CCAAT / Enhancer-binding Protein ( C / EBP ) Is Acetylated at Multiple Lysines ACETYLATION OF C / EBP AT LYSINE 39 MODULATES ITS ABILITY TO ACTIVATE TRANSCRIPTION *

Teresa I. Ceseña, Jean-Rene Cardinaux, Roland Kwok, and Jessica Schwartz 2 From the Cellular and Molecular Biology Program, the Departments of Obstetrics/Gynecology and Biological Chemistry and the Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109 and Center for Psychiatric Neuroscience, Department of Psychiatry, University of Lausanne, CH-1008 Prilly-Lausanne, Switzerland

Acetylation of nuclear proteins was first detected in histones and is viewed as a part of a mechanism allowing DNA to become accessible to transcription regulatory machinery (1,2).It is now recognized that many cellular proteins are acetylated.In the nucleus, acetylation of several transcription factors is reported to have broad impact on their function.For example, acetylation of p53 stabilizes it by preventing its ubiquitination by Mdm2, allowing p53 to enter the nucleus to activate target genes (3).Acetylation of p53 at lysines 320, 373, and 382 increases its binding to cognate DNA (4,5).Acetylation is also reported to increase nuclear localization of NF-B (6), which is essential for its transcription factor function.Acetylation of GATA-1 was found to increase its binding to DNA, thereby stimulating GATA-1-dependent transcription (7).Other functional consequences of acetylation include promoting interaction of the nuclear import factor importin-␣ with importin-␤ (8).Ku70 is unable to bind and sequester pro-apoptotic BAX when Ku70 is acetylated (9).Acetylation in the DNA-binding domain of HMGI(Y) is inhibitory, decreasing its DNA-binding ability and weakening its transcriptional potency (10).Thus, acetylation modifies the function of a variety of cellular proteins.
The function of C/EBP␤ is modulated by its phosphorylation at several sites.For example, phosphorylation of human C/EBP␤ at a mitogen-activated protein kinase (MAPK) substrate site at Thr-235 (which corresponds to Thr-188 in mouse C/EBP␤) (29) alters its ability to activate transcription of a vari-ety of downstream target genes, including c-fos (21,30).The phosphorylation of C/EBP␤ at Thr-188 is rapidly increased in an extracellular signal-regulated kinase (ERK)-dependent manner by factors such as growth hormone (GH) (30), interleukin-1 ␤ (31), and interferon-␥ (32).Phosphorylation of C/EBP␤ at Thr-188 is also increased during adipogenesis in preadipocytes and NIH-3T3 cells (33)(34)(35).Murine C/EBP␤ has been observed to relocalize to heterochromatin within the nucleus in response to GH in a manner dependent on its phosphorylation at Thr-188 (36).Phosphorylation of rat C/EBP␤ at Ser-105 or of mouse C/EBP␤ at Thr-217 by p90 ribosomal S6 kinase stimulates proliferation in differentiated hepatocytes induced by transforming growth factor-␣ (37).Protein kinase A-or protein kinase C-mediated phosphorylation at Ser-240 of rat C/EBP␤ is reported to attenuate DNA binding (38).Furthermore, GH induces a delayed de-phosphorylation at a GSK-3 site at Ser-184 of mouse C/EBP␤, which may interfere with its binding to the c-fos promoter (39).Phosphorylation of C/EBP␤ at Ser-184 also contributes to adipogenesis and activation of adipocyte genes such as C/ebp␣ and aP2 (35).
C/EBP␤ has been shown to interact with p300 (40), a nuclear coactivator with intrinsic acetyltransferase activity (41).C/EBP␤ also associates with cAMP response-element-binding protein-binding protein, a coactivator and acetyltransferase homologous to p300 (42,43).Because of the association between C/EBP␤ and these acetyltransferases, this study investigates the acetylation of C/EBP␤ and its functional consequences.This report indicates that C/EBP␤ is acetylated, in agreement with several recent reports (44,45).Multiple acetylation sites are identified, including a novel acetylation site on C/EBP␤ at Lys-39 (numbering is based on the murine sequence of C/EBP␤ unless indicated otherwise).Importantly, mutation of Lys-39 decreases the ability of C/EBP␤ to mediate transcriptional activation of target gene promoters.Using an antiacetyl-lysine antibody that recognizes acetylated Lys-39, endogenous C/EBP␤ was found to be acetylated in preadipocytes, in which C/EBP␤ is an important factor during adipogenesis.C/EBP␤ is a critical mediator of GH-regulated transcription of c-fos (46,47).A nonacetylatable mutation at Lys-39 of C/EBP␤ impaired GH-stimulated c-fos promoter activation, and GH was found to increase acetylation of C/EBP␤.Taken together, this study identifies acetylation at Lys-39 as novel modification of C/EBP␤ that is regulated and contributes, alone and in combination with other acetylatable lysines, to C/EBP␤mediated transcription.

EXPERIMENTAL PROCEDURES
Plasmids and Antibodies-The numbering used to designate residues in C/EBP␤ is based on the mouse C/EBP␤ sequence (GenBank TM accession number NM009883).The following C/EBP␤ plasmids were used.The plasmid encoding full-length C/EBP␤ (LAP1) driven by the cytomegalovirus promoter (CMV-C/EBP␤) was a gift from Dr. U. Schibler (University of Geneva) and Dr. L. Sealy (Vanderbilt University).The plasmid HA-C/EBP␤ encodes C/EBP␤ (residues 22-296, also known as LAP2) tagged with HA at the N terminus (42).HA-C/EBP␤ is able to activate the c-fos promoter at least as well as full-length CMV-C/EBP␤ (data not shown).LAP2 is a prominent active form of C/EBP␤ in GH-responsive 3T3-F442A cells (21).Hereafter, C/EBP␤ will be designated as LAP2 unless otherwise indicated.Mutations were introduced into HA-C/EBP␤ at indicated residues using Stratagene QuikChange XL site-directed mutagenesis kit.All mutations were confirmed by sequencing.The mutated forms of C/EBP␤ are referred to as follows: K39R, K39A, K39Q, K117R, and K215R/K216R; TM refers to combined mutations K39R/K117R/K215R/K216R.As additional controls for mutation of Lys-39 in the transcriptional activation domain of C/EBP␤, arginine 42 was mutated to alanine (R42A) and lysine 98 was mutated to arginine (K98R).HA-C/EBP␤ mutated to alanine at phosphorylation sites Thr-188 (T188A) or Ser-184 (S184A) was similarly generated.GST-C/EBP␤ plasmids encode fusion proteins of GST with truncated forms of C/EBP␤ (residues 22-227, 22-193, and 22-103) as described previously (42).HIS-C/EBP␤ encodes C/EBP␤ tagged with six histidine residues at the N terminus (48).Recombinant HIS-C/ EBP␤ was expressed and purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen).
Plasmids for full-length, N-terminally FLAG-tagged p300 (p300) and N-terminally FLAG-tagged P/CAF (P/CAF) were prepared, expressed, and purified as described previously (49,50).The plasmid 5XC/EBP-luc encodes a luciferase reporter gene driven by five copies of a consensus C/EBP site (42).The plasmid C/EBP␣-luc was a gift from Dr. O. MacDougald (University of Michigan) (34).The plasmid for c-fos/luciferase (c-fos-luc), which contains the mouse c-fos promoter (Ϫ379 to ϩ1) upstream of luciferase, was a gift from Dr. W. Wharton (University of South Florida) and Dr. B. Cochran (Tufts University) (51).A plasmid encoding rat growth hormone receptor (GHR) was provided by Dr. C. Carter-Su (University of Michigan) (52).RSV-␤-galactosidase was provided by Dr. M. Uhler (University of Michigan).pcDNA3.1 vector, used to normalize the total amount of DNA in transfections, was purchased from Clontech.
The following antibodies were used: anti-HA (Covance) and anti-C/EBP␤ (specific for the C terminus of C/EBP␤; Santa Cruz Biotechnology) were used at dilutions of 1:100 for immunoprecipitations and 1:1000 for immunoblotting.Anti-acetyl-lysine (anti-Ac-K (Upstate); monoclonal antibody 4G12 that detects acetylated lysines on histones and p53) was used at a dilution of 1:500 for immunoblotting.An antibody against a peptide corresponding to human C/EBP␤ phosphorylated at Thr-235 (homologous to Thr-188 of mouse C/EBP␤) (anti-pC/EBP␤, Cell Signaling) was used at a dilution of 1:1000 for immunoblotting.
In Vitro Acetylation of C/EBP␤-Acetylation assays were performed using Acetylase Buffer.To test the acetylation of C/EBP␤ in vitro, purified HIS-C/EBP␤ (3 g) or the purified GST-C/EBP␤ fusion proteins (3 g each) were incubated in Acetylase Buffer alone or with added purified p300 or P/CAF (1 g each), and 1 l of [ 14 C]acetyl-CoA (55mCi/mmol; ICN).Samples were incubated for 1 h at 30 °C, separated by SDS-PAGE (8%), and analyzed by autoradiography (Kodak X-Omat Blue XB-1).To examine acetylation of expressed C/EBP␤, CMV-C/EBP␤ (LAP1) was expressed in 293T cells to obtain high protein expression.C/EBP␤ was immunoprecipitated using anti-C/EBP␤ and used in the acetylase assay described above.Protein levels were assessed by staining the gel with Coomassie Brilliant Blue G-250 (Bio-Rad).For expressed CMV-C/ EBP␤, a duplicate immunoblot was probed with anti-C/EBP␤ to evaluate migration and protein loading.
In Vivo Acetylation of C/EBP␤-CMV-C/EBP␤ (LAP1) was expressed in 293T cells, and 48 h later cells were incubated with 200 l of [ 3 H]sodium acetate (2.90 Ci/mmol; ICN) for 1 h.C/EBP␤ was immunoprecipitated with antibodies against C/EBP␤ or rabbit IgG (Sigma), which served as a control.Samples were separated by SDS-PAGE (8%) and analyzed by autoradiography.
To assess the acetylation of WT HA-C/EBP␤ or of HA-C/ EBP␤ mutated at various lysines, appropriate plasmids were coexpressed with or without plasmids for p300 (2.2 g) or P/CAF (0.2 g) in 293T cells.pcDNA3 was used to control for total amount of DNA transfected.24 h later, cells were incubated overnight with TSA (1 M) and NAM (5 mM) in serumfree DMEM containing 1% BSA.Cell lysates were subjected to immunoprecipitation with anti-HA; samples were separated by SDS-PAGE (4 -20%), transferred to polyvinylidene difluoride membrane, and probed with either anti-Ac-K or anti-HA.Phosphorylation of WT or mutated HA-C/EBP␤ was similarly analyzed, except immunoblots were probed with anti-pC/EBP␤ instead of anti-Ac-K.
To examine the regulation of acetylation of C/EBP␤, 293T cells were transfected with plasmids for WT HA-C/EBP␤ and GHR, and then 24 h later, they were incubated overnight with TSA (1 M) and NAM (5 mM) in serum-free DMEM containing 1% BSA.48 h after transfection, cells were treated with 250 ng/ml (11.5 nM) human GH (recombinant GH kindly provided by Lilly) for 15 min prior to lysis.Cells were lysed in Lysis Buffer; samples were immunoprecipitated with anti-HA as described and analyzed by immunoblotting with anti-Ac-K and anti-C/ EBP␤.Membranes were scanned using the Odyssey infrared scanning system (47), and bands were quantified using Odyssey software.Acetylation of C/EBP␤ was calculated using values for acetylation (anti-Ac-K) divided by total C/EBP␤ (anti-C/EBP␤ or anti-HA).Statistical analysis of results from three or more experiments was performed using Student's t test (Excel) or 1-way analysis of variance and Bonferroni's multiple comparison test (Prism version 3).
Transcription Assays-WT HA-C/EBP␤ or HA-C/EBP␤ mutated at various residues alone or in combination (each 400 ng/35-mm well) were coexpressed with reporter plasmids 5XC/ EBP-luc, C/EBP␣-luc, or c-fos-luc each (400 ng/well) in CHO-GHR cells, a reliable system to assess reporter gene activation with or without GH.␤-Galactosidase (300 ng/well) was coexpressed to normalize for transfection efficiency.24 h after transfection, cells were deprived of serum and lysed for luciferase assay 24 h after that, as described previously (56).In some experiments, cells were treated with vehicle or GH (500 ng/ml, 23 nM) for 4 h before lysates were prepared.Transcriptional activation was determined by luciferase output as measured using an Opticomp luminometer and is expressed as relative luciferase units/␤-galactosidase.Each condition was analyzed in triplicate for each experiment.Statistical analysis of results from replicate, independent experiments was performed using 1-way analysis of variance and Bonferroni's multiple comparison test (Prism version 3).

RESULTS
C/EBP␤ Is Acetylated in Vitro and in Vivo-Because C/EBP␤ and several acetyltransferases interact to activate transcription (40,42,47), the acetylation of C/EBP␤ was examined.The ability of p300 or P/CAF to acetylate C/EBP␤ was tested in vitro using purified C/EBP␤ in the presence of purified p300 or P/CAF and [ 14 C]acetyl-CoA.A prominent labeled band (Fig. 1A, upper panel, arrowhead) was detected when p300 or P/CAF was present but not in their absence.Additional evidence suggesting that C/EBP␤ is acetylated was obtained by expressing C/EBP␤ in 293T cells and subjecting lysates to immunoprecipitation with antibodies against C/EBP␤.When immunoprecipitates were incubated with [ 14 C]acetyl-CoA in the presence of purified p300 or P/CAF, label was incorporated into bands (Fig. 1B, upper panel, arrowhead) that comigrate with C/EBP␤ (Fig. 1B, lower panel), indicating that C/EBP␤ is acetylated by both p300 and P/CAF.Autoacetylation of p300 and P/CAF was also observed (41,(57)(58)(59).
To determine whether C/EBP␤ is acetylated in vivo, 293T cells expressing C/EBP␤ were incubated with [ 3 H]sodium acetate, and C/EBP␤ was immunoprecipitated using anti-C/EBP␤.A labeled band (Fig. 2A, arrowhead) migrating at a size that corresponds to C/EBP␤ (data not shown) suggests that C/EBP␤ is also acetylated in vivo.In agreement, an antibody specific for acetylated lysine (anti-Ac-K) detected acetylation of C/EBP␤ in vivo.When HA-C/EBP␤ was expressed in 293T cells and C/EBP␤ was immunoprecipitated using anti-HA, basal acetylation of C/EBP␤ was detected on immunoblots using anti-Ac-K (Fig. 2B).The acetylation of C/EBP␤ increased when p300 was coexpressed, which is consistent with the in vitro acetylation observed.
Acetylation of endogenous C/EBP␤ is detected using the anti-Ac-K antibody when C/EBP␤ is immunoprecipitated from lysates of 3T3-F442A preadipocytes using anti-C/EBP␤ (Fig. 2C, lane 3, arrowhead) but not in controls incubated without antibody or with IgG (lanes 1 and 2, respectively).These results indicate that endogenous C/EBP␤ in 3T3-F442A cells is acetylated.
C/EBP␤ Contains Multiple Acetylation Sites-Full-length murine C/EBP␤ (LAP1) is a 296-residue protein that contains 21 lysines (Fig. 3A).The acetylation of several forms of truncated C/EBP␤ (residues 22-227, 22-193, and 22-103) (bottom of Fig. 3A) fused to GST was tested.The GST-C/EBP␤ fusion proteins were incubated in vitro with [ 14 C]acetyl-CoA and p300 or P/CAF (Fig. 3B).Label was incorporated into all three forms of GST-C/EBP␤, in the presence of p300 (Fig. 3B, lanes 2, 5, and 8) or P/CAF (lanes 3, 6, and 9) but not in their absence (lanes 1, 4, and 7).The acetylation of GST-C/EBP␤ (residues 22-227) agrees with a previous report that C/EBP␤ is acetylated at   (15, 17, 60 -63).As a first approach to examine which lysines within C/EBP␤ are acetylated, seven peptides corresponding to C/EBP␤ sequences containing candidate lysines primarily in transcription regulatory domains (Fig. 3A) were synthesized, and their acetylation was determined in vitro using [ 14 C]acetyl-CoA and p300 or P/CAF (Fig. 4A).Peptides corresponding to lysines 39, 117, and 215/216 of C/EBP␤ were acetylated in the presence of p300 (black bars).The C/EBP␤ peptide containing lysines 215 and 216 also contains three other lysines at positions 211, 213, and 220; the acetylation sites in the peptide are referred to here as Lys-215 and Lys-216 based on a previous report that these two lysines of C/EBP␤ are acetylated (45).Interestingly, p300, but not P/CAF, acetylated these peptides, although P/CAF as well as p300 increased acetylation of a histone H3 peptide control.
To test whether the candidate lysines within the C/EBP␤ molecule are acetylated, lysines 39, 117, and the combination of 215 and 216 were mutated within HA-C/EBP␤ to nonacetylatable arginine residues (3,64), alone or in combination (Fig. 4B).WT HA-C/EBP␤ or each of its mutated forms was expressed  without or with p300 or P/CAF in 293T cells.C/EBP␤ in cell lysates was immunoprecipitated with anti-HA and immunoblotted with anti-Ac-K.Acetylation of WT C/EBP␤ was slightly visible in the absence of histone acetyltransferases (Fig. 4B, lane 3), and the ability of p300 or P/CAF to increase acetylation of WT C/EBP␤ is clearly evident (lanes 4 and 5).In contrast, when Lys-39 was mutated, acetylation of C/EBP␤ was almost completely obliterated, even in the presence of p300 or P/CAF (Fig. 4B, lanes 6 -8).On the other hand, acetylation of K117R C/EBP␤ (Fig. 4B, lanes 10 and 11) and K215R/K216R C/EBP␤ (lanes 13 and 14) was induced by p300 and P/CAF comparably to WT C/EBP␤.These findings suggest that anti-Ac-K detects acetylation of C/EBP␤ at Lys-39 but not at the other sites tested.Furthermore, when Lys-39 was mutated in combination with Lys-117, Lys-215, and Lys-216 (Fig. 4B, TM, lanes [15][16][17], acetylated C/EBP␤ was also undetectable.Mutation of Lys-39 of C/EBP␤ in various combinations with the other candidate lysines (K39R/K117R, K39R/K215R/K216R, or K39R/K117R/ K215R/K216R) also prevented acetylation, even in the presence of p300 (supplemental Fig. 1).
The specificity of the decrease in acetylation of Lys-39 C/EBP␤ was assessed by several other comparisons.Because Lys-39 lies in the transcriptional activation domain, it is relevant that mutations at nearby arginine 42 (Fig. 4B, R42A, lane 21) and at lysine 98 (K98R, lane 22), the nearest lysine residue to Lys-39, which all lie in the transcriptional activation domain of C/EBP␤, did not interfere with acetylation as K39R did (lane 20).This suggests that mutation at Lys-39 does not simply disrupt the integrity of the transcriptional activation domain.Furthermore, mutation K39A C/EBP␤ (Fig. 4B, lane 26), like K39R (lane 25), also disrupted acetylation.These studies indicate that Lys-39 of C/EBP␤ is acetylated and support the specificity of acetylation of C/EBP␤ at Lys-39.These findings also suggest that the anti-Ac-K antibody used may detect acetylation only at Lys-39 among the multiple sites tested in C/EBP␤.The apparent specificity of anti-Ac-K for Lys-39 and the fact that Lys-39 lies in the transcriptional activation domain of C/EBP␤ led to selection of Lys-39 as an acetylation site in C/EBP␤ that warranted further analysis.
C/EBP␤ Is Acetylated at Lys-39 despite Mutations at Phosphorylation Sites Thr-188 and Ser-184-Acetylation has been linked to phosphorylation for several proteins.For example, prior phosphorylation of histone H3 at serine 10 is required for its acetylation at lysine 14 (65)(66)(67).C/EBP␤ is phosphorylated at several sites, including Thr-188, which is a substrate for ERK1/2, and Ser-184, which is a substrate of GSK-3 (29,30,35,37,39,68).The possible dependence of acetylation of C/EBP␤ on its phosphorylation was examined by expressing WT C/EBP␤ or C/EBP␤ with mutations in two of the regulated phosphorylation sites, Ser-184 or Thr-188, with or without p300.C/EBP␤ was immunoprecipitated with anti-HA and immunoblotted with anti-Ac-K to determine whether mutating phosphorylation sites of C/EBP␤ would alter its acetylation at Lys-39 (Fig. 5).Basal acetylation of WT C/EBP␤ was slightly detectable (Fig. 5, lane 2), and increased in the presence of p300 (lanes 3), as expected.Mutating Ser-184 (Fig. 5, lane 4) or Thr-188 (lane 6) in C/EBP␤ did not impair basal acetylation of Lys-39 as detected by anti-Ac-K.Furthermore, acetylation of C/EBP␤ mutated at these phosphorylation sites increased in the presence of p300 as it does as for WT C/EBP␤ (Fig. 5, lanes 5 and 7).Blotting with an antibody specific for C/EBP␤ phosphorylated at Thr-188 (anti-pC/EBP␤) indicates that WT, Ser-184, and TM C/EBP␤, but not T188A C/EBP␤, were phosphorylated at Thr-188.Phosphorylation of WT and S184A C/EBP␤ at Thr-188 was similar in the absence or presence of p300 (Fig. 5, middle panel, lanes 2-5).These findings indicate that for the sites and conditions tested, C/EBP␤ is acetylated at Lys-39 even when phosphorylation at Ser-184 or Thr-188 in C/EBP␤ is prevented by mutation.Conversely, anti-pC/EBP␤ recognizes WT C/EBP␤, and mutation of C/EBP␤ at candidate acetylation sites at Lys-39, Lys-117, or Lys-215/216, alone or in combination, did not alter its phosphorylation at Thr-188 compared with WT C/EBP␤ (supplemental Fig. 2).Furthermore, phosphorylation of C/EBP␤ was not detectably altered by p300 for WT C/EBP␤ or any of the acetylation site mutants.Together, these data suggest that acetylation of C/EBP␤ at Lys-39 and its phosphorylation at Thr-188 are independent of each other.
Mutation of Lys-39 in C/EBP␤ Impairs Its Ability to Activate Transcription at a Consensus C/EBP Site-Lys-39 of C/EBP␤ lies within its transcriptional activation domain (69).To test whether Lys-39 of C/EBP␤ contributes to its ability to activate transcription, activation of a consensus C/EBP site (5XC/EBPluc) by WT C/EBP␤ or K39R C/EBP␤ was compared in CHO-GHR cells (Fig. 6A).WT C/EBP␤ significantly increased promoter activity compared with control cells transfected with vector alone, as expected (42).Mutation K39R completely blocked the ability of C/EBP␤ to mediate transcriptional activation via the consensus C/EBP site.The specificity of the decrease in activation by K39R C/EBP␤ was assessed by comparing C/EBP␤ mutated at nearby arginine 42 (R42A) or at lysine 98 (K98R), which also lie in the transcriptional activation domain of C/EBP␤.R42A and K98R C/EBP␤ increased promoter activity to the same extent as WT C/EBP␤, consistent with the observation that they are acetylated comparably to WT C/EBP␤ (Fig. 4B).Expression of the various forms of C/EBP␤ was comparable (Fig. 6A, lower panel), indicating that the decrease in promoter activation by K39R C/EBP␤ does not reflect a difference in expression.These findings indicate that impairment of acetylation as well as impaired transcriptional activation by mutation of Lys-39 in C/EBP␤ is specific for Lys-39 and does not reflect a disabled transcriptional activation domain.Mutating Lys-39 to alanine decreased C/EBP␤-mediated transcription as effectively as K39R (Fig. 6B), indicating that the lysine per se, rather than the charge, determines the function of C/EBP␤ (50).This is also consistent with a lack of acetylation of both K39A and K39R C/EBP␤ (Fig. 4B).Conversely, mutating lysine to glutamine is thought to mimic acetylation functionally (3).Consistent with this, K39Q C/EBP␤ increased activation via the consensus C/EBP site to the same extent as WT C/EBP␤ (Fig. 6B).Together, these findings indicate that acetylation at Lys-39 contributes to the ability of C/EBP␤ to mediate transcriptional activation via a consensus C/EBP site.
Mutation of Lys-39 of C/EBP␤ Impairs Its Ability to Activate the C/EBP␣ Promoter-C/EBP␤ activates the C/EBP␣ promoter as part of a transcription factor cascade during adipogenesis (25,70,71).The ability of WT and K39R C/EBP␤ to activate the C/EBP␣ promoter was compared.WT C/EBP␤ increased activation of the C/EBP␣ promoter more than six times above control (Fig. 7A).Mutation K39R in C/EBP␤ significantly reduced its ability to activate the C/EBP␣ promoter by more than 60%.In contrast, R42A and K98R C/EBP␤ increased promoter activity to the same extent as WT C/EBP␤.These results indicate that the impairment in the ability of K39R C/EBP␤ to activate the C/EBP␣ promoter is not secondary to general disruption of the transcriptional activation domain.Mutation K39A, like K39R, inhibited the ability of C/EBP␤ to activate the C/EBP␣ promoter (Fig. 7B).Interestingly, mutation of Lys-39  to glutamine, which mimics acetylation, increased C/EBP␣ promoter activation and was as effective as WT C/EBP␤.Together, these findings support the functional importance of acetylation of C/EBP␤ at Lys-39 in its transcriptional activation of C/EBP␣.
Activation of the c-fos Promoter Depends on Acetylation of C/EBP␤ at Lys-39-c-fos is a well studied gene whose promoter can be activated by C/EBP␤ (21,46,47,72).To test whether Lys-39 of C/EBP␤ contributes to its ability to activate c-fos, WT C/EBP␤ or C/EBP␤ mutated at Lys-39 was coexpressed with c-fos-luc (Fig. 8A).WT C/EBP␤ significantly increased promoter activity above control, as expected (21,30).In contrast, mutation K39R in C/EBP␤ completely blocked its ability to activate the c-fos promoter.C/EBP␤ with other mutations in the transcriptional activation domain, R42A and K98R C/EBP␤, were comparable with WT C/EBP␤ in their ability to activate the c-fos promoter, supporting specificity of Lys-39 in the ability of C/EBP␤ to activate c-fos transcription.Neverthe-less, mutating Lys-39 to Ala, which also impairs acetylation of C/EBP␤, decreased c-fos activation comparably to K39R (Fig. 8B), indicating a critical role for Lys-39 in the function of C/EBP␤ (50).K39Q C/EBP␤ increased c-fos promoter activation to the same extent as WT C/EBP␤, consistent with glutamine mimicking acetylation.Together, these findings indicate that acetylation at Lys-39 contributes to transcriptional activation of c-fos.
Lys-39 of C/EBP␤ Contributes to GH-mediated Activation of c-fos-C/EBP␤ is a critical mediator of GH-stimulated c-fos expression, because knockdown of C/EBP␤ prevents stimulation of c-fos RNA and promoter activation by GH (47).Because GH also increases the occupancy of both C/EBP␤ and p300 on the c-fos promoter (47), it was reasoned that GH may regulate acetylation of C/EBP␤.GH was found to increase acetylation of C/EBP␤ in 293T cells expressing WT C/EBP␤ and GH receptor, as detected with the anti-Ac-K antibody that recognizes acetylation at Lys-39 (Fig. 9A).The increase suggests that acetylation of C/EBP␤ in response to GH may play a role in its ability to activate transcription.
Because mutation of C/EBP␤ at Lys-39 alters the ability of C/EBP␤ to activate c-fos, and because GH increases acetylation of C/EBP␤ at Lys-39, the role of Lys-39 of C/EBP␤ in GHstimulated c-fos transcription was examined.GH stimulated the c-fos promoter in the presence of WT C/EBP␤ (Fig. 9B), as shown previously (21).However, in cells expressing K39R C/EBP␤, the response to GH was blunted and was not significant, even in the context of reduced basal promoter activity.The reduced response to GH when Lys-39 in C/EBP␤ is mutated suggests that GH increases acetylation of C/EBP␤ and also that acetylation of C/EBP␤ at Lys-39 contributes to activation of c-fos by GH.Not only is Lys-39 a novel acetylation site on C/EBP␤ that mediates activation of its gene targets, but its acetylation also appears to be regulated.Different Patterns of Acetylatable Lysines in C/EBP␤ Differentially Activate C/EBP␤-regulated Promoters-The pattern of acetylatable lysines in C/EBP␤, which mediate c-fos activation, was found to be distinct from the patterns observed for the other promoters tested.For c-fos, mutation of C/EBP␤ at Lys-117 or Lys-215/216 failed to impair activation when compared with WT C/EBP␤ (Fig. 10A), in contrast to the inhibition seen with K39R or K39A.Reinforcing the importance of Lys-39, c-fos promoter activation was also reduced when C/EBP␤ was mutated at Lys-39 in combination with Lys-117 and Lys-215/ 216 (TM).These findings suggest that the integrity of Lys-39 is a major contributor to the ability of C/EBP␤ to activate the c-fos promoter.
Whether Lys-39 is the only acetylatable lysine that contributes to transcriptional activation by C/EBP␤ was tested by comparing other promoters to c-fos.Mutations K39R, K117R, and TM (which contains K39R) C/EBP␤, but not K215R/K216R, decreased the ability of C/EBP␤ to activate a C/EBP consensus site compared with WT C/EBP␤ (Fig. 10B).Thus, for this C/EBP consensus site, acetylation at Lys-117 as well as Lys-39 contributes to activation by C/EBP␤.A different pattern was observed for the C/EBP␣ promoter (Fig. 10C), on which mutation of each of the four lysines tested significantly decreased transcription activation.These findings emphasize that different patterns of acetylatable lysines within C/EBP␤ are differentially required to activate C/EBP␤-regulated promoters.

DISCUSSION
Lysine 39 Is a Novel Acetylation Site in C/EBP␤-These studies show that C/EBP␤ is acetylated at Lys-39 and that the ability of C/EBP␤ to activate transcription is dependent on its integrity at Lys-39.Acetylation of endogenous, as well as expressed, C/EBP␤ was detected in vivo.Lysine 39 of C/EBP␤ was initially identified as a candidate by its acetylation in C/EBP␤ peptides incubated with p300 in vitro.Acetylation at this site by p300 and P/CAF in intact C/EBP␤ was substantiated using an antibody that recognizes acetylated WT C/EBP␤ but not C/EBP␤ mutated at Lys-39.Acetylation at Lys-39 is of note because this lysine lies in the transcriptional activation domain of C/EBP␤ (69).
The anti-Ac-K antibody appears to be specific for detecting acetylation of Lys-39 in C/EBP␤, because mutation of Lys-39 almost completely obliterated the anti-Ac-K signal, whereas mutation at other candidate (117, 215, and 216) and noncandidate (98) lysines did not interfere with detecting the signal.The anti-Ac-K antibody also detected a decrease in acetylation when Lys-39 was mutated in combination with Lys-117, Lys-215, and Lys-216 (TM).Several other anti-Ac-K antibodies were not effective (data not shown).The present work using the anti-Ac-K antibody and mutated C/EBP␤ strongly supports that Lys-39 of C/EBP␤ is acetylated.
C/EBP␤ Is Acetylated at Multiple Lysines-Although acetylation of C/EBP␤ has been reported (44,45), acetylation of Lys-39 as a specific acetylation site important for transcriptional activation is novel.Nevertheless, Lys-39 is only one of multiple acetylatable lysines in C/EBP␤.A previous report that Lys-215 and Lys-216 are acetylated is supported by the present observation that a C/EBP␤ peptide corresponding to Lys-215/ 216 was acetylated when incubated with p300 in vitro.Deacety-lation of C/EBP␤ at these residues allowed transcriptional activation of the Id-1 gene, mediated by recruitment of HDAC1 by Stat5 in response to IL-3 in Ba/F3 cells (45).In this study, muta- latable lysines tested appears to be required for transcription.The functional importance of each acetylation site does not appear to parallel the extent of acetylation at each lysine.Experiments with truncations of C/EBP␤ suggest that relative acetylation of C/EBP␤ exhibited by Lys-39 represents at most 50% of the acetylation among the four lysines tested.However, for c-fos, acetylation of Lys-39 alone appears to be sufficient for transcription.For the Id-1 promoter, it is not clear whether Lys-215 and Lys-216 in C/EBP␤ are the only residues whose regulation alters transcriptional activation.Overall, the relative degree of acetylation does not appear to be the determinant of function.Rather, differences in the patterns of acetylation among acetylatable lysines in C/EBP␤ appear to confer varying effectiveness for transcriptional activation on different promoters.
Acetylation, like phosphorylation (80), may serve as a regulated molecular switch for transcription (10).The regulation of acetylation, as well as phosphorylation, of C/EBP␤ introduces greater opportunity for specificity among its range of functions.This is of note with a transcription factor such as C/EBP␤ that modulates genes involved in a wide variety of processes, including immune responses, liver regeneration, and adipocyte differentiation (12-14, 19, 25, 81).Overall understanding of the functional importance of acetylation of C/EBP␤ can provide insight into regulatory networks for a variety of cellular functions mediated by changes in gene transcription.

FIGURE 1 .
FIGURE 1. C/EBP␤ is acetylated in vitro.A, purified HIS-C/EBP␤ was incubated alone, with purified p300 or purified P/CAF as indicated, in the presence of [ 14 C]acetyl-CoA.Autoradiograph (upper panel) shows acetylation of C/EBP␤ (arrowhead) in the presence of p300 and P/CAF.Migration of autoacetylated p300 and P/CAF are indicated by dashes on the right.Lower panel shows protein labeled with Coomassie Blue to indicate loading of C/EBP␤ in all lanes.Apparent molecular mass is indicated in the margin.Similar results were obtained in two other experiments.B, CMV-C/EBP␤ expressed in 293T cells was immunoprecipitated using anti-C/EBP␤ and used in an acetylation assay without or with purified p300 or P/CAF.Acetylated C/EBP␤ (arrowhead) and autoacetylated p300 and P/CAF (dashes on left) are shown in the autoradiograph (upper panel).Immunoblot (IB) probed with anti-C/EBP␤ indicates expression of C/EBP␤ (lower panel).Similar results were obtained in two other experiments.

FIGURE 2 .
FIGURE 2. C/EBP␤ is acetylated in vivo.A, 293T cells expressing CMV-C/EBP␤ were incubated with [ 3 H]acetate.Cell lysates immunoprecipitated (IP) using anti-C/EBP␤ (␤) or rabbit IgG (IgG) are shown in an autoradiograph.Arrowhead indicates acetylated C/EBP␤.B, WT HA-C/EBP␤ was expressed in 293T cells in the absence (Ϫ) or presence (ϩ) of p300.Cell lysates were subjected to immunoprecipitation using anti-HA and used for immunoblotting (IB) with either anti-Ac-K (upper panel) or anti-HA (lower panel).Similar results were obtained in two other experiments.C, endogenous C/EBP␤ was immunoprecipitated from 3T3-F442A preadipocytes with anti-C/EBP␤.Samples incubated without antibody (lane 1) or with IgG (lane 2) served as controls.Samples were used for immunoblotting with anti-Ac-K (upper panel, arrowhead) or anti-C/EBP␤ (lower panel).Similar results were obtained in three experiments.

FIGURE 6 .
FIGURE 6.Mutations of Lys-39 of C/EBP␤ impair transcriptional activation of a consensus C/EBP site.A, plasmids for WT HA-C/EBP␤, K39R, R42A, K98R C/EBP␤, or pcDNA3.1 vector (C) were coexpressed with 5XC/EBP-luc in CHO-GHR cells.Cells were lysed 48 h later, and luciferase activity was measured.Transcriptional activation (relative luciferase units (RLU)) is expressed relative to C ϭ 1, and bars represent mean Ϯ S.E. in this and subsequent figures.S.E. of control is too small to be visible (n ϭ 3 independent experiments).A representative immunoblot (IB) of lysates used for luciferase assay, probed with anti-HA, shows relative expression of C/EBP␤ (lower panel in this and subsequent figures).Activation in the presence of K39R is significantly less (p Ͻ 0.05, see asterisk) than with WT C/EBP␤.Transcriptional activation by WT C/EBP␤ is significantly greater (p Ͻ 0.05) than control, and activation by R42A and K98R C/EBP␤ is not significantly different from WT C/EBP␤.B, plasmid for 5XC/EBP-luc was coexpressed with plasmids for C/EBP␤ without (WT) or with the indicated mutations of Lys-39, and luciferase activity was measured (n ϭ 3 experiments).Activation of 5XC/EBP-luc is significantly lower (p Ͻ 0.001, see asterisks) with K39R and K39A than WT C/EBP␤.Transcriptional activation of 5XC/EBP-luc by WT C/EBP␤ is significantly greater (p Ͻ 0.001) than control, and activation by K39Q C/EBP␤ is not significantly different from WT C/EBP␤.RLU, relative luciferase units.

FIGURE 7 .
FIGURE 7. Mutations of Lys-39 of C/EBP␤ impair transcriptional activation of the C/EBP␣ promoter.A, C/EBP␣-luc was coexpressed with C/EBP␤ plasmids as in Fig. 6A, and luciferase activity was measured (n ϭ 3 experiments).Activation of the C/EBP␣ promoter by K39R C/EBP␤ is significantly lower (p Ͻ 0.01, see asterisk) than by WT C/EBP␤.Transcriptional activation by WT C/EBP␤ is significantly greater (p Ͻ 0.01) than control, and activation by R42A and K98R C/EBP␤ is not significantly different from WT C/EBP␤.B, plasmid for C/EBP␣-luc was coexpressed with plasmids for C/EBP␤ without (WT) or with mutations in Lys-39, and luciferase activity was measured (n ϭ 3 experiments).Activation of the C/EBP␣ promoter is significantly lower (p Ͻ 0.01, asterisks) with K39R and K39A C/EBP␤ than with WT C/EBP␤.Transcriptional activation of C/EBP␣-luc by WT C/EBP␤ is significantly greater (p Ͻ 0.001) than control (C).Activation by K39Q C/EBP␤ is not significantly different from WT C/EBP␤.IB, immunoblot; RLU, relative luciferase units.

FIGURE 8 .
FIGURE 8.The integrity of Lys-39 of C/EBP␤ contributes to its ability to activate the c-fos promoter.A, plasmid c-fos-luc was coexpressed with C/EBP␤ plasmids with the indicated mutations at Lys-39, Arg-42, or Lys-98, as in Fig. 6A, and luciferase activity was measured (n ϭ 3 experiments).Activation of the c-fos promoter by K39R C/EBP␤ is significantly lower (p Ͻ 0.001, asterisk) than by WT C/EBP␤.Transcriptional activation by WT C/EBP␤ is significantly greater (p Ͻ 0.001) than control, and activation by R42A or K98R C/EBP␤ is not significantly different from WT C/EBP␤.B, plasmid for c-fos-luc was coexpressed with plasmids for C/EBP␤ without (WT) or with the indicated mutations at Lys-39, and luciferase activity was measured (n ϭ 3 experiments).Activation of c-fos is significantly lower (p Ͻ 0.001, asterisks) with K39R and K39A C/EBP␤ than WT C/EBP␤.Transcriptional activation of the c-fos promoter by WT C/EBP␤ is significantly greater (p Ͻ 0.001) than control (C), and activation by K39Q C/EBP␤ is not significantly different from WT C/EBP␤.IB, immunoblot; RLU, relative luciferase units.

FIGURE 9 .
FIGURE 9. Lys-39 of C/EBP␤ contributes to GH-mediated activation of c-fos.A, WT HA-C/EBP␤ and GHR were coexpressed in 293T cells, and cells were treated with GH (250 ng/ml) for 15 min.C/EBP␤ was immunoprecipitated with anti-HA and used for immunoblotting (IB) with anti-Ac-K (upper panel) or anti-HA (lower panel).Arrowhead indicates acetylated C/EBP␤.Acetylation of C/EBP␤, calculated as Ac-K/C/EBP␤ (see "Materials and Methods"), increased 2.2 times Ϯ 0.37 control (C) (p Ͻ 0.04) in response to GH in three experiments.B, plasmids for WT HA-C/EBP␤ or K39R C/EBP␤, and for GHR were coexpressed with c-fos-luc in CHO-GHR cells.48 h later, cells were treated with GH (500 ng/ml) for 4 h and lysed, and luciferase activity was measured.Bars (Ϯ S.E.) represent luciferase activity in cells treated with vehicle control (C, open bars) or with GH (hatched bars) (n ϭ 4 experiments).Activation of c-fos in response to GH was significantly greater than control with WT C/EBP␤ (p Ͻ 0.001, asterisk) but not with K39R C/EBP␤.In the absence of GH, basal activation of c-fos by K39R C/EBP␤ was significantly decreased (p Ͻ 0.05) compared with WT C/EBP␤.RLU, relative luciferase units.

FIGURE 10 .
FIGURE 10.Different patterns of lysine mutations impair transcription on different C/EBP␤-responsive promoters.A, c-fos promoter.The plasmid for c-fos-luc was coexpressed with plasmids for C/EBP␤ without (WT) or with mutations K39R, K117R, or K215R/K216R alone or in combination (TM), and luciferase activity was measured (n ϭ 3 experiments).Cells were lysed 48 h later, and luciferase activity was measured (n ϭ 3 independent experiments).Activation of c-fos by K39R and TM C/EBP␤ is significantly lower (p Ͻ 0.001, see asterisks) than WT C/EBP␤.Transcriptional activation of the c-fos promoter by WT C/EBP␤ is significantly greater (p Ͻ 0.001) than control, and activation by K117R or K215R/K216R C/EBP␤ is not significantly different from WT C/EBP␤.B, consensus C/EBP site.The plasmid for 5XC/EBP-luc was coexpressed with C/EBP␤ plasmids as in A, and luciferase activity was measured (n ϭ 5).Transcriptional activation by WT C/EBP␤ is significantly (p Ͻ 0.001) greater than control.Activation by K39R (p Ͻ 0.0001), K117R (p Ͻ 0.01), and TM (p Ͻ 0.001) is significantly less (asterisks) than WT C/EBP␤.C, C/EBP␣ promoter.The plasmid for C/EBP␣-luc was coexpressed with C/EBP␤ plasmids as in A, and luciferase activity was measured (n ϭ 3).Transcriptional activation by WT C/EBP␤ is significantly (p Ͻ 0.001) greater than control.Activation of the C/EBP␣ promoter is significantly lower (asterisks) than WT C/EBP␤ for K39R (p Ͻ 0.001), K117R (p Ͻ 0.01), K215R/K216R (p Ͻ 0.05), and TM (p Ͻ 0.001).RLU, relative luciferase units.