1 David X. Liu1,3,4,*, Dongmeng Qian1,5, Bin Wang5, Jin-Ming

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p300-dependent ATF5 acetylation is essential for Egr-1 gene activation and cell proliferation and 1

survival 2

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David X. Liu1,3,4,*, Dongmeng Qian1,5, Bin Wang5, Jin-Ming Yang2,3,, and Zhimin Lu6 4

Departments of 1Neural and Behavioral Sciences and 2Pharmacology, 3Penn State Cancer Institute, and 5

4Penn State Hershey Neuroscience Institute, Penn State University College of Medicine, Hershey, PA 6

17033, USA; 5Department of Microbiology, Qingdao University Medical College, Qingdao, Shandong 7

266071, China; 6Brain Tumor Center and Department of Neuro-Oncology, The University of Texas MD 8

Anderson Cancer Center, Houston, TX 77030, USA 9

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*Correspondence to: [email protected] 12

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Key words: ATF5; p300/CBP; Elk-1; Egr-1; cell survival and proliferation 14

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Running title: Regulating ATF5 function by p300-dependent acetylation 17

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.05887-11 MCB Accepts, published online ahead of print on 26 July 2011

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ABSTRACT 20

ATF5 has been shown to be a critical regulator of cell proliferation and survival; however, the 21

underlying mechanism remains largely unknown. We demonstrate here that ATF5 interacts with 22

transcriptional coactivator p300, which acetylates ATF5 at Lysine-29 (K29), which in turn enhances the 23

interaction between ATF5 and p300 and binding of the ATF5/p300 complex to the ARE region of the 24

Egr-1 promoter. ARE-bound ATF5/p300 acetylates Lysine-14 (K14) of nucleosomal histone H3 at both 25

the ARE and SRE of the Egr-1 promoter, which facilitates binding of ERK-phosphorylated Elk-1 to the 26

SRE, activating the Egr-1 promoter. Interference of p300-dependent acetylation of ATF5 or nucleosomal 27

histone H3 or blockade of ERK-dependent Elk-1 phosphorylation abrogates ATF5-dependent Egr-1 28

activation and cell proliferation and survival. These findings assign a central role for the ATF5/p300 29

complex in ATF5 function and suggest that coordinated actions by ATF5, p300, Elk-1 and ERK/MAPK 30

are essential for ATF5-dependent Egr-1 activation and cell proliferation and survival. 31

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INTRODUCTION 41

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Uncontrolled cell proliferation and enhanced cell survival are hallmarks of cancer (22), which often 43

results from aberrant gene expression. Transcriptional factors and their downstream genes essential for 44

cancer progression are potential targets for cancer therapies. The activating transcription factor 5 (ATF5), a 45

member of the ATF/CREB protein family of basic-region leucine zipper (bZIP) transcription factors (20), 46

plays an important role in the regulation of a variety of cellular functions including cell proliferation, survival, 47

and stress response (18). ATF5 is highly expressed in many types of cancer, including breast cancer, glioma, 48

neuroblastoma, medulloblastoma, thyroid follicular carcinoma, and B-cell chronic lymphocytic leukemia 49

(18). ATF5 is upregulated by growth factors and downregulated by growth factor deprivation. Exogenous 50

expression of ATF5 suppresses apoptosis induced by trophic withdrawal (12, 41), whereas interference of 51

ATF5 function induces apoptosis of several types of cancer cells (2, 12, 38, 41). On the other hand, ATF5 52

expression in neural progenitors and pheochromocytoma PC12 cells maintains them in a proliferative state and 53

blocks their differentiation, whereas ATF5 loss-of-function in these cells causes pre-mature differentiation (3, 54

4, 36), suggesting that functions of ATF5 differ from cell type to cell type. ATF5 overexpression elevates 55

expression of Hsp27, Cyclin D3, and CYP2B6 (a member of the P450 family). However, whether these 56

genes are ATF5 targets mediating ATF5-dependent cell survival and proliferation remains unclear (18). 57

Two recent studies indicated that Bcl-2 and the myeloid leukemia cell differentiation protein (Mcl-1), a 58

member of the Bcl-2 family of pro-survival factors, may contribute to ATF5-promoted survival function in 59

glioma and MCF-7 breast cancer cells; it is understood, however, that additional ATF5 targets are yet to be 60

identified (12, 45). 61

The E1A binding protein p300 (p300) and its homolog CBP (CREB binding protein) are 62

transcriptional coactivator proteins that regulate gene expression through interaction with transcriptional 63

factors and, in part, through acetylation of both histone and nonhistone substrates (17, 52). Mice completely 64


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lacking either p300 or CBP protein, or being heterozygous for both p300 and CBP, die early in 65

embryogenesis (50, 54), indicating that expression level of p300 and CBP protein is critical for their 66

functions. p300/CBP interact with several ATF/CREB family members including CREB (16) and ATF4 (27). 67

Recruitment of p300/CBP by transcription factors leads to histone hyperacetylation and appears to promote 68

changes in chromatin architecture that are permissive to transcriptional activation (46). In addition, p300/CBP-69

dependent acetylation of transcription factors leads to stabilization of transcription factor-p300/CBP complexes 70

or increased affinity of transcription factor-p300/CBP complexes to targeted promoters (7, 19, 28), stimulating 71

gene transcription. 72

Early growth response 1 (Egr-1) is a member of the immediate early gene group of transcription factors 73

(47) and, like ATF5, plays an essential role in regulation of cell proliferation, differentiation, and survival. Egr-74

1 is overexpressed in human prostate cancers (14) and acts as an important pro-survival factor in prostate 75

cancer cells during tumorigenesis (1, 8, 33). Egr-1 downregulation inhibits vascular smooth muscle cell 76

proliferation in rat (21) and sensitizes human breast carcinoma cells to apoptosis (21, 40). Egr-1 transactivates 77

a number of genes that include those coding for growth factors such as insulin-like growth factor-II, platelet-78

derived growth factor A and B, transforming growth factor-β1, and vascular endothelial growth factor-α (25, 79

49). On the other hand, expression of Egr-1 can be activated by a wide range of stimuli, including growth 80

factors, cytokines, and stress signals, suggesting that Egr-1 is part of a positive feedback loop promoting cell 81

proliferation and survival. The Egr-1 promoter, which contains 5 adjacent serum response elements (SRE), is 82

subject to elaborate transcription control that involves both serum response factor (SRF) and ternary complex 83

factors (TCFs) (23, 31, 53). Binding of both the SRF and a TCF to the SRE is required for activation of the 84

Egr-1 promoter (11, 35). TCFs, which include Elk-1 (a member of the ETS oncogene family), Sap-1, and Sap-85

2/Net/Erp, can be phosphorylated by extracellular-signal-regulated kinases (ERKs), which are members of 86

mitogen-activated protein kinases (MAPKs). Phosphorylation of Elk-1 promotes its binding to SRE and 87

thereby enhances Egr-1 transcription (42, 53). 88


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We recently identified two ATF5 response elements (AREs) in the Egr-1 promoter that are located 89

about 1 kb upstream of the SRE sites. The sequences and positions of these AREs are conserved in rat, mouse, 90

and human (29). In this report, we show that Egr-1 is a downstream target of ATF5 that contributes to ATF5-91

promoted cell proliferation and survival in cancer cells. ATF5 interacts with p300 and is acetylated at Lysine-92

29 (K29) by p300. ATF5 acetylation stabilizes the ATF5/p300 complex formation at the ARE and is essential 93

for ATF5-dependent Egr-1 expression. ARE-bound ATF5/p300 acetylates Lysine-14 (K14) of nucleosomal 94

histone H3, in both the ARE and SRE regions. ATF5-dependent H3K14 acetylation at the SRE enhances 95

binding of ERK-phosphorylated Elk-1 to the SRE, activating ATF5-dependent of Egr-1 transcription. 96

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RESULTS 98

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Egr-1 is a downstream target of ATF5 that mediates ATF5-dependent cell proliferation, 100

tumorigenic transformation, and survival 101

To determine the role of ATF5 in Egr-1 expression, we first showed that expression of an shRNA 102

against ATF5 (shRNA-ATF5) is effective in downregulating the mRNA and protein levels of endogenous 103

ATF5 in C6 rat glioma cells (Fig. 1A). Depletion of ATF5 downregulated a luciferase reporter activity 104

driven by an ATF5-specific DNA regulatory element (ARE) from the rat Egr-1 promoter (Fig. 1B) and 105

depleted both mRNA and protein levels of endogenous Egr-1 in C6 cells (Figs. 1C and 1D). Notably, 106

expression levels of Egr-2 and Egr-3, two other members of the Egr family, were not downregulated by 107

shRNA-ATF5 (Fig. 1D). Similar downregulation of the mRNA and protein levels of Egr-1 was observed 108

in MCF-7 human breast cancer cells transfected with a dominant-negative (dn) ATF5 (an ATF5 109

truncation mutant with deletion of its activation domain) that is known to interfere with ATF5 function 110

(3) (Fig. E). These results indicated that ATF5 loss-of-function specifically inhibits Egr-1 expression in 111

C6 glioma and MCF-7 breast cancer cells. 112


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To determine whether Egr-1 mediates ATF5 function, we generated a C6 cell line that stably 113

expresses FLAG-HA-tagged rat ATF5 at a level comparable to endogenous ATF5 (C6-FLAG-HA-ATF5) 114

(29). The C6-FLAG-HA-ATF5 cell line, as compared to the control C6 cell line containing the empty 115

vector (C6-pCIN4), showed a significant increase in Egr-1 expression and proliferation rate (Fig. 1F) and 116

transforming ability (Fig. 1G). In contrast, depletion of Egr-1 in the C6-FLAG-HA-ATF5 cells inhibited 117

ATF5-dependent cell proliferation and transformation potential (Figs. 1F and 1G). In addition, Egr-1 118

depletion accelerated cell death provoked by serum deprivation (SD) (Fig. 1H) and abrogated the anti-119

apoptotic effect induced by ATF5 expression (Fig. 1I). On the other hand, overexpression of Egr-1 120

rescued cells from apoptosis evoked by ATF5 depletion in C6 (Fig. 1J) and by ATF5 interference in 121

MCF-7 cells (Fig. 1K). Together, these data indicate that Egr-1 is a downstream target of ATF5 and it 122

mediates ATF5’s functions in promoting cell proliferation, tumorigenic transformation, and survival in 123

C6 and MCF-7 cells. 124

125

p300 binds to and acetylates ATF5 at K29 126

To determine the mechanism underlying ATF5-dependent Egr-1 transcription, we examined 127

whether ATF5 interacts with p300, a transcription co-activator that was previously shown to interact with 128

several ATF/CREB family members including CREB and ATF4 (16, 27). Immunoblotting of the 129

immunoprecipitated p300 from C6 cells with an anti-ATF5 antibody showed that endogenous ATF5 was 130

co-precipitated with p300 (Fig. 2A) while a control IgG failed to bring down either p300 or ATF5 data 131

not shown). In addition, the physical association between ATF5 and p300 was interrupted 24 h after 132

serum deprivation or staurosporine (STS) treatment (Fig. 2A), raising the possibility that this interaction 133

plays a role in regulation of cell proliferation and survival. Immunoblotting of the immunoprecipitated 134

GFP-ATF5 fusion proteins from HEK293 cells with an anti-FLAG antibody showed that GFP-ATF5 but 135

not GFP-dnATF5 associated with FLAG-p300 (Fig. 2B), indicating that p300 specifically interacts with 136


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the N-terminal transactivation domain of ATF5. To investigate whether ATF5 is acetylated by p300, we 137

cotransfected C6 cells with ATF5 and FLAG-p300, or with ATF5 and siRNAs against p300 and CBP in 138

the presence or absence of TSA, a histone deacetylase (HDAC) inhibitor. Expression of FLAG-p300 or 139

TSA treatment dramatically enhanced ATF5 acetylation whereas depletion of p300/CBP by siRNAs 140

abrogated the effect induced by TSA (Fig. 2C). These results suggest that ATF5 is subject to p300/CBP-141

dependent acetylation. 142

p300/CBP can acetylate protein substrates at a Gly-Lys (GK) consensus motif (6). Analysis of the 143

amino acid sequences of ATF5 revealed a GK motif (28-GK-29) conserved in human, rat, and mouse 144

(Fig. 2D). To test whether the K29 in the ATF5 GK motif is the specific acetylation site of p300, we 145

transiently transfected C6 cells with p300 and wild-type (WT) ATF5 or ATF5(K29R) in which K29 was 146

mutated into Arginine. Immunoblotting analysis showed that WT ATF5 but not ATF5(K29R) was 147

acetylated by p300 (Fig. 2E). To further demonstrate that p300 is responsible for ATF5 K29 acetylation, 148

we performed an in vitro acetylation assay using recombinant p300, GST-ATF5 and GST-ATF5(K29R) 149

purified from bacteria. As shown in Fig. 2F, purified p300 specifically acetylates GST-ATF5 but failed to 150

acetylate GST-ATF5(K29R). Taken together, these data indicate that ATF5 K29 is a specific target for 151

p300 acetylation. 152

153

ATF5 acetylation by p300/CBP is essential for ATF5/p300 binding to ARE and ATF5-dependent 154

Egr-1 promoter activation 155

To investigate the roles of ATF5 interaction with p300 and p300-dependent ATF5 acetylation in 156

regulation of Egr-1 expression, we performed a chromatin immunoprecipitation (ChIP) assay with or 157

without depletion of ATF5 or p300/CBP. As shown in Fig. 3A, both ATF5 and p300 are associated with 158

the ARE region of the Egr-1 promoter in C6 cells. Depletion of ATF5 blocked the binding of p300 to the 159

ARE, indicating a required role of ATF5 in p300 association with the ARE. Unexpectedly however, 160


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depletion of either p300 or CBP significantly weakened the binding of ATF5 to the ARE, and depletion 161

of both p300 and CBP completely abolished ATF5 binding (Fig. 3A). These results indicate that ATF5 162

and p300/CBP depend on each other for binding to the ARE. We also examined whether a similar inter-163

dependent mechanism governs binding of ATF5 and p300/CPB to the ARE of the Bcl-2 (P2) promoter, 164

which we recently found to be regulated by ATF5 (12). This analysis confirmed that binding of the 165

ATF5-p300/CBP complex to the Bcl-2 (P2) promoter was significantly compromised when ATF5, or 166

either p300 or CBP was depleted, while depletion of both p300 and CBP completely abolished ATF5 167

binding (Fig. 3B). As a control, no MCL-1 promoter DNA was detected to be associated with ATF5 in 168

the same ChIP materials, consistent with our previous findings that MCL-1 is not regulated by ATF5 in 169

C6 and MCF-7 cells (12). p300 was detected to be associated with distal but not proximal region of the 170

MCL-1 promoter in an ATF5-independent manner (Fig. 3B). To further examine the role of ATF5 171

acetylation in ATF5 binding to the ARE, we performed a ChIP assay using the C6-FLAG-HA-ATF5 172

cells. FLAG-HA-ATF5 protein–DNA complex was immunoprecipitated with a FLAG antibody and was 173

eluted with FLAG peptide. Eluted FLAG-HA-ATF5 protein–DNA complex was incubated with an anti-174

acetylated lysine (Ac-K) antibody for either depletion or re-immunoprecipitation of acetylated protein 175

from the complex. Whereas depletion of the acetylated protein removed all ATF5-bound ARE, re-176

immunoprecipitation of acetylated ATF5 recovered ATF5-bound ARE (Fig. 3C). These results indicate 177

an essential role of ATF5 acetylation in ATF5 binding to ARE. To determine whether p300/CBP-178

dependent acetylation of ATF5 regulates Egr-1 promoter activity, we cotransfected Egr-1 promoter-179

luciferase reporter and p300 with or without WT ATF5 or ATF5(K29R) into C6 cells. Quantitative real 180

time PCR (qPCR) analysis showed that expression of WT ATF5 increased Egr-1 promoter activity by 4- 181

to 5-fold and co-expression with p300 potentiated the stimulatory effect of WT ATF5, raising Egr-1 182

promoter activity by more than 20-fold (Fig. 3D). Significantly, expression of ATF5(K29R) with or 183

without co-expression of p300 did not increase Egr-1 promoter activity (Fig. 3D). In further confirmation 184

of the requirement of p300/CBP-dependent ATF5 acetylation in ATF5-promoted Egr-1 activation, 185

depletion of p300/CBP by siRNA, as treatment of C6 cells with anacardic acid (Fig. 3E) or garcinol (Fig. 186


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3F), two drugs that are known to inhibit the acetyl transferase activity of p300/CBP (5, 48), blocked 187

ATF5-stimulated Egr-1 activation. These results indicate that p300/CBP-dependent ATF5 acetylation is 188

essential for ATF5 binding to the ARE and ATF5-dependent Egr-1 promoter activation. 189

190

ATF5/p300 binding to ARE is required for p300-dependent K14 acetylation of nucleosomal histone 191

H3 at ARE and SRE and for activation of Egr-1 promoter 192

SRE in the Egr-1 promoter is a focal point for regulation (23, 31, 53), which is located about 1 kb 193

downstream of the ARE sites (29). To investigate how the ARE-bound ATF5/p300 complex regulate Egr-194

1 promoter activity and whether the ARE-bound ATF5/p300 complex regulate Egr-1 promoter by 195

affecting the function of the SRE, we transfected FLAG-p300 with WT GFP-AFT5, GFP-dnATF5, or 196

GFP-ATF5(K29R) into C6 cells. As shown in Fig. 4A, a ChIP assay with antibodies against GFP, FLAG, 197

or acetylated H3 K14 (H3K14ac), which is a product of p300/CBP acetylation and is associated with 198

active promoters (10, 32, 37), showed that both WT ATF5 and p300 bind to the ARE and that their 199

binding correlates with increased H3K14ac at the ARE (Fig. 4A, upper panel, lanes 3, 4 and 5). In 200

contrast, expression of dnATF5 or ATF5(K29R) resulted in a loss of DNA binding by both ATF5 and 201

p300 and decreased H3K14ac at the ARE (Fig. 4A, middle panels, lanes 3, 4 and 5). Interestingly, while 202

neither WT ATF5 nor p300 associated with the SRE directly, elevated nucleosomal H3K14ac at the SRE 203

region was evident (Fig. 4A, upper panel, lanes 7, 8 and 9), which was not observed in cells expressing 204

p300-unassociatable dnATF5 or ATF5(K29R) (Fig. 4A, lanes middle panels, 7, 8 and 9). These results 205

suggest that binding of ATF5-associated p300 at ARE is required for accumulation of nucleosomal 206

H3K14ac at both the ARE and SRE regions. Accordingly, depletion of p300/CBP, as expression of 207

ATF5(K29R), abrogated H3K14 acetylation at both the ARE and SRE regions (Fig. 4B). 208

To further examine the role of ARE-bound ATF5/p300 complex in SRE acetylation and Egr-1 209

promoter activity, we stably transfected luciferase reporter vectors driven by WT, or ARE- or SRE-210


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deleted mutant of Egr-1 promoters in 293 cells. Transient expression of WT ATF5 or ATF5(K29R) in 211

these cells and ChIP assays with an anti-H3K14ac antibody showed that expression of WT ATF5 but not 212

ATF5(K29R) increased nucleosomal H3K14ac at the ARE and the SRE regions of the Egr-1 promoter 213

(Fig 4C, WT panel). In addition, deletion of the ARE blocked the increase of H3K14ac at both sites (Fig. 214

4C, DA panel); deletion of the SRE abolished the increase of H3K14ac at the SRE but not at the ARE 215

region (Fig. 4C, DS panel). These results strongly support the conclusion that ARE-bound ATF5/p300 216

complex is required for H3 K14 acetylation at the SRE. qPCR analyses showed that ARE deletion 217

completely, whereas SRE deletion significantly, abrogated ATF5-promoted transactivation of the Egr-1 218

promoter (Fig. 4D). These results indicate that ATF5/p300 binding to ARE is required for p300/CBP-219

dependent H3 K14 acetylation at both the ARE and SRE regions and that stimulation of the SRE by 220

means of H3 K14 acetylation promoted by ARE-bound ATF5/p300 complex plays a major role in Egr-1 221

activation. 222

223

ARE-bound ATF5/p300 promotes binding of ERK-phosphorylated Elk-1 to SRE, which is essential 224

for EGF-induced Egr-1 expression 225

Elk-1 phosphorylation by ERK/MAPK is known to increase its binding to SRE of the Egr-1 226

promoter and enhance Egr-1 transcription (42, 53). To determine whether stimulation of SRE by ARE-227

bound ATF5/p300 involves ERK-phosphorylated Elk-1, we used epidermal growth factor (EGF) to 228

stimulate C6 cells with or without expressing shRNA-ATF5, WT ATF5 or ATF5(K29R), and in the 229

presence or absence of the MEK/ERK inhibitor PD098059. As shown in Fig 5A, immunoblotting of the 230

immunoprecipitated ATF5 with antibodies against acetylated lysine (Ac-K) and p300 showed that EGF 231

induced acetylation of ATF5 and the interaction between ATF5 and p300 (Fig. 5A, IP:ATF5 panels). In 232

addition, a ChIP assay with anti-H3K14ac and anti-phosphorylated Elk-1 (Elk-1-p) showed that EGF 233

treatment resulted in acetylation of H3K14 and binding of Elk-1-p at the SRE region. Depletion of ATF5, 234


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which abolished H3 K14 acetylation at SRE but did not affect Elk-1 phosphorylation, or inhibition of Elk-235

1 phosphorylation by PD098059, which did not affect either ATF5 acetylation or H3 K14 acetylation at 236

SRE, abrogated binding of Elk-1-p to the SRE (Figs. 5A, ChIP panels). In further support of a required 237

role of p300/CBP-dependent ATF5 acetylation in binding of Elk-1-p to the SRE, treatment of cells with 238

the p300/CBP inhibitor anacardic acid or overexpression of acetylation-deficient ATF5(K29R) abolished 239

EGF-induced association between ATF5 and p300 and binding of Elk-1-p to the SRE (Fig. 5B). These 240

results indicate that Elk-1 binding to the SRE of the Egr-1 promoter is dependent on ATF5 acetylation 241

and Elk-1 phosphorylation. 242

We next examined Egr-1 expression in EGF-stimulated C6 cells with or without overexpressing 243

WT ATF5, ATF5(K29R), or shRNA-ATF5, and in the presence or absence of PD098059. Expressing WT 244

ATF5 but not ATF5(K29R) dramatically increased EGF-induced Egr-1 expression (Fig. 5C). In addition, 245

inhibition of ERK activity by PD098059 blocked ATF5-promoted Egr-1 expression (Fig. 5C). Together, 246

these results demonstrate that both the accumulation of H3K14ac at SRE, which is dependent on p300-247

promoted ATF5 acetylation, and the elevation of Elk-1-p, which is generated upon ERK activation, are 248

essential for EGF-promoted Egr-1 expression. 249

250

p300-acetylated ATF5 cooperates with ERK-phosphorylated Elk-1 to promote EGF-induced cell 251

proliferation and survival 252

To determine the roles of ATF5 acetylation- and Elk-1-phosphorylation-dependent Egr-1 activation in 253

EGF-induced cell proliferation, we examined the rate of bromodeoxyuridine (BrdU) incorporation in C6 254

cells transfected with WT ATF5, ATF5(K29R), or shRNA-ATF5 in the absence or presence of EGF 255

and/or PD098059. As shown in Fig. 6A, ATF5 depletion, expression of ATF5(K29R), or ERK inhibition 256

blocked EGF-induced BrdU incorporation whereas overexpression of WT ATF5 enhanced the EGF-257

induced effects, which was largely abrogated by expression of siRNA-Egr-1. On the other hand, 258


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overexpression of Egr-1 mitigated ATF5(K29R)-induced blockage on EGF-promoted BrdU incorporation 259

(Fig. 6A). These observations indicate that both p300/CBP-dependent ATF5 acetylation and ERK-260

dependent Elk-1 phosphorylation are required for EGF-induced cell proliferation that is mediated by Egr-261

1 gene activation. 262

To examine the roles of ATF5 acetylation- and Elk-1-phosphorylation-dependent Egr-1 activation in 263

serum deprivation-induced apoptosis, we transiently transfected p300 or siRNAs against p300 and CBP with 264

WT ATF5 or ATF5(K29R) into C6 cells subject to serum deprivation in the presence or absence of 265

anacardic acid or PD098059. Expression of p300 with WT ATF5 but not ATF5(K29R) blocked the serum 266

deprivation-induced cell death as indicated by reduced presence of apoptotic nuclei visualized by Hoechst 267

33342 staining (Fig. 6B). Consistent with Egr-1 being the downstream mediator for ATF5-promoted cell 268

survival, depletion of Egr-1 abrogated ATF5’s pro-survival effect while overexpression of Egr-1 269

mitigated the defective ATF5(K29R) (Fig. 6B). In further support of a required role for p300/CBP-270

dependent ATF5 acetylation and ERK-promoted Elk-1 phosphorylation in ATF5-promoted cell survival, 271

expression of siRNA against CBP and p300, or treatment with anacardic acid or PD098059 reversed the 272

survival effect of ATF5 in C6 cells subject to serum deprivation (Fig. 6C). Significantly, overexpression 273

of Egr-1 in these cells protected them from apoptotic death (Fig. 6C). These results indicate that both 274

p300/CBP-dependent ATF5 acetylation and ERK-dependent Elk-1 phosphorylation are required for 275

ATF5-dependent cell survival mediated by Egr-1. 276

277

DISCUSSION 278

279

ATF5 plays a critical role in regulating cell proliferation, differentiation, and survival (18); 280

however, the downstream targets that mediate its function and the mechanism by which it regulates target 281

genes remain largely unknown. Here, we demonstrate that 1) Egr-1 is an ATF5 downstream target that 282


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medicates ATF5’s function in cell proliferation and survival in C6 glioma and MCF-7 breast cancer cells; 283

2) ATF5 interacts with p300 and is acetylated at K29 by p300; ATF5 acetylation is required for ATF5 to 284

promote Egr-1 expression and cell proliferation and survival; 3) p300-dependent ATF5 acetylation is 285

essential for interaction between ATF5 and p300 and binding of the ATF5/p300 complex to the ARE of 286

the Egr-1 promoter. A similar inter-dependent role for ATF5 and p300 was observed in the binding of 287

ATF5/p300 complex to the ARE of the Bcl-2 promoter; 4) ARE-bound ATF5/p300 complex promotes 288

the acetylation of nucleosomal histone H3K14 at both the ARE and SRE regions; 5) ATF5-dependent 289

enrichment of H3K14ac at the SRE, a hallmark of gene activation, is accompanied with binding of ERK-290

phosphorylated Elk-1 to SRE and ATF5-dependent Egr-1 activation. Thus, activation of the ERK/MAPK 291

pathway is obligatory for ATF5 to activate Egr-1 expression and to promote cell proliferation and 292

survival. These observations reveal a novel pathway for ATF5 activation and function, and provide an 293

example of synergistic interplay between two transcription factors, i.e., the p300-acetylation-regulated 294

ATF5 and the ERK-phosphorylation-regulated Elk-1, via two far-away promoter enhancers, i.e., ARE and 295

SRE. These results have significant implications regarding the molecular mechanisms by which ATF5, as 296

an emerging transcription regulator, controls various cellular functions. 297

p300/CBP bind to the proline-rich domains of several proteins such as p53 and the Notch co-298

activator MAML1 (12, 44). The N-terminal transactivation domain of ATF5 contains about 200 amino 299

acids, of which prolines are more than 25%. Deletion of the N-terminal transactivation domain of ATF5 300

abolished ATF5 binding to p300 (Fig. 2B). This proline-rich N-terminal domain is also targeted by 301

HSP70 for protection against both proteosome- and caspase-dependent protein degradation processes 302

(29a). p300 acetylates ATF5 at K29 (Fig. 2E), which in turn enhances the association of p300 with ATF5 303

and the binding of the ATF5/p300 complex to the AREs of the Egr-1 promoter (Figs. 2C and 3D). Thus, 304

ATF5 acetylation is an indispensible event for ATF5-mediated activation of Egr-1 and subsequent cell 305

proliferation and survival. Although the mechanism of ATF5 acetylation-promoted interaction between 306

ATF5 and p300 and the binding of this protein complex to the ARE warrant further investigation, this 307


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regulation is reminiscent of p300/CBP-dependent p53 regulation, in which p300/CBP acetylate p53 and 308

increases p53 binding to the p21 gene promoter leading to a transcriptional activation of the p21 gene (19, 309

43). Thus, acetylation of ATF5 may constitute a major regulatory mechanism that modulates ATF5’s 310

activity in regulation of target genes. This is supported by our observation that loss-of-function of either 311

ATF5 or p300/CBP compromises binding of ATF5/p300 complex to the ARE of the Bcl-2 promoter (Fig. 312

3B), another ATF5-regulated gene (12). 313

Previous studies showed that p300/CBP, by serving as a scaffold for “bridging” or “looping” 314

chromatin, can act from a distal enhancer site to acetylate components of the basal transcriptional 315

machinery (e.g. TATA box-binding protein) and stabilize the transcriptional complex (9, 17, 24, 51). A 316

similar mechanism may govern the acetylation of the far-away downstream nucleosomal histone H3 at the 317

SRE of the Egr-1 promoter by ARE-bound p300/CBP. 318

Several observations suggest that the chromatin modifications triggered by ATF5-tethered 319

p300/CBP are likely more pervasive than the acetylation of H3K14 at the ARE and SRE. First, 320

p300/CBP is known to acetylate all four core histones in the nucleosomes (39), simultaneous 321

modifications at other sites on H3 and on other histones are likely. Notably, acetylation of N-termini 322

almost invariably associates with weakening of the DNA-histone and internucleosomal interactions, 323

increased binding of transcription factors to DNA, and activation of gene transcription (9, 26, 30). 324

Second, in addition to and accompanying with H3K14 acetylation, we also detected changes in K9-325

methylated-H3 (H3K9me) and K9-phosphorylated-K14-acetylated-H3 (H3K9pK14ac) at the SRE of the 326

Egr-1 promoter (data not shown), indicating participation of Lysine methyltransferase(s) and 327

Serine/Threonine protein kinase(s) (26, 34) in the ATF5/p300-promoted chromatin alterations. It is 328

conceivable that these chromatin alterations, initiated by the acetyl transferase activity of the ARE-bound 329

ATF5/p300, collectively determine ATF5’s stimulatory effect on Egr-1 transactivation. 330


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Interestingly, ATF5 and Egr-1 share several cellular functions and both regulate cell proliferation 331

and survival in a manner that is highly dependent on cell type (12, 18, 55). However, their functions do 332

not seem to overlap in all types of cells. One possible reason for ATF5 and Egr-1 to function differently 333

in certain cells may come from the fact that ATF5 regulates other genes in addition to Egr-1. For instance, 334

recent studies identified Bcl-2 and Mcl-1 as ATF5 targets contributing to ATF5’s pro-survival function in 335

several types of cells (12, 45). 336

Our study illustrates a central role of p300-acetylated ATF5 in ATF5-dependent Egr-1 activation 337

(Fig. 7). Our work reveals sequential and indispensible actions of p300 in ATF5 acetylation, of acetylated 338

ATF5 in maintaining ARE-bound ATF5/p300 complex, of ARE-bound ATF5/p300 complex in 339

enrichment of nucleosomal H3K14ac at the SRE, and of H3K14ac-enriched SRE in binding with ERK-340

phosphorylated Elk-1 and subsequent ATF5-dependent Egr-1 activation. These findings reveal the 341

dynamic feature of the ATF5/p300-regulated mechanisms and suggest that activation of the ERK/MAPK 342

pathway is critically involved in ATF5-dependent Egr-1 transactivation and subsequent cell proliferation 343

and survival. 344

345

MATERIALS AND METHODS 346

DNA Constructs, shRNAs, siRNAs and retroviruses 347

pCMS-EGFP-FLAG-ATF5, pCMS-EGFP-FLAG-dnATF5, pLeGFP-FLAG-ATF5 and pLeGFP-FLAG-348

dnATF5 (3) and pCIN4 and pCIN4-FLAG-HA-ATF5 (29) were described elsewhere. Mammalian 349

expression vector FLAG-p300 and Egr-1 were obtained from W. Gu (Columbia University) and Dan 350

Liebermann (Temple University), respectively. pCIN4-FLAG-HA-ATF5(K29R) was created using 351

QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) following manufacturer’s protocol 352


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with pCIN4-FLAG-HA-ATF5 as template and mutation-containing oligoes 5’-353

CTCGTAGACTATGGGAGACTCCCCCTGGCCCC-3’ 354

and 5’-GGGGCCAGGGGGAGTCTCCCATAGTCTACGAG-3’. 355

pSIREN-RetroQ-ZsGreen-ATF5 and pSIREN-RetroQ-ZsGreen-luciferase (non-silencing control shRNA) 356

were described previously (3). pSIREN-RetroQ-ZsGreen-Egr-1 was created similarly using oligoes 5’-357

GATCCGATGAACGCAAGAGGCATATTCAAGAGAAGATAGTCAGGGATCATGGTTTTTTACG358

CGTG-3’ and 5’-359

AATTCACGCGTAAAAAACCATGATCCCTGACTATCTTCTCTTGAAAGATAGTCAGGGATCAT360

GGCG-3’ and the pSIREN-RetroQ-ZsGreen vector (Clontech), following the manufacturer’s instructions. 361

To generate the Egr1 promoter-luciferase reporter construct (pGL3-Egr-1 Prom(WT)-luc), a DNA 362

fragment from (-1971) to (-1) of the rat Egr1 gene was amplified by PCR using the whole genomic DNA 363

as template, which was inserted into a luciferase reporter vector pGL3-basic with KpnI and XhoI. 364

Numbering of the Egr1 sequence is relative to the translation start site. The oligonucleotide sequences 365

used as primers are as follows: 5’-GGGGTACCCCCCGATCTTCCTTCTTCTG-3’ and 5’-366

CCGCTCGAGGTGGGTGAGTGAGGAAAGGA-3’. Deletions of ARE and SRE regions from the 367

pGL3-Egr-1 Prom(WT)-luc were made by PCR fragment cloning. For pGL3-Egr-1 Prom(DA)-luc in 368

which the ARE was deleted, the primers 5’-GGGGTACCGGTTGCTTCGGAGATAGGG-3’ and 5’-369

CCGCTCGAGGTGGGTGAGTGAGGAAAGGA-3’ were used. For pGL3-Egr-1 Prom(DS)-luc, the 370

primers 5’-GGGGTACCCTTCTGTCTCTCAATCTCCTTCCA-3’ and 5’-371

CCGCTCGAGGTGGGTGAGTGAGGAAAGGA-3’ were used. Wild type Egr1 expression plasmid 372

MSCV-puro/Egr-1 is a gift from Dr. B. Hoffman (Temple University). siRNA against CBP and p300 373

were purchased from Dharmacon, Inc. (Chicago, IL). Detailed information on construction of pLeGFP-374

ATF5, pLeGFP-dnATF5, pLeGFP-ATF5(K29R), GST-ATF5, and GST-ATF5(K29R) is available upon 375

request. All plasmids were confirmed by sequencing. 376


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377

Cell Culture, Transfection, Retrovirus Infection and Stable Cell Lines 378

C6, MCF-7 and HEK293 cells were grown in DMEM medium (Invitrogen) supplemented with 10% fetal 379

bovine serum (FBS) (Gemini Bio-Products), 100 μg/ml streptomycin, and 100 IU/ml penicillin. For 380

serum deprivation, cells were washed with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 381

mM KH2PO4, pH 7.4.) and maintained in serum free DMEM medium. For EGF stimulation, 1-day-382

transfected C6 cells were grown in medium without serum for 24 h prior to EGF (50 ng/ml) addition. 383

When used, PD98059 (Sigma) and anacardic acid (Enzo Life Sciences) were added to cells at a final 384

concentration of 50μM and 30μM, respectively, 60 min prior to stimulation by EGF. Cell transfection was 385

carried out using FuGENE 6 (Roche) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's 386

instructions. Replication-defective retroviruses were generated by transfection of retroviral vectors 387

expressing intended genes with pVSV-G in HEK293 cells as described (3). Stable C6-FLAG-HA-ATF5 388

and C6-pCIN4 cell lines were described previously (29). Stable cell lines HEK293-Prom(WT)-luc, -389

Prom(DA)-luc, and -Prom(DS)-luc were selected in 500μg/ml of G418 (Clontech) after transfection of 390

HEK293 cells with pcDNA3.1 with luciferase reporter vectors in which luciferase gene is driven by WT, 391

or ARE- or SRE-deleted mutation of Egr-1, namely pGL3-Egr-1 Prom(WT)-luc, pGL3-Egr-1 Prom(DA)-392

luc and pGL3-Egr-1 Prom(DS)-luc. 393

394

PCR, RT-PCR and qPCR 395

Primers 5’-TCTGACGACCCTGATCTTCC-3’ and 5’-TTTCATTCACTGCTTGCGTC-3’ were used for 396

detection of ARE (200 bp) of the Egr-1 promoter in regular PCR and qPCR. Primers 5’-397

ACTGCCGCTGTTCCAATACT-3’ and 5’-GTGAAGACCTCCCATCCAAG-3’ were for detection of 398

SRE (106 bp), 5’-GTCCAAGAATGCAAAGCACA-3’ and 5’-CCTTCCCAGAGGAAAAGCAA-3’ for 399

Bcl-2 (P2) promoter. Two pairs of primers, 5’-CCTCGCTTGCGTCAGAGCGG-3’ and 5’-400


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AGGCCGCGACTCCAGACTCG-3’, and 5’-TGCTGGGGGTCAACCTGGGA-3’ and 5’-401

CTTCCCCAGTCCCGGGGAGG-3’, were used to cover both the proximal (-64 to +29) and distal (-1021 402

to -888) regions of the MCL-1 promoter with respect to transcription start site (+1). For mRNA 403

abundance determination, total cellular RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, 404

CA) following the manufactures’ instructions. Two µg of total RNA was used as a template for cDNA 405

synthesis using SuperScript II (Invitrogen). Either regular PCR within linear amplification range or qPCR 406

using Applied Biosystems 7300 and/or iCycler (Bio-Rad Laboratories, Hercules, CA) were employed to 407

quantify relative cDNA abundance. Specific primers used were 5’-TGCACCCACCTTTCCTACTC-3’ 408

and 5’-AGGTCTCCCTGTTGTTGTGG-3’for Egr-1; 5’-GGTGATGCTGGTGCTGAGTA-3’ and 5’-409

ACTGTGGTCATGAGCCCTTC-3’ for β-actin; and 5’-CGCTGCTGGTGCCAACCCT-3’ and 5’-410

GGCGTTGGTCGCTTCCGGA-3’ for luciferase. 411

412

Antibodies 413

Antibodies used for immunoblotting and immunoprecipitation were anti-ATF5 (Abcam, Cambridge, 414

United Kingdom, and an in-house produced rabbit anti-ATF5 antibody from L. Greene), anti-β-actin (BD 415

Biosciences), anti-CBP, anti-Egr-1, anti-Egr-2, anti-Egr-3, anti-GFP, anti-p300 (Santa Cruz 416

Biotechnology, Santa Cruz, CA), anti-acetylated-Lysine (Ac-K) (Cell Signaling Technology), anti-Elk-1 417

and anti-phosphorylated Elk-1 (Elk-1-p) (Cell Signaling Technology, Danvers, MA), anti-FLAG (Sigma, 418

St. Louis, MO), and anti-HA (Roche, Basel, Switzerland). Antibodies used for chromatin 419

immunoprecipitation were anti-ATF5, anti-GFP and anti-p300 (Santa Cruz Biotechnology), anti-420

acetylated-Lysine (Ac-K), anti-acetylated-H3K14 (H3K14ac) and anti-phosphorylated Elk-1 (Elk-1-p) 421

(Cell Signaling Technology), and anti-FLAG (Sigma). 422

423


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Immunoblotting, Immunoprecipitation, Chromatin Immunoprecipitation, Chromatin 424

Immunodepletion, and Chromatin Re-immunoprecipitation 425

Immunoblotting (IB) and immunoprecipitation (IP) were performed as described previously (29, 32). 426

Chromatin immunoprecipitation (ChIP) was performed essentially as previously described (32). Briefly, 427

cells were incubated in culture media containing 1% formaldehyde with gentle shaking for 10 min at 428

room temperature, and crosslinking was stopped by addition of 2.5 M glycine to a final concentration of 429

0.125 M glycine. After two washes with cold PBS, cells were harvested in ice cold lysis buffer (10 mM 430

Tris-Cl [pH 8.0], 85 mM KCl, 0.5% NP-40, 5 mM EDTA, and fresh proteinase inhibitor cocktail) and 431

incubated on ice for 10 min. Nuclei were collected, suspended in cold RIPA buffer (10 mM Tris-Cl (pH 432

8.0), 150 mM NaCl, 0.1% SDS, 0.1% DOC, 1% Triton X-100, 5 mM EDTA, and fresh proteinase 433

inhibitor cocktail), and sonicated to shear the genomic DNA to an average of 300 bp. Cleared extracts 434

were blocked with protein A/G beads (Upstate Biotechnology), and aliquots of the supernatants were used 435

for immunoprecipitation by various antibodies. After seven washes by RIPA buffer with gentle rotation 436

for 5 min each time, 80% of each pellet was de-crosslinked in sample buffer and the samples were 437

resolved by SDS-PAGE and analyzed by Western immunoblotting with indicated antibodies. 20% of the 438

ChIP pellet was used to recover DNA by phenol extraction and ethanol precipitation after reversal of 439

crosslinking. The purified DNA was then analyzed either by PCR within linear amplification range 440

followed by agarose gel electrophoresis or by quantitative real-time PCR using Applied Biosystems 7300 441

and/or iCycler (Bio-Rad Laboratories). For chromatin immunodepletion (ChID), FLAG-ChIPed material 442

from C6-FLGA-HA-ATF5 cell was released from agarose beads with 3xFLAG peptide (Sigma) and the 443

elution was split into 2 aliquots. One aliquot was subject to 2 rounds of immunodepletion using beads-444

bound anti-Ac-K (2 μg each time); the other aliquot was mock-immunodepleted with IgG. Re-ChIP was 445

performed essentially as previously described (32). Briefly, FLAG-ChIPed material from C6-FLGA-HA-446

ATF5 cell was released from agarose beads with 3xFLAG peptide and the elution was split into 2 equal 447

aliquots which were subject to immunoprecipitation using beads-bound anti-Ac-K (2 μg) or IgG as mock 448


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control. For input controls, 20% of ChIP samples were used for Western analysis and 0.1% for PCR 449

analysis. 450

451

In vitro acetylation assay 452

In vitro acetylation assays were carried out according to the published method (19). Briefly, recombinant 453

p300 protein was immunoprecipitated from 100 µg of nuclear extract (NE) (32) using anti-HA antibody 454

plus Protein A-agarose (Sigma). Immunoprecipitates were washed twice in phosphate buffered saline 455

containing 0.1% Tween 20 (PBS-T), and once in acetyl-transferase assay buffer (50 mM Tris-Cl pH 8, 456

10% glycerol, 10 mM butyric acid, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF). Individual reactions 457

contained immunoprecipitated proteins from 20 µg of NE in 25 µl of assay buffer containing 10 µM 458

[Acetyl-1-14C] CoA (New England Nuclear, NEC-313; 51 mCi/mmole, 0.4 mM) and 2 µg GST-ATF5 or 459

GST-ATF5(K29R), purified using a Pierce Glutathione Agarose GST-purification Kits (Pierce). 460

Acetylation reactions were incubated for 1 h at 30°C on a rotating platform, followed by addition of SDS-461

PAGE sample buffer, electrophoresis through 10% SDS-PAGE gels, and transfer to NC membranes. 462

Proteins were detected by immunoblotting using antibodies against p300 and ATF5 while [14C]-acetylated 463

ATF5 and p300 were visualized, after application of a commercial fluorography enhancing solution 464

(Amplify, Amersham) as previously (32), by autoradiography at −70°C for 2 days. 465

466

Cell proliferation assays 467

For direct cell counting, C6-FLAG-HA-ATF5 or C6-pCIN4 cells were plated at a density of 1×106 468

cells/well in 6-well tissue culture plates and infected with retroviruses empty or expressing shRNA-Egr-1. 469

The infected cells were counted and seeded into 24-well plates (1×104 cells/well) in DMEM containing 470

10% FBS. Cells were collected daily by trypsin digestion and cell numbers were determined manually 471


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using a hemacytometer. For BrdU incorportation assay, C6 cells transfected with GFP-expressing vectors 472

were labeled with 10 µM BrdU (Sigma) for 5 h and subsequently fixed with 4% paraformaldehyde for 5 473

min at 4°C and blocked by incubation in 10% Normal Goat Serum for 60 min at RT. Cultures were 474

immunolabeled with a mouse monoclonal anti-BrdU (Boehringer, Mannheim, DE) and a polyclonal anti-475

GFP (Clontech, Mountain View, CA) antibody overnight at 4°C. The cells were then incubated with a 476

FITC- or Alexa 488-conjugated anti-rabbit and TRITC- or Alexa 568-conjugated goat antimouse IgG2A 477

(Southern Biotechnology, Birmingham, AL) for 60 min, counterstained with DAPI, cover-slipped, and 478

examined. 479

480

Hoechst 33342 staining for quantitative assessment of cell death and in vitro clonogenic assay 481

Hoechst 33342 staining (12, 29, 32) and in vitro clonegenic assay (15) were performed essentially as 482

described previously. 483

484

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44. Saint Just Ribeiro, M., M. L. Hansson, and A. E. Wallberg. 2007. A proline repeat 611 domain in the Notch co-activator MAML1 is important for the p300-mediated acetylation 612 of MAML1. Biochemical Journal 404:289-98. 613

45. Sheng, Z., L. Li, L. J. Zhu, T. W. Smith, A. Demers, A. H. Ross, R. P. Moser, and M. 614 R. Green. 2010. A genome-wide RNA interference screen reveals an essential 615 CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic 616 implications. Nature Medicine 16:671-7. 617

46. Sterner, D. E., and S. L. Berger. 2000. Acetylation of histones and transcription-related 618 factors. Microbiology & Molecular Biology Reviews 64:435-59. 619

47. Sukhatme, V. P., X. M. Cao, L. C. Chang, C. H. Tsai-Morris, D. Stamenkovich, P. 620 C. Ferreira, D. R. Cohen, S. A. Edwards, T. B. Shows, and T. Curran. 1988. A zinc 621 finger-encoding gene coregulated with c-fos during growth and differentiation, and after 622 cellular depolarization. Cell 53:37-43. 623

48. Sun, Y., X. Jiang, S. Chen, and B. D. Price. 2006. Inhibition of histone 624 acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation. 625 FEBS Letters 580:4353-6. 626


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49. Svaren, J., T. Ehrig, S. A. Abdulkadir, M. U. Ehrengruber, M. A. Watson, and J. 627 Milbrandt. 2000. EGR1 target genes in prostate carcinoma cells identified by microarray 628 analysis. Journal of Biological Chemistry 275:38524-31. 629

50. Tanaka, Y., I. Naruse, T. Hongo, M. Xu, T. Nakahata, T. Maekawa, and S. Ishii. 630 2000. Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of 631 CREB-binding protein. Mechanisms of Development 95:133-45. 632

51. Tolhuis, B., R. J. Palstra, E. Splinter, F. Grosveld, and W. de Laat. 2002. Looping 633 and interaction between hypersensitive sites in the active beta-globin locus. Molecular 634 Cell 10:1453-65. 635

52. Vo, N., and R. H. Goodman. 2001. CREB-binding protein and p300 in transcriptional 636 regulation. Journal of Biological Chemistry 276:13505-8. 637

53. Whitmarsh, A. J., P. Shore, A. D. Sharrocks, and R. J. Davis. 1995. Integration of 638 MAP kinase signal transduction pathways at the serum response element. Science 639 269:403-7. 640

54. Yao, T. P., S. P. Oh, M. Fuchs, N. D. Zhou, L. E. Ch'ng, D. Newsome, R. T. Bronson, 641 E. Li, D. M. Livingston, and R. Eckner. 1998. Gene dosage-dependent embryonic 642 development and proliferation defects in mice lacking the transcriptional integrator p300. 643 Cell 93:361-72. 644

55. Yu, J., I. de Belle, H. Liang, and E. D. Adamson. 2004. Coactivating factors p300 and 645 CBP are transcriptionally crossregulated by Egr1 in prostate cells, leading to divergent 646 responses. Molecular Cell 15:83-94. 647

648 649 650 651 652 ACKNOWLEDGEMENTS 653

654

We thank J. Angelastro, J. Goldman, M. Green, L. Greene, W. Gu and B. Hoffman for reagents 655

and I. Zagon for comments on this manuscript. We also thank D. Guan, G. Li and Y. Xu for 656

technical assistance. This work was supported in part by American Cancer Society Research 657

Scholar Awards RSG-08-288-01-GMC (D.X.L.) and RSG-09-277-01-CSM (Z.L.), Department 658


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of Defense grants BC085617 (D.X.L.) and BC050789 (J.M.Y.), National Cancer Institute grants 659

CA135038 (J.M.Y.), CA 66077 (J.M.Y.), and 5R01CA109035 (Z.L.), and National Natural 660

Science Foundation of China 30770105 (B.W.). 661

662

663

FIGURE LEGENDS 664

665

Fig. 1. Egr-1 is an ATF5 downstream target that mediates ATF5’s function in cell proliferation and 666

survival. 667

A) C6 glioma cells were transiently transfected with a non-silencing (NS) shRNA or shRNA against 668

ATF5. Total cellular RNA and whole cell extract were prepared 3 d later for RT-PCR (left panels) and 669

immunoblotting (right panels) analyses with primers corresponding to ATF5 and β-actin (control) or 670

antibodies against ATF5 and β-actin (control). 671

B) Luciferase reporter assay in C6 cells transiently transfected with indicated constructs and a luciferase 672

reporter that is driven by an ATF5-specific DNA regulatory element from the rat Egr-1 promoter. 673

C) Semi-quantitative RT-PCR analysis monitoring expression levels of Egr-1 and β-actin (control) 674

mRNAs in C6 cells transiently transfected with indicated constructs. 675

D) Immunoblotting analysis monitoring Egr-1, Egr-2, Egr-3, and β-actin (control) abundance in C6 cells 676

transiently transfected with shRNA-ATF5 for indicated times. 677

E) MCF-7 breast cancer cells were transiently transfected with a vector empty (vector) or expressing 678

dnATF5. Total cellular RNA and whole cell extract were prepared 3 d later for RT-PCR (left panels) and 679

immunoblotting (right panels) analyses as in (A). 680


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F) C6 cells stably expressing FLAG-HA-ATF5 (C6-FH-ATF5) or empty vector (C6-pCIN4) infected 681

with retroviruses expressing control shRNA (-) or shRNA-Egr-1 (+). Expression of Egr-1 and β-actin 682

(control) was monitored by immunoblotting using antibody against Egr-1 and β-actin (upper panels); cell 683

proliferation was measured by direct cell counting (lower panel). 684

G) Soft agar clonogenic assay monitoring transformation potential of C6-pCIN4 and C6-ATF5 cells 685

infected with retroviruses expressing a non-silencing (NS) shRNA or shRNA against Egr-1. 686

H) MCF-7 cells transiently transfected with a non-silencing (NS) shRNA or shRNA against Egr-1 were 687

serum-starved (SD) for 2 d. Transfected (GFP+) cells were scored for the presence of apoptotic nuclei 688

visualized by Hoechst 33342 staining. 689

I) C6 cells transiently transfected with vector empty (EGFP) or expressing ATF5 with or without shRNA 690

to Egr-1 were serum-starved (SD) for indicated times. Transfected (GFP+) cells were scored for the 691

presence of apoptotic nuclei visualized by Hoechst 33342 staining. 692

J) C6 cells transiently transfected as in (I) except GFP-expressing shRNA constructs empty (vector) or 693

against ATF5 (shRNA-ATF5) and a construct expressing WT Egr-1 (wtEgr-1) were used. Expression of 694

Egr-1 and β-actin (control) was monitored by immunoblotting as in (F) and apoptotic cells were 695

determined as in (I). 696

K) MCF-7 cells transiently transfected a pLeGFP vector empty (-) or expressing dnATF5 and a vector 697

empty (-) or expressing Egr-1 as indicated. Two days later, transfected (GFP+) cells were scored for the 698

presence of apoptotic nuclei visualized by Hoechst 33342 staining as (H). All experiments with statistical 699

analyses were performed at least three times, and error bars depict means ± SEM. 700

701

Fig. 2. p300 interacts with ATF5 and acetylates ATF5 at K29. 702


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A) Anti-p300-immunoprecipitates from C6 cells serum-deprived (upper panel) or staurosporine-treated 703

(lower panel) for 24 h were immunoblotted with antibodies against p300 or ATF5. 704

B) Anti-GFP-immunoprecipitates from HEK293 cells transiently transfected with indicated constructs 705

were immunoblotted with antibodies against FLAG or GFP. 706

C) C6 cells were transiently transfected with indicated constructs and followed with or without TSA (20 707

nM) treatment for 24 h. Immunoblotting analyses of immunoprecipitates using indicated antibodies were 708

performed. Top panel (Input) shows expression of FLAG-HA-ATF5 expression in transfected cells. 709

D) Amino acids sequences flanking the GK motifs in human, rat, and mouse ATF5. 710

E) Anti-FLAG-immunoprecipitates from C6 cells transiently transfected with indicated constructs were 711

immunoblotted with antibodies against HA or Ac-K. 712

F) In vitro acetylation of GST-ATF5 at K29 by p300. Upper panel: acetylation assay was performed as 713

described in Materials and Methods. [14C]-acetyl-CoA labeled GST-ATF5by p300 (and p300 self-714

acetylation) was visualized by autoradiography. Lower panel: Western blotting analysis showing input of 715

the purified GST-ATF5, GST-ATF5(K29R), and recombinant FLAG-p300 in each reaction. 716

717

Fig. 3. Acetylation of ATF5 by p300 increases ATF5 binding to ARE leading to Egr-1 activation. 718

A) Chromatin from C6 cells transiently transfected with indicated shRNAs or siRNAs was 719

immunoprecipitated with antibodies against ATF5, p300 or CBP. The amount of associated proteins and 720

Egr-1 promoter (ARE) DNA were monitored by immunoblotting with antibodies against ATF5, CBP, and 721

p300 and by PCR using primers specific to the ARE region of the rat Egr-1 promoter. Inputs in this and 722

all following experiments used 20% of ChIP materials for Western blotting analysis and 0.1% for PCR 723

analysis. 724


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B) C6 cell transfection with the shRNAs or siRNAs and ChIP analysis using antibodies against ATF5 or 725

p300 were performed as in (A). Bcl-2 (P2) and MCL-1 promoter proximal (-64 to +29) and distal (-1021 726

to -888) regions were analyzed in PCR reactions. 727

C) FLAG-HA-ATF5 protein-DNA complex from C6-FLAG-HA-ATF5 cells was immunoprecipitated 728

with a FLAG antibody and was eluted with FLAG peptide. Eluted FLAG-HA-ATF5 protein-DNA 729

complex was incubated with an anti-Ac-K antibody for either depletion or re-immunoprecipitation, which 730

was followed by immunoblotting analysis with an anti-HA antibody (upper panel) or PCR assay with 731

primers specific to the ARE region (lower panel). An irrelevant antibody (anti-B-Raf) was used as 732

controls (Mock) in the depletion and re-ChIP experiments. 733

D and E) qPCR analyses of Egr-1 expression in C6 cells transiently transfected with indicated constructs 734

in the absence or presence of anacardic acid (30μM). Data are presented as means ± SEM (n=3). 735

F) qPCR analyses of Egr-1 expression in C6 cells transiently transfected with vector empty (vector) or 736

expressing ATF5 in the absence or presence of anacardic acid (30μM) or garcinol (20μM). Data are 737

presented as means ± SEM (n=3). 738

739

Fig. 4. ARE-bound ATF5/p300 complex promotes enrichment of acetylated H3K14 at both ARE 740

and SRE sites. 741

A) ChIP analyses monitoring binding of ATF5 and p300 to ARE and SRE and enrichment of acetylated 742

H3K14 at ARE and SRE, in C6 cells transfected with indicated constructs. Input was 5% of the pre-ChIP 743

DNA. An irrelevant antibody (anti-B-Raf) was used as a negative control. 744

B) qPCR analyses of associated ARE and SRE DNA in anti-H3K14ac-precipitated ChIP materials. 745

Chromatin was prepared from C6 cells transfected with indicated constructs followed with serum 746

deprivation for 24 h. H3K14ac-associated ARE in ATF5-transfected cell was arbitrarily set at 100%. 747


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C) Chromatin preparation and qPCR analyses of H3K14ac-associated ARE and SRE DNA were 748

performed as in (B). HEK293 cells stably transfected with luciferase reporter vectors driven by WT, or 749

ARE-deleted (DA) or SRE-deleted (DS) mutant Egr-1 promoters were transiently transfected with 750

indicated constructs. H3K14ac-associated ARE in 293-Egr-1 Prom(WT)-luc cells transfected with ATF5 751

was arbitrarily set at 100%. 752

D) qPCR determination of reporter luciferase mRNA expression in the 293 cells as described in (C). All 753

statistical data are presented as means ± SEM (n=3). 754

755

Fig. 5. p300/CBP-dependent ATF5 acetylation is required for EGF-induced Elk-1 binding to SRE 756

and Egr-1 activation. 757

A) C6 cells transfected with vector empty (-) or expressing siRNA-ATF5 (+) or in the absence (-) or 758

presence (+) of PD098059 (50μM) were treated without or with EGF (50 ng/ml) for 10 h. Top two panels: 759

Immunoblotting of cell extracts monitoring abundance of Elk-1 and phosphorylated Elk-1. Middle panels: 760

Immunoblotting of ATF5-immunoprecipitates with antibodies against ATF5, acetylated Lysine (Ac-K) or 761

p300. Bottom four panels: ChIP analyses monitoring acetylated H3K14 (H3K14ac)-associated and 762

phosphorylated Elk-1 (Elk-1p)-associated SRE DNA using primers specific to the SRE region of the rat 763

Egr-1 promoter. 764

B) C6 cells transiently transfected with indicated constructs and/or in the absence or presence of 765

anacardic acid (30μM ) were treated with EGF (50 ng/ml) for 10 h. GFP-precipitated ChIP materials were 766

immunoblotted with antibodies against ATF5, Ac-K or p300 (upper three panels) and ChIP analyses 767

monitoring Elk-1p-associated SRE was performed as in (A). 768


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C) qPCR analyses of Egr-1 expression in C6 cells transiently transfected with indicated constructs and/or 769

in the absence or presence of PD098059 (50μM) and/or EGF (50 ng/ml) for 10 h. Data are presented as 770

means ± SEM (n=3). 771

772

Fig 6. p300/CBP-acetylated ATF5 cooperates with ERK-phosphorylated Elk-1 to enhance EGF-773

induced cell proliferation and to promote survival of C6 cell subject to serum deprivation. 774

A) C6 cells transiently transfected with indicated constructs were untreated or treated with EGF (50 775

ng/ml) in the absence or presence of PD098059 (50μM) for 24 h. Cell proliferation was monitored by 776

BrdU incorporation. 777

B) C6 cells cotransfected with a construct expressing GFP and constructs containing indicated genes were 778

serum-starved for 48 h. GFP+ nuclei were scored for the presence of apoptotic nuclei visualized by 779

Hoechst 33342 staining. 780

C) Apoptotic assay was performed as in (B) except cells were transient transfected with indicated 781

constructs followed by serum deprivation for 48 h in the absence or presence of anacardic acid (30μM) or 782

PD098059 (50μM). All experiments with statistical analyses were performed at least three times, and 783

error bars depict means ± SEM. 784

785

Fig 7. A model for cell proliferation and survival promoted by ATF5-dependent Egr-1 activation. 786

A-C) ATF5 interacts with p300/CBP and is acetylated at K29, which further enhances interaction 787

between ATF5 and p300/CBP and binding of ATF5-p300/CBP complex to the ARE of the Egr-1 788

promoter. ARE-bound ATF5-p300/CBP complex acetylates K14 of nucleosomal histone H3 at both ARE 789

and SRE regions of the Egr-1 promoter. 790


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D and E) Enrichment of acetylated H3K14 at SRE primes the SRE for binding by ERK-phosphorylated 791

Elk-1, resulting in Egr-1 gene activation. ATF5-dependent Egr-1 activation leads to cell proliferation and 792

survival. 793

794


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Fig 1Vector

shRNA-NS

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Fig 2


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Fig 3A

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Fig 4

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Fig 5

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Fig 6

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Fig 7


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Documents

1 David X. Liu1,3,4,*, Dongmeng Qian1,5, Bin Wang5, Jin-Ming