inhibition of HDAC1 (Figure 202 4B). This confirms that Api5 undergoes acetylation and de-acetylation by p300 and HDAC1 203 respectively. Earlier studies by Han and group have shown that Api5 is acetylated at lysine 204 251 [2]. To identify whether Api5 acetylation and deacetylation by p300 and HDAC1 205 respectively occurs at the lysine 251 residue, K251 mutants of Api5 were generated using 206 site-directed mutagenesis. mVenus-tagged wild type, K251A (acetylation-deficient mutant), 207 K251R (charge-mimic mutant) and K251Q (constitutive acetylation mimic) mutants of Api5 208 were ectopically expressed in cells. The expression levels of mVenus, which is a proxy for 209 ectopic expression of Api5 K251 mutants were analyzed in the presence and absence of p300i 210 and romidepsin using GFP-specific antibody. It was observed that levels of wild-type Api5 211 were reduced while the levels of K251A, K251Q and K251R mutants of Api5 remained 212 unaffected upon p300 inhibition (Figure 4C and D). However, the levels of wild-type Api5 213 increased upon inhibition of HDAC1 by romidepsin whereas that of the other mutants 214 remained unchanged (Figure 4E and F). Therefore, it was established that p300 acetylates 215 while HDAC1 de-acetylates Api5 at the lysine 251 residue.

216 Acetylation of Api5 by p300 is the signal for its nuclear localization while de-acetylation 217 leads to the translocation of Api5 to the cytoplasm.

218 From the previous results, it can be inferred that p300 acetylates Api5 at lysine 251, thereby 219 maintaining its stability. It has earlier been reported that Api5 is a nuclear protein. To 220 investigate the role of acetylation on Api5 function, cells were treated with the acetylation 221 and deacetylation inhibitors and immunofluorescence was performed. In control cells, Api5 222 was observed to be present in the nucleus while upon inhibition of p300, the percentage of 223 cells showing Api5 to be present in the nucleus was negligible. Moreover, most cells showed 224 nuclear and cytoplasmic localization of Api5 (Figure 5A-B). Interestingly, 24% of cells 225 showed only cytoplasmic presence of Api5 upon p300 inhibition which was not observed in 226 the control cells (Figure 5B). Api5 localization was also investigated upon inhibition of 227 HDAC1. We found that, upon HDAC1 inhibition, majority of the cells (67%) showed both 228 nuclear and cytoplasmic localization of Api5, whereas 29% of cells showed only nuclear 229 localization of Api5 (Figure 5C-D). Only 4% of cells showed cytoplasmic localization of 230 Api5 which is negligible. To follow the compartmentalization switch of Api5, both p300 and

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231 HDAC1 were inhibited. The cytoplasmic localization of Api5 that was observed upon p300 232 inhibition as well as the nuclear localization observed upon HDAC1 inhibition was reduced 233 upon inhibition of both p300 and HDAC1. 92% of cells showed both nuclear and cytoplasmic 234 localization of Api5 (Figure 5E-F). Interestingly it was observed that upon inhibition of 235 HDAC1, 73% of cells showed Api5 nuclear foci while the foci were reduced to 46% when 236 both p300 and HDAC1 inhibitors were added in concert (Figure 5G). Therefore, it was 237 concluded that Api5 acetylation at lysine 251 by p300 stabilizes Api5 inside the nucleus 238 while de-acetylation by HDAC1 at the same lysine residue leads to Api5 to locate to the 239 cytoplasm.

240 Acetylation of Api5 is critical for normal cell cycle progression

241 Lacazette and group have reported Api5 protein levels to peak during the G1 and S phase of 242 cell cycle while the transcript levels remained unaltered [9]. Our data as well as data from 243 Han’s group [2] have shown acetylation to be responsible for maintaining the stability of 244 Api5. It may be hypothesized that the stability of Api5 during the G1 and S phases of the cell 245 cycle may be due to acetylation of Api5. To demonstrate whether Api5 acetylation affected 246 cell cycle profile, cells were transfected with the K251 mutants of Api5 in an Api5 knock 247 down background and then analyzed. Interestingly, cells transfected with the K251R mutant 248 of Api5 were observed to be arrested at the S phase of the cell cycle (Figure 6A). PCNA, a S- 249 phase cell cycle marker was also found to be upregulated in the Api5-K251R transfected cells 250 (Figure 6B-D) while the other cell cycle markers remained unaffected (Figure 6B, E and F). 251 This result was further corroborated with the observation that the Api5-K251R mutant 252 expressing cells showed higher proliferation when compared to the other Api5 acetylation 253 mutants (Figure 6G). This increase in proliferation may possibly be due to an alteration in the 254 cell cycle functioning resulting from the change in acetylation status of Api5. Therefore it 255 was confirmed that the acetylation status of Api5 also regulated proliferation and cell cycle 256 progression.

257 AMPK and Akt regulate the p300 mediated stability of Api5

258 It has already been reported that AMPK can either directly or indirectly regulate G1/S cell 259 cycle transition regulators such as p53 and p21 [47]. AMPK requires the activity of AKT to 260 regulate other cell cycle regulators [59]. It has been previously reported that activated Akt 261 can activate p300 by phosphorylating it at serine 1834 residue [60]. Therefore to discern 262 whether this signaling cascade led to the stability of Api5, both AMPK and Akt were

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263 inhibited using small molecule inhibitors. Api5 levels were observed to be reduced upon 264 inhibition of both AMPK (Figure 7A-B) and Akt (Figure 7C-D) independently. It was also 265 observed that upon inhibition of AMPK, Akt phosphorylation at serine 473 decreased which 266 is known to be required to activate p300 (Figure 7E-F). Our experiments suggested that 267 inhibition of AMPK inhibits the activation of Akt. Akt is the kinase that has been reported to 268 phosphorylate and activate p300 histone acetyl transferase. E2F1, a transcription factor is 269 known to be acetylated by p300. This p300-mediated acetylation of E2F1 has been shown to 270 regulate stability of the protein. Reduced levels of E2F1 upon inhibition of AMPK and Akt 271 separately confirms that both AMPK and Akt are involved in regulating the histone acetyl 272 transferase activity of p300 (Figure 7E-I). Inhibition of activation of Akt and reduction in 273 Api5 protein levels upon inhibition of AMPK confirms that p300 mediated stability of Api5 274 is achieved through the AMPK-Akt pathway as illustrated in Figure 8A.

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275 Discussion

276 In this study we have shown that p300, a histone acetyltransferase to be responsible for 277 acetylating and maintaining the stability of Api5. p300 is one of the most studied 278 acetyltransferases which not only acetylates histone proteins but also other non-histone 279 proteins too [23]. E2F1 is another known protein that is stabilized upon acetylation by p300 280 [28]. E2F1 is a transcription factor that plays a critical role during the G1/S transition of the 281 cell cycle [29]. E2F1 levels vary during the different cell cycle phases, similar to that of Api5 282 [9]. E2F1 protein levels peak during the G1 phase of cell cycle and starts to degrade as the 283 cell crosses the S phase. p53, the master regulator of the cell is also acetylated by the p300 284 [26]. There are a number of studies that show that upon acetylation, p53 stabilizes and thus, is 285 able to perform its different functions like activation of DNA damage response and repair 286 pathway, cell cycle arrest and apoptosis [42].

287 Histone deacetylases are one of the members of the deacetylase family of enzymes that 288 remove the acetylation from targeted proteins. Initially HDACs were identified and reported 289 to de-acetylate histone proteins [40]. But extensive studies have shown that HDACs can also 290 de-acetylate non-histone proteins. Both E2F1 and p53, which are acetylated by p300, are also 291 de-acetylated by Class 1 HDACs [29; 61]. E2F1 and p53 upon deacetylation become unstable 292 and lose their activity. HDAC1 mediated de-acetylation of E2F1 also aids in its localization 293 shift from nucleus to cytoplasm.

294 Earlier studies have shown Api5 acetylation at K251 residue to stabilize Api5 inside the 295 nucleus [2]. However, the enzymes involved in the acetylation process were unknown. Our 296 study, is the first to identify p300 and HDAC1 as the acetylating and de-acetylating enzymes 297 of Api5 respectively, that work in concert, thus leading to the stability or instability of the 298 protein during normal cell functioning. In this study, we report that Api5, a nuclear protein 299 under normal physiological conditions is acetylated by histone acetyltransferase p300. p300 300 is an essential regulator of Api5 as the acetylated protein is localized in the nucleus while the 301 de-acetylated protein switches its localization to the cytoplasm. As Api5 levels reduce upon 302 inhibition of p300, it suggests that Api5 might be undergoing post-translational degradation 303 in the cytoplasm. As the acetylated Api5 is stable in the nucleus and is not transported to the 304 cytoplasm, it may also be inferred that acetylation prevents Api5 from binding to 305 translocator/transporter proteins that are responsible for its translocation/ transportation from

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306 the nuclear compartment to the cytoplasm. However, de-acetylation might be exposing Api5 307 binding site to transporter protein(s) that enables the protein to translocate to the cytoplasm.

308 As mentioned earlier, Api5 levels peak during G1 and S phase of cell cycle and is thereby 309 able to play a role in the regulation of cell cycle progression as well as proliferation of cells 310 by possibly functioning as a transcription factor. During G1 and S phases of the cell cycle, 311 Api5 may possibly be interacting with chromatin to transcribe those genes that regulate 312 progression of the cell cycle. Transcription of those genes may also regulate the proliferation 313 of cells. Thus it may be inferred that acetylation of Api5 by p300 possibly provides additional 314 functionality to Api5 that includes transcription of cell cycle regulators.

315 It has also been reported that Api5 is overexpressed in many cancers. This overexpression of 316 Api5 promotes angiogenesis and metastasis in cancerous cells. Oncogenic properties of tumor 317 promoting proteins can also be considered as an outcome of their stability. In this case, Api5 318 is being stabilized by the action of p300 histone acetyltransferase. Inhibition of p300 and 319 induction of HDAC1 can reduce the oncogenic potential of Api5. Thus inhibitors of p300 and 320 inducers of HDAC1 have therapeutic implications in the treatment of cancers that have high 321 expression of Api5.

322 As K251 acetylation of Api5 is the only known post-translational modification which is 323 conserved from protists to mammals [2], suggesting that conservation of this acetylation may 324 play a key role during normal development. AMPK and Akt are important regulators of 325 various cellular processes like cell division, transcription regulation and apoptosis [44; 45; 326 46] [49; 50]. Reduced levels of Api5 upon inhibition of these key regulators also indicates its 327 role in normal physiological and developmental processes. Although our study suggests that 328 p300 is the acetylating and stabilizing regulator of Api5 while HDAC1 is the de-acetylating 329 and negative regulator of stability, one cannot ignore the possibility of other enzymes too 330 playing a role in the regulation of Api5 stability.

331 Thus taken together, our data suggests that p300 and HDAC1 are the novel regulators of Api5 332 which not only interact but also mediate the localization and activity of Api5 by acetylating 333 and de-acetylating at lysine 251 respectively as illustrated in Figure 8B.

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334 Materials and methods

335 Chemicals and antibodies

336 p300 inhibitor (382113), protease inhibitor cocktail (P2714), dimethyl sulfoxide (DMSO) and 337 propidium iodide (PI) (P4170) were purchased from Sigma-Aldrich and romidepsin from 338 Seleck Biochem. Lipofectamine 2000, Alexa flour 488 goat anti-rabbit and Phalloidin 568 339 were purchased from Invitrogen. GFP (ab290), RFP (ab62341) and p300 (ab10485) 340 antibodies were obtained from Abcam, HDAC1 (sc-8410) from Santacrutz, Api5 (PAB7951) 341 from Abnova, acetyl lysine (05-515) from Merck Millipore and GAPDH (G9545) from 342 Sigma-Aldrich.

343 Plasmids

344 CSII-EF-MCS plasmid was a gift from Dr. Sourav Banerjee, NBRC, Manesar, India. pCAG- 345 HIVgp and pCMV-VSV-G-RSV-Rev plasmids were purchased from RIKEN BioResource 346 Centre. mVenusC1 was gifted by Jennifer Lippincott-Schwartz, NIH, USA in which Api5 347 was cloned. The Api5 was made siRNA-resistant and K251 mutants were generated using 348 site directed mutagenesis.

349 Cell culture and drug treatments

350 MCF-7 cells were obtained from the European Collection of Cell Cultures (ECACC). 351 HEK293T cells were a gift from Dr. Jomon Joseph (NCCS, Pune). The cells were maintained 352 in 100 mm dishes (Eppendorf or Corning) and were grown in high glucose Dulbecco’s 353 Modified Eagle Medium (DMEM; Lonza) containing 10% heat inactivated FBS (Invitrogen) 354 and 100 units/mL penicillin-streptomycin (Invitrogen) and incubated at 37°C humidified 5%

355 CO2 incubators (Eppendorf or Thermo Scientific).

356 7 x 105 cell were seeded on 35mm dishes 16 hrs prior to treatment of p300i or HDACi and 357 incubated for 4 hrs and 12 hrs respectively.

358 MCF-7 cells stably expressing mCherry-tagged Api5 were generated using lentiviral- 359 mediated transduction. Briefly, 7.5 x 105 HEK293T cells were seeded on 35mm dish and 360 transfected with 1 µg of mCherry-CSII-EF-MCS or mCherry-CSII-EF-MCS-Api5 plasmid 361 along with 1 µg of pCAG-HIVgp and 0.5 µg pCMV-VSV-G-RSV-Rev packaging plasmids 362 using Lipofectamin 2000. 1 ml DMEM containing 30% FBS was added to the cells 24 hours 363 post transfection. 5 x 105 MCF-7 cells were seeded on a 35 mm dish for transduction. Viral

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364 supernatant was collected 48 hours post transfection and filtered through a 0.45 µm filter to 365 get rid of the cell debris. Filtered viral supernatant containing media along with 1 ml fresh 366 media was added to the MCF-7 cells. 4 µg polybrene was added to the cells to increase the 367 transduction efficiency. Cells were replenished with fresh medium 48 hours post 368 transduction. Transduced MCF-7 cells were passaged and used for further experiments.

369 For transfections, 6 x 105 cells were seeded in 35mm dishes and incubated at 37°C overnight. 370 siRNA duplexes targeting Api5 and LacZ were purchased from Dharmacon (Thermo 371 Scientific). Sense sequences of the siRNA are: Api5, 5′-GACCUAGAACAGACCUUCAUU- 372 3′, LacZ, 5′-CGUACGCGGAAUA CUUCGAdTdT-3′. siRNA against LacZ and Api5 were 373 transfected as described earlier [62]. Api5 K251 mutants which were generated by site 374 directed mutagenesis were transfected 24 hrs post knockdown of Api5.

375 Immunoblotting and Immunoprecipitation

376 mCherry-tagged Api5 over-expressing stable cells with or without treatments were lyzed in 377 cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 5 mM EDTA, 250

378 mM NaCl, 50 mM NaF, 0.1 mM Na3VO4 and protease inhibitors. Immunoprecipitations were 379 performed as described [63] using GFP, RFP, p300, or HDAC1 specific antibodies. Western 380 blotting was performed with Api5, GFP, acetyl lysine and GAPDH antibodies as previously 381 mentioned [62].

382 Semi-quantitative PCR

383 RNA extraction and cDNA synthesis of p300i and HDACi treated cells were performed as 384 described previously [64]. Semi-quantitative PCR was performed using API5 specific 385 forward (5'-CGAGTGGCAGATATACTAACGC-3') and reverse (5'- 386 TCCTCTCCTTGAAGTATTTGGC-3') primers. GAPDH was used as endogenous control 387 and was amplified using forward (5'-ACCACAGTCCATGCCATCAC-3') and reverse (5'- 388 TCCACACCCTGTTGCTGTA-3') primers. The following PCR cycle was used for the 389 amplification: 95˚C for 60 sec, 58˚C for 45 sec, 72˚C for 60 sec and final extension for 3 min. 390 The experiments were repeated three times to confirm API5 levels

391 Immunofluorescence

392 6 x 105 cells were seeded on coverslips placed in 35mm dishes. After treatment of p300i or 393 HDACi, cells were fixed in 4% paraformaldehyde and permeabilized using 0.5% TritonX 394 after PBS washes. After PBS-Glycine wash, cells were blocked using 10% FBS for 1 hr and

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395 incubated overnight with Api5 antibody at 4°C. Cells were incubated with secondary 396 antibodies conjugated with Alexa Flour for 1 hr at room temperature along with phalloidin. 397 Nuclei was stained using Hoechst 33258 for 5 mins and washed twice using PBS. The cells 398 were mounted using mounting media and imaged under 63X oil immersion objective of the 399 SP8 confocal microscope (Leica, Germany).

400 Cell proliferation assay

401 6 x 105 MCF-7 cells were seeded in 35mm dishes and incubated at 37°C overnight. mVenus- 402 tagged WT and K251 mutants of Api5 plasmids were transfected as mentioned before. 48 hrs 403 post transfection, cells were trypsinized and pelleted down by centrifugation at 1000 rpm for 404 5 mins. Cell pellet was suspended in 1X PBS. 1:10 dilution of the suspended cell pellet was 405 prepared in 1X PBS and the cell count was measured using 60 µm sensor connected to 406 SceptarTM hand held automated cell counter (Millipore, PHCC00000).

407 Flow cytometry

408 MCF-7 cells after transfection were trypsinized using 1X trypsin and collected in media. 409 After pelleting, the cells were washed with cold PBS twice to remove excess media. The cells 410 were then fixed in 70% ethanol overnight at 4°C and stained with 1 mg/ml Propidium Iodide 411 in the presence of RNase A (20 mg/ml) at 37°C for 1 hr. Cell cycle profile was analyzed on 412 BD Accuri™ C6 flow cytometer (BD Biosciences).

413 Statistical analysis

414 Densitometry analysis of western blots were performed using ImageJ software. Results were 415 analyzed from a minimumm of three independent experiments and plotted using GraphPad 416 Prism (GraphPad Software, La Jolla, CA, USA). Student’s t-test and Mann Whitney U test 417 was used to test the significance in difference of Api5 levels between two samples and One- 418 way Anova and Dunnett’s multiple comparisons test for more than two samples. p value > 419 0.05 was considered non-significant. *, ** and *** correspond to p<0.05, p<0.001, and 420 p<0.001 respectively.

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421 Conflict of Interest

422 The authors declare that they have no competing interests.

423 Author Contributions

424 V.K.S and M.L conceived and conceptualized the project. V.K.S designed, performed and 425 analyzed the experiments. V.K.S and M.L wrote the paper.

426 Funding

427 This study is supported by a grant from Science and Engineering Research Board (SERB), 428 Govt. of India (EMR/2016/001974) and partly by IISER, Pune Core funding. V.K.S was 429 funded by IISER Pune Core funding.

430 Acknowledgments

431 We thank Drs Richa Rikhy, Nagaraj Balasubramanium (IISER Pune, India) and Manas 432 Kumar Santra (NCCS, Pune, India) for their useful suggestions. We also thank Dr. 433 Krishanpal Karmodiya for the HDAC inhibitor and Dr. Gayathri Panangat as well as Sanjana 434 Nair (IISER Pune, India) for help with the in-silico analysis. The authors acknowledge the 435 IISER Pune Microscopy Facility for access to equipment and infrastructure. We thank Lahiri 436 Lab members Rintu Umesh for help with stable cell line preparation and Aishwarya 437 Venkataravi for designing the schematics in Figure 8. We also thank Lahiri lab members for 438 helpful comments and discussions

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439 References

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633

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634 Figure Legends

635 Figure 1. Api5 interacts with p300 in silico

636 A) Various acetyl transferases and their probability to acetylate lysine 251 of Api5 637 represented as p-value using ASEB webserver, where lower p-value corresponds to a higher 638 probability. B) The lowest-energy docking position of Api5 and p300 analyzed using 639 HADDOCK webserver and output analysed using PIZSA represented as a cartoon diagram. 640 C) Interaction between Lysine 251 of Api5 with p300. D) Various amino acid interactions 641 between Api5 and p300.

642 Figure 2. p300 interacts with and regulates Api5

643 A) MCF-7 cells treated with varying concentrations of p300 inhibitor for 4 hrs were lyzed 644 and Api5 levels were analyzed using western blotting. B) Quantification showing the fold 645 change in Api5 protein levels after normalization with GAPDH. C) API5 transcript levels 646 upon p300 inhibition was analyzed using semi-quantitative PCR. D) quantification of 647 transcript levels after normalizing to GAPDH. MCF-7 cells stably expressing mCherry-Api5 648 were lyzed and immunoprecipitations were performed using E) GFP and F) p300 specfic 649 antibodies.

650 Figure 3: HDAC1 interacts with and regulates Api5

651 A) MCF-7 cells treated with varying concentrations of the HDAC1 inhibitor, romidepsin for 652 16 hrs were lyzed and Api5 levels were analyzed using western blotting. B) Quantification 653 showing the fold change in Api5 protein levels after normalization with GAPDH. C) API5 654 transcript levels upon romidepsin treatment was analyzed using semi-quantitative PCR and 655 D) quantified after normalizing with GAPDH. MCF7 cells stably expressing mCherry-Api5 656 were lyzed and immunoprecipitation was carried out using E) GFP and F) HDAC1 specfic 657 antibodies.

658 Figure 4: p300 and HDAC1 acetylate and deacetylate Api5 at K251 respectively

659 mCherry-tagged Api5 over-expressing MCF-7 cells treated with A) 7.5 µM of p300i for 4 hrs 660 and B) 0.1 nM of romidepsin for 16 hrs were lyzed and immunoprecipitation was performed 661 using RFP-specific antibody. Western blotting analysis was performed to ascertain the 662 acetylation status of Api5 using acetyl lysine specific antibody. MCF-7 cells were transfected 663 with K251 mutant constructs and treated with C) 7.5 µM of p300i and E) 0.1 nM of

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

664 romidepsin post 24 hrs of transfection for 4 and 16 hrs respectively. Western blotting was 665 performed to check for the levels of mutant Api5 using GFP specific antibody. D) and F) 666 Quantification showing the fold change in Api5 protein levels after GAPDH normalization.

667 Figure 5: p300 and HDAC1 regulate Api5 localization

668 MCF-7 cells were treated with A-B) 7.5 µM p300i, C-D) 0.1 nM HDACi and E-F) 7.5 µM 669 p300 and 0.1 nM HDACi for 4 hrs and 16 hrs respectively. Immunofluorescence assay was 670 performed using Api5 specific antibody and cells with different Api5 staining pattern were 671 manually counted and analyzed. G) Percentage of cells showing Api5 foci formation 672 following the different inhibitor treatments.

673 Figure 6: Acetylation status of Api5 affects cell cycle.

674 MCF-7 cells transfected with siRNA-resistant Api5-K251 mutants 24 hrs post Api5 knock 675 down. Cells were fixed in ethanol and stained with propidium iodide and cell cycle profile 676 was monitored. A) Percentage of cells in the different cell cycle phases were analyzed in a 677 flow cytometer and plotted as box plots. B) Western blotting was performed to check the 678 levels of B) Api5, PCNA, Cyclin A and Cyclin B1 and C-F) quantified after normalizing to 679 the loading control GAPDH. G) MCF7 cells transfected with mVenus-tagged Api5 WT and 680 K251 mutants were counted using SceptarTM to check for cell proliferation.

681 Figure 7: AMPK and AKT regulate Api5 stability

682 MCF-7 cells were treated with A) 20 µM AMPKi, and C) 5 µM AKTi for 4 hrs and 24 hrs 683 respectively. Immunoblotting analysis was performed using Api5 specific antibody. GAPDH 684 was used as loading control. B and D) Quantification showing the fold change in Api5 levels 685 after normalization to GAPDH. A) p21 and C) pAkt S373 were used as positive control to 686 confirm AMPK and Akt inhibition respectively. E and F) pAkt S473 and E and G) E2F1 687 levels upon AMPK inhibition. H and I) E2F1 protein levels were analyzed following Akt 688 inhibition using immunoblotting.

689 Figure 8: Schematic representation depicting the regulation of Api5 following acetylation and 690 deacetylation

691 A) Model predicting the regulation of Api5 via the AMPK, Akt and p300 signaling cascade. 692 B) p300 acetylation of Api5 at lysine residue 251 stabilizes the protein in the nucleus;

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

693 whereas de-acetylation of Api5 at K251 by HDAC1 de-stabilizes the protein which enables it 694 to get translocated to the cytoplasm.

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 1 A Probability of Acetyl transferase acetylation at 251 p value CBP/ p300 0.28 GCN5/PCAF 1 TIP60/MYST1/2/3/4 0.99

B C

Api5 Api5

p300

Api5

p300 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Figure 2 A B 0 5 7.5 10 12.5 p300i (�M) *** *** *** ** Api5 55 kDa

GAPDH 35 kDa Relative Api5 levels Relative

p300i (�M) C D 2.0 - + p300i (7.5 �M) ns API5 1.5

150 bp 1.0

GAPDH 0.5 450 bp

Relative API5 levels Relative 0.0 p300i - +

E F IP IgG GFP IP IgG p300

p300 GFP 300 kDa 80 kDa

IP-p300 p300 IP-GFP GFP 300 kDa 80 kDa

p300 GFP 300 kDa 80 kDa

p300

GFP Input Input 300 kDa 80 kDa

GAPDH GAPDH 35 kDa 35 kDa bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 3

A B 0 0.05 0.1 HDACi (nM) *** 2.0 **

1.5 Api5 55 kDa 1.0

GAPDH 0.5 35 kDa Relative Api5 levels Relative 0.0 0 0.05 0.1 HDACi (nM) C D - + HDACi (0.1 nM) 1.5 ns

API5 1.0 150 bp

GAPDH 0.5 450 bp

Relative API5 levels Relative 0.0 HDACi - + E F IP IgG GFP IP IgG HDAC1

HDAC1 GFP 55 kDa 80 kDa IP-GFP

GFP IP-HDAC1 HDAC1 80 kDa 55 kDa HDAC1 GFP 55 kDa 80 kDa GFP HDAC1

80 kDa Input Input 55 kDa GAPDH GAPDH 35 kDa 35 kDa bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Figure 4

A B IP IgG RFP IP IgG RFP + - + p300i (7.5 �M) + - + HDACi (0.1 nM)

Acetyl lysine Acetyl lysine 80 kDa 80 kDa

RFP RFP 80 kDa 80 kDa

C D WT K251A K251Q K251R - + - + - + - + p300i (7.5 �M) GFP 80 kDa

GAPDH 35 kDa Relative Api5 levels Relative p300i - + - + - + - + WT K251A K251Q K251R E F WT K251A K251Q K251R - + - + - + - + HDACi (0.1 nM) GFP 80 kDa

GAPDH 35 kDa Relative Api5 levels Relative HDACi - + - + - + - + WT K251A K251Q K251R Figure 5bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A Hoechst 33258 Api5 Phalloidin Merged B

Nucleus + Cytoplasm Cytoplasm Nucleus

Control 100

74 50 98 % of cells

p300i 24 2 0 p300i - + C D Hoechst 33258 Api5 Phalloidin Merged Nucleus + Cytoplasm Cytoplasm Nucleus

Control 100

67 50 97

% of cells 4 29

HDACi 0 HDACi - +

E Hoechst 33258 Api5 Phalloidin Merged F Nucleus + Cytoplasm Cytoplasm Nucleus 2 100 Control

92 50 98 % of cells + 4 p300i 0 HDACi p300i - + + HDACi

G 80 73

60 46 40

20 % of cells (foci) 5 3 0 p300i - + - + HDACi - - + +

A G2/M C 1.5 S Endogenous Api5

G1 1.0

100 7.5 13.2 14.3 11.2 13.3 0.5 23.0 20.0 19.7 22.6 49.2 0.0 Relative protein levels 50 WT K251Q K251R 69.5 66.8 66.0 66.2 siLacZ siApi5 37.5 % of cells is cell cycle phase 0 D WT K251Q K251R 15 PCNA siLacZ siApi5

B 10 siLacZ siApi5

WT K251Q K251R 5 GFP

80 kDa Relative protein levels 0 WT K251Q K251R Api5 siLacZ siApi5 55 kDa

PCNA E 29 kDa 1.5 Cyclin B1 GAPDH 35 kDa 1.0 Cyclin A 52 kDa 0.5 Cyclin B1 55 kDa 0.0 Relative protein levels WT K251Q K251R GAPDH siLacZ siApi5 35 kDa

F G 1.5 * Cyclin A 14 ns )

4 ns

1.0 12

0.5 10

8 0.0 Relative protein levels

WT K251Q K251R Number of cells (10 siLacZ siApi5 6 WT K251A K251Q K251R bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Figure 7 perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A - + AMPKi (20 �M) B Api5 Api5 55 kDa *** GAPDH 35 kDa 1.0

p21 0.5 21 kDa

0.0 Relative Api5 levels Relative GAPDH AMPKi - + 35 kDa

C - + Akti (5 �M) Api5 D Api5 55 kDa *** GAPDH 35 kDa 1.0

pAkt S473 0.5 63 kDa

Akt 0.0 Relative Api5 levels Relative 63 kDa Akti - + GAPDH 35 kDa

F G E - + AMPKi (20 �M) 1.5 1.5 pAkt S473 E2F1 pAkt S473 1.0 63 kDa 1.0 Akt 0.5 0.5 63 kDa Relative E2F1 levels GAPDH Relative pAkt levels 0.0 0.0 35 kDa AMPKi - + AMPKi - + E2F1 48 kDa GAPDH 35 kDa I

H 1.5 - + Akti (5 �M) E2F1 1.0 E2F1 48 kDa 0.5 GAPDH 35 kDa Relative E2F1 levels 0.0 Akti - + bioRxiv preprint doi: https://doi.org/10.1101/2020.11.22.393256; this version posted November 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Figure 8 A

CYTOPLASM

AMPK

Akt p300

Ac K251 Api5 Api5 NUCLEUS

CYTOPLASM Translocates Api5 to cytoplasm

p300 HDAC1

Ac K251 Ac Api5 Api5 K251 Ac Api5 Api5 Stable Unstable NUCLEUS