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p300 Acetyltransferase Activity Differentially Regulates the 1
Localization and Activity of the FOXO Homologues in Skeletal 2
Muscle 3
Sarah M. Senf1, Pooja B. Sandesara2, Sarah A. Reed2, Andrew R. Judge1,2 4
1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida, 5
USA; 2 Department of Physical Therapy, University of Florida, Gainesville, Florida, USA 6
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Corresponding author: 8
Andrew R. Judge, 9
Department of Physical Therapy 10
J303, Biomedical Sciences Building 11
1275 Center Drive 12
University of Florida, 13
Gainesville, FL 32610 14
USA 15
Tel: 352-273-9220 16
Fax: 352-273-6109 17
E-mail: [email protected] 18
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Running title: p300 HAT activity represses FOXO in muscle 25
Articles in PresS. Am J Physiol Cell Physiol (March 9, 2011). doi:10.1152/ajpcell.00255.2010
Copyright © 2011 by the American Physiological Society.
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ABSTRACT 26
The Forkhead Box O (FOXO) transcription factors regulate diverse cellular processes, and in 27
skeletal muscle are both necessary and sufficient for muscle atrophy. Although the regulation of 28
FOXO by Akt is well evidenced in skeletal muscle, the current study demonstrates that FOXO is 29
also regulated in muscle via the acetyltransferase (HAT) activities of p300/CBP. Transfection of 30
rat soleus muscle with a dominant-negative (d.n.) p300, which lacks HAT activity and inhibits 31
endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription 32
from the promoter of a bona fide FOXO target gene, atrogin-1. Conversely, increased HAT 33
activity via transfection of either WT p300 or WT CBP repressed FOXO activation in vivo in 34
response to muscle disuse, and in C2C12 cells in response to dexamethasone & acute starvation. 35
Importantly, manipulation of HAT activity differentially regulated the expression of various 36
FOXO target-genes. Co-transfection of FOXO1, FOXO3a or FOXO4 with the p300 constructs 37
further identified p300 HAT activity to also differentially regulate the activity of the FOXO 38
homologues. Markedly, decreased HAT activity strongly increased FOXO3a transcriptional 39
activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear 40
localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear 41
localization. In summary, this study provides the first evidence to support the acetyltransferase 42
activities of p300/CBP in regulating FOXO signaling in skeletal muscle, and suggests that 43
acetylation may be an important mechanism to differentially regulate the FOXO homologues and 44
dictate which FOXO target-genes are activated in response to varying atrophic stimuli. 45
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Key Words: HAT, muscle atrophy, disuse, cachexia, gene regulation, atrogin-1 47
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INTRODUCTION 49
FOXO signaling has been implicated in skeletal muscle atrophy associated with sepsis 50
(9), starvation (24, 27), diabetes (28), cancer (27), aging (16, 27) and heart failure (44). More 51
direct work, using genetic approaches, demonstrate that at least two of the FOXO homologues, 52
FOXO1 (23) and FOXO3a (43), are sufficient to cause skeletal muscle atrophy, in vivo. Perhaps 53
more importantly, blocking FOXO transactivation prevents at least 40% of disuse muscle fiber 54
atrophy (39, 45), further demonstrating the requirement of FOXO for the normal atrophy 55
phenotype in a physiological model of muscle atrophy. Given the significance of FOXO in the 56
regulation of muscle mass, identifying the immediate upstream regulators of FOXO may lead to 57
the development of specific countermeasures to prevent muscle wasting. 58
The regulation of FOXO signaling by Akt has been extensively characterized in a variety 59
of cell types, including skeletal muscle (2, 26, 43). In response to growth conditions or growth 60
factor stimuli the IGF-1/PI3K/Akt pathway is activated, which leads to FOXO phosphorylation 61
by Akt on specific residues, which promotes the cytosolic retention and inactivation of FOXO (2, 62
42). In skeletal muscle, direct evidence to support the IGF-1/PI3K/Akt pathway in regulating 63
FOXO can be found in several studies (26, 43, 48). Decreases in this signaling pathway in 64
skeletal muscle during physiological conditions of muscle atrophy such as starvation and muscle 65
disuse are thought to contribute to FOXO activation. However, increasing evidence demonstrate 66
FOXO-signaling to be controlled via additional post-translational modifications and protein-67
protein interactions which are distinct from Akt-mediated phosphorylation (19, 51). Yet, many of 68
these additional regulatory mechanisms have yet to be thoroughly explored in skeletal muscle. If 69
similar control mechanisms indeed exist in skeletal muscle to modulate FOXO activity, this 70
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could potentially open up new avenues for therapeutically blocking FOXO function and the 71
associated muscle atrophy during physiological conditions of muscle wasting. 72
One such mechanism of FOXO regulation identified in multiple cell types, involves the 73
regulation of FOXO-dependent transcription by the histone acetyltransferase (HAT) proteins, 74
p300 and CREB binding protein (CBP) (11, 14, 35). These HAT proteins each possess an 75
intrinsic acetyltransferase activity which catalyzes the transfer of an acetyl group to specific 76
lysine residues on target proteins (10, 11, 32). Although HATs are most well known for 77
regulating gene transcription through histone acetylation and relaxation of chromatin structure at 78
gene promoters (12, 31), HATs also play an important role in regulating the activity of a variety 79
of transcription factors, including p53, MyoD, HIF-1alpha, as well as the FOXO transcription 80
factors (47, 52). HATs may regulate transcription factor activity through various mechanisms 81
which include interaction and recruitment of factors to target gene promoters, via acting as 82
adaptor molecules facilitating protein-protein interactions, and through direct acetylation of 83
transcription factors or other necessary co-factors which thereby alter transcription factor activity 84
(25). Evidence for HAT-mediated regulation of the FOXO transcription factors can be found in 85
multiple cell types. Interestingly however, the resulting effect of HATs on FOXO appears to be 86
cell-type specific and/or specific to the FOXO homologue. For example, p300 increases 87
FOXO1-dependent transcription from the IGFBP-1 promoter reporter in H4IIE rat hepatoma 88
cells, which requires p300 HAT activity (35). Similarly, p300 increases FOXO3a-dependent 89
transcription from the Bim promoter in human embryonic kindey cells (HEK293T) (34). In 90
contrast, p300 represses FOXO4-induced transcription of GADD45, p27, p21 and MnSOD in 91
HEK293 cells (11). Therefore depending on the cell type, FOXO homologue, and target gene 92
measured, HATs may either repress or activate FOXO-induced transcription, which may reflect 93
5
an important fine-tuning mechanism of FOXO-target gene regulation. However, despite the 94
importance of understanding the mechanisms which lead to FOXO-dependent transcription in 95
skeletal muscle due to its known role in causing muscle atrophy, no data currently exist to 96
suggest whether the acetyltransferase activities of p300/CBP regulate FOXO in skeletal muscle. 97
Therefore, the purpose of the current study was to determine whether HAT proteins regulate the 98
FOXO transcription factors in skeletal muscle, and whether this is altered during conditions of 99
muscle wasting. 100
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MATERIALS AND METHODS 117
Animals 118
Sprague-Dawley male rats (200-225g) were ordered from Charles River Laboratories 119
(Wilmington, MA, USA). The University of Florida Institutional Animal Care and Use 120
Committee approved all animal procedures. UF is accredited by the Association for Assessment 121
and Accreditation of Laboratory Animal Care (#A3377-01). Animals were maintained in a 122
temperature and humidity-controlled facility with a 12-hour light/dark cycle. Water and standard 123
diet were provided ad libitum. 124
Plasmids and Reporter Gene Assays 125
Expression plasmids for WT p300 and the dominant negative (d.n.) p300 mutant (which lacks 126
acetyltransferase activity due to an inactivating point mutation, converting aspartic acid 1399 to 127
tyrosine ) were obtained from Dr. Tso-Pang Yao (Duke University, Durham, NC, USA) and have 128
been previously described (21). The FOXO1 expression plasmid was a gift from Dr. Akiyoshi 129
Fukamizu (University of Tsukuba, Ibaraki, Japan) and has previously been used and described 130
(32). The tagged FOXO1-EGFP plasmid was obtained from Addgene (plasmid 9022), and was 131
deposited by Dr. Domenico Accili (Columbia University, New York, NY, USA) and has 132
previously been described (13). The FOXO4 expression plasmid was a gift from Dr. Boudewijn 133
Burgering (University Medical Center, Utrecht, The Netherlands) and has been previously used 134
and described (53). The FOXO3a expression plasmid was obtained from Addgene (plasmid 135
10710), was deposited by Dr. William Sellers (Novartis, Cambridge, MA) and has been 136
previously used and described (37). The WT FOXO3a-DsRed fusion construct was created via 137
PCR amplification of the FOXO3a cDNA out of the parent vector using primers to create 138
HindIII and SalI restriction sites on the 5’ and 3’ ends of the FOXO3a coding region, 139
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respectively. FOXO3a cDNA was then sub-cloned, in frame, into the DsRed2-c1 plasmid. 140
Verification that FOXO3a cDNA was in frame was confirmed via DNA sequencing (DNA 141
Sequencing Core, University of Florida). The d.n.Akt and c.a.Akt expression plasmids were 142
obtained from Addgene (plasmids 12643 & 16244, respectively) and were deposited by Dr. 143
Mien-Chie Hung (The University of Texas, M. D. Anderson Cancer Center, Houston, TX) and 144
have previously been described (58). The DAF-16/FOXO responsive reporter plasmid, the 145
atrogin-1-GL2 promoter reporter plasmid and the d.n.FOXO construct have also been previously 146
used and described (46). pRL-TK-Renilla was purchased from Promega. Plasmid DNA was 147
amplified and isolated from bacterial cultures using Endotoxin-Free Maxi or Mega Prep Kits 148
(Qiagen, Valencia, CA, USA), precipitated in ethanol and re-suspended in 1X sterile filtered 149
phosphate buffered saline (PBS) for in vivo transfections, or Tris-EDTA (TE) buffer for 150
transfections in culture. 151
In vivo, Plasmid Injection and Electroporation. 152
Transfection of plasmid DNA into skeletal muscle, in vivo, has been detailed previously (22, 46). 153
For rat experiments, 10μg each of the expression or control plasmid(s) and 40μg of the reporter 154
plasmid were diluted in a total of 50μl 1XPBS for each solei injection. Standard procedures 155
were used to determine luciferase activity on skeletal muscle homogenates using a Modulus 156
single tube multimode reader (Promega) and have been described previously (46). 157
Animal Models and Muscle Preparation 158
Disuse muscle atrophy via cast immobilization of both hind limbs was induced in rats four days 159
following plasmid injection and has been detailed previously (46). After three days of 160
immobilization or weight bearing activity soleus muscles were removed and processed either 161
immediately for RNA isolation or frozen in liquid nitrogen and stored at -80°C until further 162
8
biochemical analyses. For experiments using exclusively genetic manipulations, muscles were 163
harvested 7 days post-plasmid injection. 164
Cell Culture Experiments 165
C2C12 cells were cultured on 0.1% gelatin coated 6-well plates in high-glucose DMEM 166
(Invitrogen), 10% fetal bovine serum and 5% CO2. Muscle cells were transfected with plasmid 167
DNA at ~80% confluence using FuGENE® HD Transfection Reagent (Promega Corp, Madison, 168
WI, USA) at a 3.5:1 ratio of reagent to total DNA. Sixteen hours following transfection muscle 169
cells were differentiated into myotubes by incubation in differentiation medium (2% Horse 170
Serum in DMEM). For dexamethasone studies, 6-day differentiated myotubes were treated with 171
either vehicle (water) or 1uM water-soluble dexamethasone (Sigma, St. Louis, MO, USA) in 172
differentiation media for 6 hours and harvested in Passive Lysis Buffer (Promega). In the 173
nutrient deprivation groups, differentiation media was removed from 4-day differentiated cells 174
and Hanks Balanced Salt Solution (HBSS) added for either 2 hours (localization experiments) or 175
6 hours (reporter assays and gene expression) prior to harvest. To inhibit PI3Kinase, 10μM 176
LY294002 (Calbiochem, Merck KGaA, Darmstadt, Germany) or vehicle (ethanol) was added to 177
4-day differentiated myotubes for 6 hours. For reporter experiments, cells were harvested in 178
Passive Lysis Buffer (Promega) and luciferase activity determined by normalizing firefly 179
luciferase activity to pRL-TK Renilla luciferase activity using a Dual-Luciferase® Reporter 180
Assay (Promega). 181
RNA isolation, cDNA synthesis, and RT-PCR 182
RNA isolation and cDNA synthesis from whole muscle was performed using a Trizol-based 183
method as previously described (46). RNA isolation from C2C12 myotubes was performed 184
similarly, following addition of 250uL Trizol per well and vigorous scraping, as previously 185
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described (33) . cDNA was generated from 1ug of RNA and was used as a template for qRT-186
PCR using primers for atrogin-1, GenBank NM_133521; MuRF1, GenBank NM_080903; 187
Cathepsin-L, GenBank NM_013156; 4E-BP1, GenBank NM_053857; LC3b, GenBank 188
NM_022867; p21, Genbank NM_080782; Gadd45α, GenBank NM_024127; Foxo1, GenBank 189
NM_001191846; Foxo3a, GenBank NM_001106395; Foxo4, GenBank NM_001106943; or 18S, 190
GenBank X03205.1, which were ordered from Applied Biosystems (Austin, TX, USA). TaqMan 191
probe-based chemistry was used to allow detection of PCR products using a 7300 real-time PCR 192
system (Applied Biosystems), and quantification of gene expression was performed using the 193
relative standard curve method. 194
Western Blotting and Co-Immunoprecipitation Assays 195
Preparation of muscle homogenates and western blotting were performed according to standard 196
procedures and have been described previously (46). Primary antibodies for p300 (#554215, BD 197
Pharmingen, San Jose, CA, USA), FOXO1 (#9454S, Cell Signaling Technology, Boston, MA, 198
USA); phospho-FOXO1 (Ser256) (#9461, Cell Signaling Technology); FOXO3a (SC-11351, 199
Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-FOXO3a (Thr32) (SC-12357, Santa 200
Cruz Biotechnology) and FOXO4 (07-1720, Millipore, Billerica, MA), were used according to 201
manufacturer’s directions. Tubulin primary antibody, (T6074 from Sigma-Aldrich Inc, St. 202
Louis, MO, USA) was used to control for equal protein loading and protein transfer. For co-203
immunoprecipitation assays proteins, 500ug of muscle protein were incubated overnight with 204
either 4ug of Anti-Acetyl Lysine antibody (#05-515) or 4ug of anti-p300 (#05-257) using a 205
Catch and Release Reversible Immunoprecipitation System (#17-500), all from Millipore. The 206
following day, precipitated proteins were washed and subsequently eluted in denaturing buffer, 207
boiled, and western blotting performed for endogenous FOXO3a and FOXO1. 208
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Fluorescent Microscropy 209
C2C12 myoblasts were seeded on 6-well plates containing 0.1% gelatin-coated glass coverslips, 210
transfected and differentiated for 4 days. Following treatment, cells were rinsed with PBS and 211
fixed for 30 minutes in 4% paraformaldehyde. Following three washes in PBS, two drops of 212
Vectashield Mounting Medium for Fluorescence, with Dapi, (#H-1200, Vector Laboratories, Inc, 213
Burlingame, CA, USA) was added to each coverslip. A Leica DM5000B microscope (Leica 214
Microsystems Inc., Bannockburn, IL USA) containing GFP (green) and Rhodamine (red) filter 215
cubes was used to visualize FOXO1-EGFP or FOXO3a-DsRed positive myotubes, respectively. 216
A Dapi (blue) filter was used to visualize Dapi-stained nuclei. Images were captured and merged 217
using Leica Application Suite, version 3.5.0. 218
Statistics 219
Data were analyzed using a two-way ANOVA followed by Bonferroni post-hoc comparisons 220
when appropriate (GraphPad Software, San Diego, CA). All data are expressed as the mean ± 221
SEM, and significance was set at P<0.05. 222
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RESULTS 232
p300 acetyltransferase activity is necessary and sufficient to repress FOXO signaling in 233
skeletal muscle 234
To determine if p300 HAT activity regulates FOXO signaling in skeletal muscle, we injected and 235
electrotransfered a FOXO-responsive luciferase reporter plasmid plus either a control plasmid, 236
WT p300 or dominant negative (d.n.) p300 (which lacks acetyltransferase activity) expression 237
plasmid into the skeletal muscle of rats prior to either normal weight bearing activity or three 238
days of muscle disuse induced via hind limb cast immobilization. As shown in Figure 1A, 239
FOXO activity was increased (~3-fold) in response to immobilization, which is in agreement 240
with our previous findings (46). However, in muscles overexpressing WT p300, FOXO reporter 241
activation in response to immobilization was prevented. Further, expression of the d.n.p300 242
mutant, which outcompetes and inhibits endogenous p300 HAT activity, was sufficient to 243
increase FOXO activity in normal weight bearing muscle (~6-fold) and further enhanced FOXO 244
activity during immobilization (~14-fold). To determine whether another protein which 245
possesses HAT activity and is highly homologous to p300, CREB-binding protein (CBP), also 246
inhibits disuse-induced activation of the FOXO reporter we collected additional data using a 247
control or WT CBP expression plasmid. Similar to WT p300, overexpression of WT CBP 248
prevented the increase in FOXO activity during disuse (Figure 1B). 249
To test whether p300 and CBP could similarly prevent the activation of FOXO in 250
response to a stimulus distinct from muscle disuse we determined the effect of p300/CBP on 251
FOXO activation, in vitro, in response to treatment with the glucocorticoid, dexamethasone 252
(Dex). C2C12 myoblasts were transfected with the FOXO reporter and pRL-TK-renilla plus an 253
empty vector, WT p300 or WT CBP plasmid. Cells were differentiated for 6 days, and treated 254
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with either vehicle (water) or Dex, at a concentration of 1µM for 6 hrs. Dex treatment decreases 255
phospho-FOXO protein levels in C2C12 myotubes (43), which is widely used as a marker of 256
FOXO activation and, indeed, we found Dex increased FOXO reporter activity 1.7-fold over 257
control. However, this increase in FOXO activity due to Dex treatment was abolished in C2C12 258
cells transfected with either WT p300 or WT CBP (Figure 1C). These findings provide the first 259
evidence that FOXO activity is regulated in skeletal muscle via the acetyltransferase activity of 260
p300/CBP and further indicate that HAT activity is both necessary and sufficient to repress 261
FOXO activity in skeletal muscle. 262
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p300 HAT activity differentially regulates FOXO target gene transcription 264
Due to the pronounced effect that p300 HAT activity has on FOXO transcriptional activity in 265
skeletal muscle, we next determined whether p300 could also block the increased transcription of 266
a bona fide FOXO target gene, atrogin-1/MAFbx, during 3 days of muscle disuse. Atrogin-1 267
mRNA was increased 4.7-fold in response to muscle disuse, which was attenuated by 65% in 268
muscles overexpressing WT p300 (Figure 2A). This repression by p300 required its HAT 269
activity since expression of d.n.p300 did not similarly repress atrogin-1 mRNA levels. To further 270
confirm that p300 can repress atrogin-1 transcription during muscle disuse we conducted similar 271
experiments to test the effect of WT p300 or d.n.p300 on a luciferase reporter construct driven by 272
2.4 kb of the atrogin-1 promoter. As shown in Figure 2B, similar to the affect of p300 on 273
atrogin-1 mRNA, WT p300 prevented disuse-induced activation of the atrogin-1 promoter 274
reporter, which required its HAT activity. Notably, the effects of p300 HAT activity were more 275
pronounced on the atrogin-1 promoter reporter when compared to atrogin-1 mRNA levels. This 276
finding is likely explained by the muscle fiber transfection efficiency in whole muscle with the 277
13
p300 constructs, which in our hands is ~50%. Therefore, the effects of WT and d.n.p300 on 278
mRNA levels in transfected fibers are diluted by mRNA levels from non-transfected fibers. In 279
contrast, when co-injecting two plasmids (p300 and the atrogin-1 promoter reporter constructs), 280
co-transfection of a muscle fiber occurs nearly 100% of the time (38). As a result, the atrogin-1 281
promoter reporter is only reporting from those fibers which also took up the p300 (or empty 282
vector) constructs—thereby eliminating the dilution effect. 283
Since d.n.p300 was sufficient to increase both the FOXO reporter and the atrogin-1 284
promoter reporter during normal weight bearing conditions, we further determined whether the 285
increase in atrogin-1 promoter activity by d.n.p300 required active FOXO. Co-expression of 286
d.n.p300 with a d.n.FOXO construct (which inhibits FOXO activity (46)), blocked the ability of 287
d.n.p300 to activate the atrogin-1 promoter reporter (Figure 2C). Collectively these data 288
demonstrate that p300 HAT activity represses atrogin-1 transcription, which is mediated through 289
the repression of FOXO. 290
We next examined the effect of p300 HAT activity on the mRNA levels of additional 291
FOXO target-genes during normal weight bearing conditions and 3 days of immobilization. As 292
shown in Figure 2D, and in agreement with others (1, 4, 22, 30, 57), 3 days of muscle disuse 293
induced a significant increase in the mRNA levels of MuRF1 (3.2-fold), p21 (3.9-fold), 4E-BP1 294
(2-fold), Gadd45a (1.7-fold), cathepsin L (2.4-fold) and LC3b (2.1-fold). However, increasing 295
HAT activity via overexpression of WT p300 significantly repressed the disuse-induced increase 296
in MuRF1 by 55%, and the bona fide FOXO target-gene, p21, by 54% both of which required 297
p300’s HAT activity. In contrast, WT p300 further enhanced the disuse-induced increases in 4E-298
BP1 by 48% and another bona fide FOXO target-gene, Gadd45α, by 75% which required p300’s 299
HAT activity. Importantly, reducing HAT activity via expression of d.n.p300 significantly 300
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repressed the disuse-induced increases in cathepsin L by 78%, and 4E-BP1 by 45%. Alterations 301
in p300 HAT activity did not significantly affect the levels of LC3b mRNA. Three days of 302
muscle disuse also significantly increased the mRNA levels of FOXO1 (2.2-fold) and FOXO3 303
(1.9-fold), which has previously been demonstrated (40, 45) and also increased the mRNA levels 304
of FOXO4 (2.3-fold) (Figure 2E). Notably, increased p300 HAT activity had a significant 305
repressive effect on FOXO4 mRNA levels during both weight bearing and immobilization 306
conditions and on FOXO1 mRNA levels during immobilization. There was no significant effect 307
of p300 HAT activity on FOXO3a mRNA levels. Together, these data indicate that p300 HAT 308
activity differentially regulates the expression of FOXO target genes, as well as the FOXO genes 309
themselves. 310
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p300 acetylates and represses FOXO3a transcriptional activity 312
Data thus far demonstrate that p300 HAT activity represses the increase in FOXO transcriptional 313
activity in response to two different atrophic stimuli and differentially regulates the gene 314
expression of FOXO-target genes. Because the FOXO reporter may respond to each of the 315
skeletal muscle FOXO family members, the regulation of total FOXO activity and target gene 316
expression by p300 may be related to the regulation of FOXO1, FOXO3a, FOXO4, or some 317
combination of FOXO factors. We therefore tested the extent to which p300 HAT activity 318
regulates the transcriptional activity of FOXOs 1, 3a or 4 in skeletal muscle. To do this we 319
injected and electrotransfered rat solei with the FOXO reporter plasmid plus FOXO1, FOXO3a 320
or FOXO4 expression plasmids (Figure 3A), each with an empty vector, WT p300, or d.n.p300. 321
As shown earlier in Figure 1A, transfection of WT p300 alone had no affect on basal levels of 322
the FOXO reporter, while transfection of d.n.p300 was sufficient to increase FOXO activity 4.6-323
15
fold. Transfection of FOXO1 alone did not significantly increase the FOXO reporter and did 324
not significantly alter reporter activity in the presence of WT p300 or d.n.p300 (Figure 3B). In 325
contrast, the 2.5-fold increases in FOXO reporter activity induced individually by FOXO3a and 326
FOXO4 were abolished by WT p300. Moreover, the increases in FOXO reporter activity 327
induced individually by FOXO3a (2.5-fold) and d.n.p300 (4.6-fold), were synergistically 328
increased to 16-fold when FOXO3a and d.n.p300 were co-expressed together. Co-expression of 329
FOXO4 plus d.n.p300 resulted in a 7-fold increase in FOXO activity, demonstrating an additive 330
effect on FOXO activity. Together these findings show that p300 is sufficient to repress both 331
FOXO3a and FOXO4 activity via its HAT activity, and suggests that a decrease in p300 332
acetyltransferase activity potently increases the transcriptional activity of FOXO3a. 333
Due to the pronounced effect that p300 HAT activity has on repressing FOXO3a 334
transcriptional activity, we determined whether we could detect changes in endogenous FOXO3a 335
acetylation in muscles injected with the WT p300 and d.n.p300 constructs. Total acetylated 336
proteins were immunoprecipitated from equal amounts of protein extract using an anti-acetyl-337
lysine antibody and subsequently immunoblotted for FOXO3a. As shown in Figure 3C, muscles 338
injected with WT p300 demonstrated an increase in acetylated FOXO3a when compared to 339
muscles injected with an empty vector, while d.n.p300-injected muscles showed a decrease in 340
acetylated FOXO3a. These findings show that FOXO3a is a target of p300 HAT activity and 341
suggest that p300 may regulate FOXO3a transcriptional activity through direct acetylation. 342
Protein modification via acetylation has previously been demonstrated to regulate protein 343
stability (41). Therefore, we determined whether p300 HAT activity regulates FOXO3a protein 344
levels. Due to the relatively low levels of endogenous FOXO3a protein in skeletal muscle and 345
the difficulty that this poses in quantifying a p300-effect on FOXO3a, we measured the effect of 346
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either WT or d.n.p300 on ectopically expressed FOXO3a. As shown in Figure 3D, co-347
transfection of WT p300 significantly reduced FOXO3a protein, which required its HAT 348
activity, since co-tranfection of d.n.p300 did not similarly reduce FOXO3a protein levels. 349
Although the decrease in FOXO3a protein by WT p300 may explain our finding that WT p300 350
repressed FOXO3a-induced transcription of the FOXO reporter (as shown in Figure 3B), the 351
levels of FOXO3a protein in muscles co-transfected with d.n.p300 were not significantly 352
increased. Therefore, the synergistic increase in FOXO3a-induced reporter activity when HAT 353
activity was decreased (by d.n.p300) cannot be fully accounted for by higher levels of FOXO3a 354
protein. To demonstrate this we normalized FOXO reporter activity from Figure 3B (underlined 355
groups) to total FOXO3a protein levels. Data are normalized such that the ratio of FOXO 356
activity/total FOXO3a protein levels in muscles transfected with FOXO3a alone is set to baseline 357
(Figure 3E). Since co-transfection of WT p300 with FOXO3a blocked FOXO3a-induced 358
reporter activation and concomitantly decreased total FOXO3a levels, the ratio of FOXO 359
activity/total FOXO3a protein did not change in the presence of WT p300. However, the 16-fold 360
increase in FOXO activity in muscles transfected with FOXO3a and d.n.p300, was still increased 361
by more than 4-fold when normalized to total FOXO3a protein levels (Figure 3E). Therefore, 362
regulation of FOXO3a protein levels by p300 HAT activity cannot fully account for the effect of 363
p300 on FOXO3a transcriptional activity. In summary these data demonstrate that p300 HAT 364
activity increases FOXO3a acetylation, and represses the transcriptional capacity of FOXO3a. 365
366
p300 & CBP differentially regulate FOXO1 and FOXO3a cellular localization 367
One mechanism whereby acetylation has been shown to regulate FOXO transcriptional activity 368
is through the regulation of FOXO cellular localization (13, 36). To determine whether 369
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p300/CBP regulate FOXO cellular localization we transfected C2C12 cells with a FOXO3a-370
DsRed fusion construct plus an empty vector, WT p300 or WT CBP expression plasmid. 371
Following differentiation into myotubes we induced FOXO3a-DsRed nuclear localization via 372
nutrient deprivation for 2 hours, which has been shown previously (7, 43), and determined the 373
effect of p300 and CBP on FOXO3a-DsRed cellular localization via fluorescent microscopy 374
using a Rhodamine (red) filter. The redistribution of FOXO3a-DsRed from the cytosol to the 375
nucleus in response to nutrient deprivation (Figure 4A, B) was confirmed via Dapi staining 376
(blue), and was prevented by p300 (Figure 4D) and attenuated by CBP (Figure 4F). Similarly, 377
during control conditions, FOXO3a-DsRed was more predominantly cytosolic in myotubes co-378
expressing p300 (Figure 4C) or CBP (Figure 4E). Since FOXO1 signaling is also increased in 379
response to nutrient deprivation (15) we further tested the ability of p300 and CBP to regulate 380
FOXO1 cellular localization, using the same experimental design just described, but replacing 381
FOXO3a-DsRed with FOXO1-GFP, and using a GFP (green) filter. Two hours of nutrient 382
deprivation induced a modest, but visible increase in FOXO1-GFP nuclear localization (Figure 383
4G, H), which was potentiated by WT p300 (Figure 4J) and attenuated by WT CBP (Figure 4L). 384
Though less visible, WT p300 similarly increased FOXO1-GFP nuclear localization during 385
control conditions. This effect of p300 on FOXO1-GFP localization in response to starvation is 386
in stark contrast to its effect on FOXO3a-DsRed, further indicating that p300 differentially 387
regulates the FOXO homologues. 388
Since both p300 and CBP were found to regulate FOXO1 and FOXO3a cellular 389
localization, we further measured their ability to regulate total FOXO reporter activation in 390
response to nutrient deprivation. To do this we transfected C2C12 cells with the FOXO reporter 391
plasmid plus an empty vector, WT p300 or WT CBP plasmid. Following four days of 392
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differentiation, cells either remained in differentiation media (control conditions) or were 393
nutrient deprived by removing the media and incubating in HBSS for 6 hours. FOXO-dependent 394
luciferase activity was increased by ~20% following nutrient deprivation, and was not altered by 395
p300 during either condition (Figure 4M). In contrast, transfection of CBP reduced FOXO-396
dependent luciferase activity during both control and nutrient deprivation conditions when 397
compared to empty vector groups. To determine the physiological relevance of this decrease in 398
total FOXO activity by CBP, we measured the ability of CBP to regulate the gene transcription 399
of the atrophy related FOXO target gene, atrogin-1, following 6 hours of nutrient deprivation. 400
As shown in Figure 4N, 6 hours of HBSS treatment induced a 2.5-fold increase in atrogin-1 401
transcription, which was attenuated by ~60% in HBSS treated cells transfected with CBP. 402
Therefore overexpression of CBP can attenuate both FOXO3a and FOXO1 nuclear localization 403
in response to nutrient deprivation and prevent the full activation of atrogin-1. 404
Our findings in Figure 3B using the FOXO1 and p300 constructs suggest that alterations 405
in p300 HAT activity are not sufficient to regulate FOXO1 transcriptional activity (on the FOXO 406
reporter), in vivo. However, in our localization experiments shown in Figure 4I and 4J, p300 407
strongly induced FOXO1 nuclear localization. Since p300 is predominately localized to the 408
nucleus, we tested the extent to which FOXO1 and p300 interact, which may potentially explain 409
the increased nuclear residence of FOXO1 in p300-transfected cells. Since p300 also regulated 410
FOXO3a localization, though in an opposite manner, we also measured the extent to which p300 411
interacts with FOXO3a. To do this we used a co-immunoprecipitation assay kit in which we 412
immunoprecipitated p300 from protein extracts of soleus muscles injected with an empty vector 413
or WT p300 plasmid. Following p300 protein precipitation we subsequently immunoblotted for 414
endogenous FOXO3a or FOXO1. As shown in Figure 4O and 4P, p300 interacts with both 415
19
FOXO3a and FOXO1, and this interaction is increased when p300 is overexpressed. The 416
interaction of p300 was found to be greater with FOXO1 than FOXO3a, which may therefore 417
explain our finding that p300 potentiates FOXO1 nuclear localization during nutrient 418
deprivation. 419
420
p300/CBP repression of FOXO is mediated via Akt 421
The ability of HATs to regulate FOXO signaling has previously been shown in other cell types to 422
occur through enhancing Akt-mediated repression of FOXO. This led us to question whether 423
p300/CBP could repress FOXO activation when Akt signaling is specifically inhibited. 424
Treatment of C2C12 myotubes with 10 μM LY294002 for 6 hours to inhibit PI3K/Akt signaling 425
resulted in a 40% increase in FOXO reporter activation (Figure 5A). Neither CBP nor p300 426
were able to repress FOXO reporter activation induced by LY294002 treatment. These findings 427
suggest p300/CBP may require Akt to inhibit FOXO signaling. To further test this we transfected 428
C2C12 cells with the FOXO reporter plasmid plus an empty vector, d.n.Akt, or d.n.Akt plus WT 429
p300 to determine whether p300 can repress FOXO activation when Akt activity is directly and 430
chronically inhibited. Following 3 days of differentiation luciferase activity was measured. As 431
shown in Figure 5B, transfection of d.n.Akt was sufficient to significantly increase FOXO 432
reporter activity by ~50%, which trended toward a further increase (p=0.087) in cells co-433
transfected with WT p300. This finding confirms that p300 cannot repress FOXO activation 434
when Akt activity is chronically inhibited, and suggests that p300 HAT activity may inhibit 435
FOXO through promoting FOXO inhibition by Akt. Based on this data, we hypothesized that 436
d.n.p300-induced activation of FOXO during normal conditions, in vivo (as shown in Figure 1A) 437
may have occurred through decreasing FOXO sensitivity to Akt. To test this we determined 438
20
whether increased Akt activity could overcome FOXO activation induced by d.n.p300. To do 439
this, we electroporated rat soleus muscles with the FOXO reporter plasmid and either empty 440
vector, d.n.p300, c.a.Akt, or d.n.p300 plus c.a.Akt plasmids. As shown in Figure 5C, the 441
increase in FOXO activity induced by d.n.p300 was repressed in muscles co-transfected with 442
c.a.Akt. Collectively these data demonstrate that p300 HAT activity is necessary for the normal 443
repression of FOXO in skeletal muscle under baseline conditions and that increasing HAT 444
activity is sufficient to repress FOXO activation during atrophic conditions, both of which appear 445
to be mediated via Akt-signaling. To identify whether p300 can increase FOXO phosphorylation 446
at the known Akt sites, we measured endogenous FOXO1 and FOXO3a phosphorylation from 447
soleus muscles injected with an empty vector or WT p300 plasmid. Overexpression of p300 448
increased both FOXO1 and FOXO3a phosphorylation (Figure 5D), providing further evidence 449
that p300 may increase FOXO sensitivity to Akt. Importantly, since we found in previous 450
experiments that p300 increases FOXO3a acetylation (Figure 3C), we similarly determined 451
whether p300 also acetylates FOXO1. To do this we precipitated total acetylated proteins from 452
muscles injected with either an empty vector or WT p300 plasmid, as described previously, and 453
subsequently immunoblotted for FOXO1. Similar to the effect on FOXO3a, overexpression of 454
WT p300 increased FOXO1 acetylation (Figure 5E). 455
456
457
458
459
460
461
21
DISCUSSION 462
The current study provides the first evidence to support the acetyltransferase (HAT) activities of 463
p300/CBP in regulating FOXO signaling in skeletal muscle. We demonstrate that (1) p300 HAT 464
activity is necessary to repress FOXO transcriptional activity, in vivo, during normal 465
physiological conditions, (2) increasing CBP or p300 HAT activity is sufficient to block FOXO 466
activation, in vivo, in response to skeletal muscle disuse and in C2C12 cells in response to 467
nutrient deprivation or dexamethasone treatment, (3) increased p300/CBP HAT activity can 468
repress the activation of a subset of FOXO target genes in response to catabolic stimuli, and (4) 469
p300 interacts with and acetylates both FOXO1 and FOXO3a, and differentially regulates their 470
cellular localization and transcriptional activity. Together, these findings are the first to identify 471
p300/CBP-mediated acetylation as a mechanism to regulate the FOXO transcription factors in 472
skeletal muscle. 473
The findings in the current study suggest that p300/CBP HAT activity plays an important 474
role in repressing FOXO activity in skeletal muscle during normal physiological conditions. 475
Importantly, since neither p300 nor CBP overexpression reduced basal levels of FOXO activity 476
during normal conditions, this suggests that endogenous HAT activity already maintains a 477
maximal inhibitory effect on total FOXO signaling. In contrast, because increased HAT activity 478
via either p300 or CBP overexpression repressed FOXO activation in vivo, in response to muscle 479
disuse and in vitro, in response to nutrient deprivation and dexamethasone treatment, these data 480
indicate that FOXO regulation by HAT activity is altered during catabolic conditions. The 481
insufficiency of endogenous HAT proteins to repress FOXO during these conditions may 482
potentially be explained by multiple scenarios, one of which could be a decrease in HAT 483
activity. Although the regulation of p300/CBP HAT activity is multi-factorial, their activity is 484
22
regulated in part via direct Akt-mediated phosphorylation (20), and active Akt is reduced during 485
muscle disuse, (6), nutrient deprivation (Ref) and Dex treatment (ref). In addition, acetyl-coA is 486
a substrate for p300/CBP-mediated acetylation of target substrates, and is a key intermediate in 487
numerous metabolic processes (25). Therefore, decreases in the availability of acetyl-coA during 488
catabolic conditions could also reduce p300-mediated acetylation of target proteins. 489
Alternatively, the insufficiency of HAT proteins to repress FOXO during catabolic 490
conditions could be due to an increase in histone deacetylase (HDAC) activity. HDAC proteins 491
counteract the activities of HATs by removing acetyl groups from target proteins. There are five 492
different classes of HDACs (Class I, Class IIa, Class IIb, Class III (Sirtuins or SIRTs) and Class 493
IV) which each contain multiple family members (17). Similarly, there are multiple proteins 494
which possess HAT activity, including p300, CBP, PCAF and GCN5. Since HAT and HDAC 495
proteins acetylate/deacetylate specific protein substrates, the inability of endogenous HAT 496
proteins to suppress FOXO during catabolic conditions may result from the altered expression, 497
cellular localization, and/or activity of any one or combination of the HAT or HDAC proteins. 498
While measurement of these variables was beyond the scope of the current study, other studies 499
have shown that SIRT1 (5), HDAC2 (55), HDAC4 (1, 8, 50), HDAC5 (8) and HDAC6 (50) are 500
increased in response to skeletal muscle disuse. Although we are unaware of any published data 501
to support FOXO regulation by any of these HDACs in skeletal muscle, there is certainly 502
evidence in other cell types to support FOXO regulation by the NAD+-dependent SIRTs (3, 10, 503
49). In response to low nutrient conditions when NAD+ levels are elevated, FOXO 504
deacetylation by the Sirtuins increases FOXO-dependent transcription of various genes involved 505
in both glucose metabolism (13, 29) as well as autophagy (18). Since the activities of both 506
SIRTs and HATs are regulated via bioenergetic factors (NAD+ and acetyl-coA, respectively), it 507
23
seems plausible that alterations in the energy state of the muscle during catabolic conditions 508
could dictate which FOXO-target genes are activated, through altering HAT/HDAC-mediated 509
regulation of the FOXO factors. 510
In the current study we measured the effects of p300 HAT activity on various FOXO-511
target genes known to be elevated during muscle disuse. Interestingly, increased p300 HAT 512
activity was found to repress the disuse-induced activation of some FOXO-targets (atrogin-1, 513
MuRF1 and p21), yet contributed to the increased expression of others (Gadd45α, 4E-BP1 and 514
cathepsin-L). Subsequent experiments further found that p300 also differentially regulated the 515
activity and localization of the FOXO homologues, which could potentially explain the 516
differential effect of p300 and its HAT activity on FOXO target gene expression. Although the 517
relative contribution of each endogenous FOXO homologue to the regulation of these genes in 518
skeletal muscle during physiological muscle wasting is not known, FOXO overexpression and 519
transgenic studies have yielded important information in this regard. Several studies have now 520
demonstrated that FOXO3a is sufficient to increase atrogin-1, MuRF1 and LC3 gene 521
transcription (43, 54), both in C2C12s and in whole muscle. In contrast, the data is conflicting 522
on whether FOXO1 is sufficient to increase atrogin-1 and/or MuRF1 in skeletal muscle (23, 48, 523
54). Importantly, however, cathepsin L and Gadd45α mRNA levels are increased in the skeletal 524
muscle of FOXO1 transgenic mice and decreased in mice transgenically expressing d.n.FOXO1 525
(56). These genetic studies therefore suggest that atrogin-1 and MuRF1 are primary targets of 526
FOXO3a and that cathepsin L and Gadd45α are primary targets of FOXO1. Given our collective 527
findings that (a) p300 HAT activity represses FOXO3a transcriptional activity and nuclear 528
localization and represses atrogin-1 and MuRF1 gene expression and (b) p300 increases FOXO1 529
nuclear localization and contributes to the increased gene expression of cathepsin-L and 530
24
Gadd45α, it may be speculated that p300 regulates these genes through differentially regulating 531
FOXO3a- and FOXO1-dependent gene transcription. 532
It is important to mention however, that p300 also regulates histone configuration, can act 533
as a transcriptional co-activator, and may regulate additional transcription factors other than 534
FOXO. Therefore the changes in mRNA levels observed with the p300 constructs may also 535
reflect changes in these variables. However, our use of the atrogin-1 promoter reporter plasmid 536
circumnavigates at least one of these issues. Since plasmid DNA constructs remain 537
extrachromosomal, their regulation does not therefore depend upon an open histone 538
configuration for active gene transcription (as does genomic DNA). Therefore, at least for 539
atrogin-1, p300-mediated regulation of its promoter activity is not mediated through changes in 540
histone configuration. 541
While the current study provides evidence that p300/CBP-mediated repression of FOXO 542
may require intact Akt signaling, p300/CBP overexpression still repressed the physiological 543
activation of FOXO during skeletal muscle disuse. Therefore, although the levels of active Akt 544
are reduced, in vivo, during periods of muscle disuse (6), these reduced levels of Akt were 545
presumably sufficient for overexpressed WT p300/CBP to repress the disuse-induced activation 546
of FOXO in the current study. Furthermore, since reduction of endogenous HAT activity via 547
expression of d.n.p300 in control muscle was sufficient to activate FOXO, this also demonstrates 548
that FOXO can be activated without directly manipulating Akt levels. Collectively, these 549
findings suggest that it may be possible to therapeutically manipulate Akt-mediated repression of 550
FOXO indirectly, via targeting HAT activity, which would have important ramifications for the 551
muscle wasting field. 552
25
In summary these findings demonstrate that p300/CBP acetyltransferase activity is both 553
necessary and sufficient to repress FOXO transcriptional activity in skeletal muscle, in vivo. 554
Furthermore, this study offers new insight into the differential regulation of the FOXO 555
homologues in skeletal muscle and highlights new therapeutic possibilities for blocking specific 556
FOXO target-genes during conditions of muscle wasting. 557
558
GRANTS 559
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin 560
Diseases Grant R03AR056418 (to A. R. Judge). S. M. Senf is supported by a T32 from the 561
National Institute of Child Health and Human Development Grant T32-HD-043730. 562
563
DISCLOSURES 564
No conflicts of interest are declared by the author(s). 565
566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582
26
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764
30
FIGURE LEGENDS 765
Figure 1- p300 acetyltransferase activity is necessary and sufficient to repress FOXO 766
transcriptional activity in skeletal muscle. A and B: Foxo-dependent luciferase reporter 767
activity from weight bearing and 3-day cast immobilized solei injected with WT p300 or 768
d.n.p300 (lacks HAT activity) (A) or WT CBP (B), or the respective control plasmids (empty 769
vector; EV). A representative western blot for p300 from EV, WT p300 and d.n.p300 injected 770
muscles is shown in A. Data are reported as mean ± SEM, normalized to the weight bearing, 771
empty vector injected group, and N=at least 5 muscles/group C: C2C12 myoblasts were 772
transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either an empty vector, WT 773
p300 or WT CBP expression plasmid. Following 6 days of differentiation myotubes were 774
treated with vehicle or 1µM dexamethasone (DEX) for 6 hours and assayed for luciferase 775
activity. Data represent N=3 and are reported as mean ± SEM, normalized to the absolute 776
control group. Experiments were repeated at least three times. Significance was established at 777
p<0.05. *Significantly different from absolute control group. †Significantly different from empty 778
vector within respective treatment group. 779
780
Figure 2- p300 HAT activity differentially regulates the transcription of FOXO-target 781
genes during muscle disuse. A: Relative atrogin-1 mRNA levels from weight bearing or 3-day 782
cast immobilized solei injected with an empty vector, WT p300 or d.n.p300 plasmid. B: 783
Atrogin-1 promoter reporter activity from weight bearing or 3-day cast immobilized rat solei 784
injected with an atrogin-1 promoter luciferase reporter plus an Empty Vector, WT p300 or 785
d.n.p300 plasmid. C: Atrogin-1 promoter reporter activity from weight bearing rat solei co-786
injected with an empty vector or d.n.p300 plus either an empty vector or a d.n.FOXO construct. 787
31
D & E: Relative mRNA levels of (D) MuRF1, p21, 4E-BP1, Gadd45α, cathepsin-L, LC3b, 18S 788
and, (E) Foxo1, Foxo3a, and Foxo4 from rat solei injected with an empty vector, WT p300 or 789
d.n.p300 and exposed to weight bearing or 3-days of cast immobilization. All data are reported 790
as mean ± SEM, normalized to the absolute control group, N=at least 5 muscles/group. 791
Significance was established at p<0.05. *Significantly different from absolute control group. 792
†Significantly different from empty vector within respective treatment group. 793
794
Figure 3- p300 HAT activity differentially regulates the transcriptional activity of the 795
FOXO homologues. A-E: Rat soleus muscles were injected and electroporated with a FOXO-796
responsive luciferase reporter plus an empty vector (EV), FOXO1, FOXO3a or FOXO4 797
expression plasmid, each with an EV, WT p300, or d.n.p300 plasmid. A: Representative western 798
blots for total FOXO1, FOXO3a and FOXO4 overexpression. B: Seven days following plasmid 799
injection muscles were removed and assayed for FOXO-dependent luciferase activity. All data 800
are reported as mean ± SEM from at least 6 muscles/group, normalized to the absolute control-801
injected group and significance was established at p<0.05. *Significantly different from absolute 802
control group (EV only). †Significantly different from FOXO + EV within respective FOXO 803
group. #Significantly different. C: Endogenous FOXO3a acetylation was determined in soleus 804
muscles injected with an empty vector (EV), WT p300 or d.n.p300 plasmid via 805
immunoprecipitation (IP) of total acetylated proteins from protein extracts using an anti-acetyl-806
lysine antibody followed by western blot for FOXO3a. Experiments were independently 807
repeated three times. D: Representative western blot showing FOXO3a protein levels in soleus 808
muscles injected with FOXO3a plus an EV, WT p300 or d.n.p300, using α-tubulin as loading 809
control. E: FOXO activity (underlined in Figure 3B) normalized to total FOXO3a protein levels 810
32
from muscles injected with FOXO3a plus either an empty vector, WT p300 or d.n.p300 (from 811
Figure 3D). 812
813
Figure 4- p300 and CBP differentially regulate FOXO3a and FOXO1 cellular localization. 814
A-L: FOXO1 and FOXO3a localization in C2C12 cells transfected with FOXO3a-DsRed or 815
FOXO1-GFP plus an empty vector (EV), WT p300 or WT CBP plasmid, differentiated for 4 816
days and either left in differentiation media (control) or nutrient deprived by replacing media 817
with HBSS for 2 hours. FOXO3a-DsRed localization was visualized using a Rhodamine (Red) 818
filter and was merged with Dapi-stained (blue) nuclei (A-F). Arrows in B point to FOXO3a-819
DsRed positive nuclei, which are pink in the merged image. FOXO1-GFP positive myotubes 820
were visualized using a GFP (green) filter and were merged with Dapi-stained (blue) nuclei (G-821
L). Arrows in H and J point to nuclei that are FOXO1-GFP positive, and which are light blue in 822
merged images. M: C2C12 myoblasts transfected with a FOXO-responsive reporter, pRL-TK-823
Renilla, and an EV, WT p300 or WT CBP expression plasmid. Following 4 days of 824
differentiation myotubes were left in differentiation media (control) or nutrient deprived (HBSS) 825
for 6 hours and harvested for luciferase activity. Data are expressed as mean ± SEM, and are 826
normalized to the absolute control group. N: Atrogin-1 gene expression following 6 hours of 827
nutrient deprivation (HBSS) of C2C12 cells. Myoblasts were transfected with either an EV or 828
WT CBP and differentiated for 4-days prior to treatment. Data are expressed as mean ± SEM, 829
and are normalized to the respective control group. All data represent N=3, and experiments 830
were independently repeated at least three times. Significance was established at p<0.05. 831
*Significantly different from the respective control group. †Significantly different from EV + 832
HBSS group. O and P: The ability of p300 to interact with endogenous FOXO3a (O) and 833
33
FOXO1 (P) was determined in soleus muscles injected with either an empty vector or WT p300 834
expression plasmid. Equal amounts of protein extract were used to immunoprecipitate (IP) p300, 835
followed by subsequent immunoblot for either FOXO3a or FOXO1. Western blots for 836
endogenous FOXO3a and FOXO1 indicate the relative input for IP experiments. Experiments 837
were independently repeated three times. 838
839
Figure 5- HAT-induced repression of FOXO is mediated via Akt. 840
A: C2C12 cells were transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either 841
an empty vector (EV), WT p300 or WT CBP expression plasmid. Following 4 days of 842
differentiation myotubes were treated with either vehicle (ethanol) or the PI3Kinase inhibitor 843
LY294002 (10uM) for 6 hours, and harvested for luciferase activity. Data are normalized to their 844
respective vehicle treated group, and are therefore expressed as (+/-) LY294002. B: C2C12 cells 845
were transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either an EV, WT 846
p300, d.n.Akt, or WT p300 + d.n.Akt. Following 3 days of differentiation, myotubes were 847
harvested for luciferase activity. Data are normalized to the absolute control group. C: Rat 848
soleus muscles were injected and electroporated with a FOXO-responsive reporter plus either an 849
EV, d.n.p300, c.a.Akt, or c.a.Akt + d.n.p300. Seven days following injections, muscles were 850
removed and harvested for luciferase activity. Data are normalized to the respective empty 851
vector group (black bars) within the EV or c.a.Akt groups. All cell culture data represent N=3 852
and are reported as mean ± SEM, and were repeated at least three times. Significance was 853
established at p<0.05. *Significantly different from absolute control group. D: Representative 854
western blots showing phospho- and total FOXO1 and FOXO3a from soleus muscles injected 855
with either an empty vector or WT p300 plasmid. E: Endogenous FOXO1 acetylation from 856
34
muscle extracts in (D) was determined via immunoprecipitation (IP) of total acetylated proteins 857
using an anti-acetyl-lysine antibody followed by western blot for FOXO1. Experiments 858
represent N=3. 859
860
Figure 6- Proposed regulation of FOXO by p300/CBP acetyltransferase (HAT) activity. 861
A: During normal physiological conditions, HAT proteins acetylate FOXO and promote FOXO 862
retention in the cytosol by Akt. B: In response to catabolic conditions, disruptions in both Akt 863
and HAT signaling contribute to FOXO nuclear localization and transcriptional activation of 864
target-genes. 865
866
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0 Empty VectorWT p300d.n.p300
WB Imm
*
* †
†
*
FOXO
(6xD
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Rep
orte
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ive
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A B
250kDa
WB:p300
Figure 1
EV WTp300 d.n.p300
Vehicle Dex0.0
0.5
1.0
1.5
2.0
2.5
*
Empty VectorWT p300
†
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†
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orte
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orte
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ive
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ity)
WB Imm WB Imm WB Imm WB Imm WB Imm WB Imm WB Imm0.0
0.5
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* * Empty VectorWT p300d.n.p300
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†
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Empty VectorWT p300d.n.p300
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gin-
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omot
er R
epor
ter
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0.0
1.0
2.0
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5.0Empty Vectord.n.p300
EV d.n.FOXO
*
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gin-
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omot
er R
epor
ter
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Figure 2
CA B
D
WB Imm WB Imm WB Imm0.0
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1.5
2.0
2.5Empty VectorWT p300d.n.p300
* **
Foxo1 Foxo3a Foxo4
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*
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*
*
Rel
ativ
e m
RN
A le
vel
E
A
FOXO1
FOXO3a
- +
- +
FOXO4 - +
100kDa
75kDa
WB:FOXO1
WB:FOXO3a
WB:FOXO4
75kDa
100kDa75kDa
D
EV FOXO1 FOXO3a FOXO40.02.04.06.08.0
10.012.014.016.018.020.0
d.n.p300
Empty VectorWT p300
*
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#
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†
FOXO
(6XD
BE)
Rep
orte
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ity)
B
50kDa
C
FOXO3a
FOXO3a
Tubulin
FOXO3aWT p300
d.n.p300
Tubulin
EV
WTp30
0
d.n.p300
0.0
1.0
2.0
3.0
4.0
5.0
FOXO
Rep
orte
r Act
ivity
/Ec
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c FO
XO3a
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tein
EEV
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d.n.p300
IP:Acetyl-K
WB:FOXO3a
Input:
WB:
FOXO3a
Figure 3
EV
EV
EV
WT p300
WT CBP
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Control6 hr HBSS
**
*
†
FOXO
(6xD
BE)
Rep
orte
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ive
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fera
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ity)
Control Nutrient Deprivation
A B
C D
DapiFOXO3a Merge Dapi Merge
DapiFOXO1 Merge DapiFOXO1 Merge
B
E
G H
I J
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WT CBP
0.0
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*
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ativ
e At
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Figure 4
IP: p300
Input
FOXO3a
-WT p300
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WTCBP
WTp300
EV
IP: p300
WT p300
WB:FOXO1
Input WB:FOXO1
O P
+ - +
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0.5
1.0
1.5
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Empty VectorWT p300
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*
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(6xD
BE)
Rep
orte
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fera
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Figure 5BA
EV c.a.Akt0.0
1.0
2.0
3.0
4.0
5.0
Empty Vectord.n.p300
*
FOXO
(6xD
BE)
Rep
orte
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ive
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fera
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ctiv
ity)
EV
WT p300
WT CBP
0.00
0.25
0.50
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1.00
1.25
1.50
1.75
(+/-) LY294002
FOXO
(6xD
BE)
Rep
orte
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ive
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fera
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ctiv
ity)
C
D100 kDa
75 kDa100 kDa
75 kDa
WB:p-FOXO3a
WB:FOXO3a
100 kDa
75 kDa
WB:p-FOXO1
WB:FOXO1
WT p300 - +
WT p300 - +
100 kDa
75 kDa
100 kDa
75 kDa
50 kDa
WB:FOXO1
IgG
IP: Acetyl-KE
AktNormal�physiological�
conditions AktCatabolic�conditions
Figure 6
A B
HATs
FOXO
X
Ac
FOXOAc
P
FOXO
Atrogin�1/MAFbxMuRF1p21
FOXO
HATs
AcAc
