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J. Biol. Chem., Vol. 279, Issue 40, 41477-41486, October 1, 2004
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¶
From the
Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria 3050 Australia and the
Division of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
Received for publication, May 10, 2004 , and in revised form, July 21, 2004.
| ABSTRACT |
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-globin locus. We have studied the role of acetylation in the regulation of the transcription factor NF-E4, a component of a protein complex that facilitates the preferential expression of the human
-globin genes in fetal erythroid cells. We have shown that NF-E4 interacts directly with, and serves as a substrate for, the acetyltransferase co-activator PCAF. Acetylation of NF-E4 is restricted to a single residue (Lys43) in the amino-terminal domain of the protein and results in two important functional consequences. Acetylation of NF-E4 prolongs the protein half-life by preventing ubiquitin-mediated degradation. This stabilization is PCAF-dependent, since enforced expression in fetal/erythroid cells of a mutant form of PCAF lacking the histone acetyltransferase domain (PCAF
HAT) decreases NF-E4 stability. Acetylation of Lys43 also reduces the interaction between NF-E4 and HDAC1, potentially maximizing the activating ability of the factor at the
-promoter. These results provide further demonstration that co-activators, such as PCAF, can influence individual transcription factor properties at multiple levels to alter their effects on gene expression. | INTRODUCTION |
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One such locus is the
-like globin cluster in which a linear array of five genes is expressed in a highly regulated tissue-specific and developmentally specific manner (20). To date, very few transcription factors that specifically influence the temporal profile of globin gene expression have been identified. One of these, EKLF is a red cell-specific activator that is critical for switching on high level adult
-globin expression (2123). EKLF binds to the CACCC element in the proximal
-promoter, leading to chromatin remodeling and transcriptional activation (2428). The transcriptional activity of EKLF is regulated by site-specific acetylation by p300 and CBP that results in an enhanced activation potential of the protein and increased binding to the SWI/SNF chromatin remodeling complex (12, 29).
NF-E4 is another globin-specific transcription factor (30). This protein forms the stage selector protein complex (SSP) with the ubiquitous transcription factor CP2 (31). The SSP facilitates the interaction of the
-globin genes with the powerful enhancer elements contained in the locus control region in fetal erythroid cells through binding to the stage selector element (SSE) in the proximal
-promoter (32). Enforced expression of NF-E4 in the human fetal/erythroid K562 cell line and human cord blood progenitors leads to increased fetal globin gene expression (30). In cord blood progenitors, where active competition between fetal and adult globin genes occurs, enforced expression of NF-E4 also leads to a reduction in
-globin gene expression (30).
In this study, we demonstrate that NF-E4 is a direct target of the co-activator PCAF. The resultant acetylation of Lys-43 in NF-E4 results in both an increase in the stability of the protein due to diminished targeting by ubiquitination and an alteration in protein-protein interactions that favor transcriptional activation. Thus, a single amino acid modification results in complementary functional changes that enhance the ability of NF-E4 to positively regulate fetal globin gene expression.
| EXPERIMENTAL PROCEDURES |
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Reagents and Antibodies[14C]Acetyl-CoA (59 mCi/mmol) and sodium [3H]acetate (5 Ci/mmol) were purchased from Amersham Biosciences and PerkinElmer Life Sciences, respectively. Tran35S-label was obtained from ICN (Costa Mesa, CA). Trichostatin A (TSA), sodium butyrate, acetyl-CoA, ubiquitin, MG132, and cycloheximide were from Sigma. Peroxidase-conjugated goat anti-mouse and monoclonal antirabbit immunoglobulin G, monoclonal anti-FLAG (M2), and anti-ubiquitin antibodies were from Sigma. Monoclonal anti-HA (12CA5) antibody was from Roche Applied Science. Monoclonal anti-acetyl lysine and anti-HDAC1 antibodies were from Upstate (Waltham, MA). Anti-PCAF and anti-tubulin antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-NF-E4-specific antibody was generated by immunizing rabbits with a C-terminal synthetic peptide with the amino acid sequence LKTDSALEQTPQQLPSLHLS coupled to keyhole limpet hemocyanin.
Plasmids, Transfections, Luciferase, and
-Galactosidase Assays The expression vectors GST-NF-E4 and GST-CP2 in pGEX were described previously (30, 31). For the GST-NF-E4 truncation constructs, PCR fragments corresponding to the shortened coding sequences were generated with BamHI and XhoI ends and were cloned into pGEX vectors. The plasmids pCX-PCAF, pCX-PCAF
HAT, MSCV-IRES-PCAF-GFP, and MSCV-IRES-PCAF
HAT-GFP were kindly provided by Dr. Steven Brandt (13). Plasmids expressing GST-PCAF-(352832) and GST-CBP-(10981877) were kindly provided by Dr. Tony Kouzarides (14). The plasmid pCDNA3.1-FLAG-ubiquitin was kindly provided by Dr. Ivan Dikic. The in vitro transcription/translation plasmid for PCAF-(1832) was cloned by inserting an EcoRI/XhoI fragment from MSCV-IRES-PCAF-GFP into vector pSP72. Mutation of GST-NF-E4 to GST-K43R was accomplished with the QuikChange mutagenesis system (Stratagene, La Jolla, CA). Retroviral vectors expressing HA-NF-E4 and HA-K43R were constructed by cloning fragments obtained by PCR amplification of coding sequences of pGEX-NF-E4 and pGEX-K43R into MSCV-IRES-GFP. The integrity of all constructs that generated fusion proteins was confirmed by DNA sequencing.
Stable K562 cell lines overexpressing HA-NF-E4, HA-K43R, PCAF, and PCAF
HAT were generated according to the protocols described previously, except that only the top 10% of GFP-positive cells were collected (30). For the co-immunoprecipitation experiment, 293T cells were transiently transfected with various plasmids by a calcium phosphate method (Invitrogen). For the reporter gene assays, native K562 cells or stable K562 cell lines overexpressing HA-NF-E4 and HA-K43R were co-transfected with HS253
-Luciferase, containing hypersensitivity site 2 of the
-globin locus control region linked to the 53
-promoter relative to the CAP site and the firefly luciferase reporter gene (32), and pCH110, containing the
-galactosidase gene driven by SV40 promoter as an internal transfection efficiency control by electroporation. For the TSA induction experiments, the transfected cells were divided into two aliquots, one of which was cultured in various concentrations of TSA for 20 h and the other in standard medium alone. For all other transfection experiments, cells were harvested after 36 h and lysed, and luciferase activity was determined on a Monolight 2001 luminometer using the luciferase assay kit (Promega).
-Galactosidase activity was measured in a spectrophotometer using the enzyme assay system (Promega), and the relative luciferase activities were calculated by dividing the luciferase activity by the
-galactosidase activity to correct for transfection efficiency. Three independent transfections were performed in duplicate.
Metabolic LabelingFor 35S labeling, K562 cells were grown in RPMI supplemented with 5% dialyzed fetal bovine serum containing Tran35S-label (20 µCi/ml; a mixture of cysteine and methionine) for 4 h. For 3H labeling, K562 cells were cultured in RPMI containing sodium [3H]acetate (1 mCi/ml) and 2 µM TSA for 2 h. Extracts were prepared and boiled after adding 2x sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2%
-mercaptoethanol) to disrupt interactions between proteins before being subjected to immunoprecipitation with specific anti-NF-E4 antibody. The immunoprecipitated samples on beads were extensively washed with stringent washing buffer (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Triton X-100, 0.1% SDS), resolved on SDS-PAGE, and analyzed by autoradiography.
Immunoprecipitation and ImmunoblottingCells were lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Nonidet P-40, 10 mM sodium butyrate) containing a protease inhibitor mixture (Roche Applied Science) and cleared by centrifugation. Immunoprecipitations were carried out by adding appropriate antibodies plus protein G-Sepharose beads, followed by incubation at 4 °C. The immunoprecipitates were washed extensively, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were incubated with various specific antibodies and then washed extensively prior to incubation with peroxidase-conjugated anti-rabbit or antimouse immunoglobulin G. After further extensive washes, the blots were visualized by using ECL reagents (Amersham Biosciences). All immunoprecipitations were performed in duplicate.
Recombinant Protein Expression and GST Pull-down AssayGST-fusion proteins were produced in BL21 Escherichia coli as described previously (30). 35S-Labeled NF-E4 and PCAF synthesized using the T7 TNT kit (Promega) and Tran35S-label (ICN) were incubated with GST fusion proteins prebound to glutathione beads at 4 °C overnight. The samples were washed extensively and subjected to SDS-PAGE. The gels were dried and analyzed by autoradiography.
Determination of Protein Half-lifeK562 cells stably expressing HA-NF-E4, HA-K43R, PCAF, and PCAF
HAT were treated with cycloheximide (100 µM) to stop protein synthesis and incubated for the indicated times. Cells were lysed, and lysates were subjected to SDS-PAGE and Western blotting probed with the indicated specific antibodies. The protein signals were scanned and quantitated by PhosphorImager analysis software (Amersham Biosciences).
In Vitro Protein Acetylation Assay23 µg of the indicated GST fusion protein or SCL and 200 ng of acetyltransferase protein were incubated in a reaction containing 50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium butyrate, and 30 µM acetyl-coenzyme A or 1 µl of [14C]acetyl-CoA for 1 h at 30 °C. The reaction mixture was subjected to SDS-PAGE and analyzed by autoradiography of dried gels or by Western blotting with anti-acetyl lysine antibody.
In Vitro Ubiquitination AssayRecombinant protein was used as the substrate in a ubiquitin reaction containing 20 mM Hepes, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 2 mM ATP, 5 µg of ubiquitin, 20 µM MG132, and 5 µl of crude rabbit reticulocyte (Promega) for 1 h at 30 °C. In some cases, GST fusion proteins were acetylated prior to the ubiquitination reaction by PCAF in the presence or absence of unlabeled acetyl-CoA. The samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes followed by determination of ubiquitination by Western blotting analysis.
In Vivo Ubiquitination Assay293T cells were transfected with pCDNA3.1-FLAG-ubiquitin, and cell lysate was prepared 36 h later. For immunoprecipitation, 1 mg of protein was incubated with
-HA antibody at 4 °C overnight before protein G beads were added for 2 h. The beads were washed twice with NaCl (1 M) in Tris-buffered saline supplemented with Nonidet P-40 (1%),
-mercaptoethanol (0.05%), and EDTA (1 mM). Proteins were loaded onto SDS-PAGE followed by immunoblot analysis with the indicated antibodies and enhanced chemiluminescence detection.
Electrophoretic Mobility Shift AssayThe electrophoretic mobility shift assay was performed as previously described (31) using recombinant proteins. In some cases, recombinant NF-E4 was acetylated in vitro prior to binding DNA. Specific antibodies against NF-E4 or CP2 or specific or nonspecific cold oligonucleotide competitors in molar excess were added to the reaction mixture for 30 min on ice before the addition of the labeled SSE oligonucleotides.
| RESULTS |
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HAT) tagged with a FLAG epitope, or vector alone (13, 14). Cellular extracts from the transfected cells were immunoprecipitated with preimmune sera (lane 1) or anti-HA antibody (lanes 24) and immunoblotted with either anti-HA antibody (
-HA) or antibody specific for acetylated lysine (
-AcK) (Fig. 1C). An increase in the level of acetylated NF-E4 was observed in the presence of PCAF compared with vector alone (compare lanes 2 and 3). The level of acetylated NF-E4 was decreased in cells transfected with PCAF
HAT, consistent with its dominant negative role (lane 4). The expression of HA-NF-E4 and FLAG-PCAF/PCAF
HAT were similar in the transfected lines (lower panels). These findings indicate that NF-E4 serves as a substrate for the acetyltransferase, PCAF.
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-AcK) (Fig. 3A, top right panel). Full-length NF-E4 (amino acids 1179) (top panel, lane 5) and NF-E4 (amino acids 148) (lane 3) were both acetylated in vitro. In contrast, NF-E4 lacking the first 48 amino acids (lane 4), NF-E4 (amino acids 125) (lane 2), and GST alone (lane 1) remained unacetylated, despite significant levels of GST fusion protein expression (Fig. 3A, bottom right panel). This acetylation pattern was duplicated when acetylated lysine was detected by incorporation of [14C]acetyl-CoA in the in vitro assay (data not shown). These results localized the site of acetylation of NF-E4 to the residues between 26 and 48 and indicated that the acetylation status of the NF-E4 protein was accurately reflected by Western analysis with the antibody specific for acetylated lysine.
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-HA antisera and immunoblotted with either
-AcK or
-HA. As shown in Fig. 3C, despite abundant expression of the mutant and wild type NF-E4 proteins as demonstrated with anti-HA antibody (
-HA), only a faint band was detected in the setting of the K43R mutation (
-AcK). In contrast, the wild-type protein displayed prominent acetylation consistent with Lys-43 acting as the sole site of in vitro acetylation and the major site of in vivo PCAF-dependent acetylation.
NF-E4 DNA Binding Is Not Affected by Acetylation in Vitro The DNA-binding properties of many transcription factors are altered by acetylation (19). To examine the effects of this modification on NF-E4 DNA binding, we performed an electrophoretic mobility shift assay with recombinant CP2 and NF-E4 and an SSE probe from the human
-promoter (Fig. 4). Under the conditions used, recombinant CP2 failed to bind the SSE probe in isolation (lane 1). Recombinant NF-E4 was preincubated with PCAF in the presence and absence of acetyl-CoA using the conditions defined in Fig. 1A. Both forms of NF-E4 failed to bind the SSE in isolation (data not shown). When increasing amounts of unacetylated NF-E4 (lanes 24) or acetylated NF-E4 (lanes 57) were combined with rCP2 and probe, a robust fast migrating complex was observed. The formation of this complex was unaffected by the acetylation status of the rNF-E4 but was altered by specific antibodies to both CP2 and NF-E4 (lanes 8 and 9). A weaker, slower migrating complex that was similarly unaffected by the acetylation status of NF-E4 was also noted. We attributed this to a higher order NF-E4·CP2 complex (30), since it was dependent on the amount of NF-E4 added and was supershifted with both antibodies. To further confirm the specificity of the protein-DNA complexes, we examined the effects of specific and nonspecific unlabeled oligonucleotides. As shown in lanes 1113, the presence of a 10100-fold molar excess of unlabeled SSE probe resulted in diminution/loss of the SSP complexes. In contrast, a 100-fold excess of an unrelated unlabeled oligonucleotide did not alter the SSP·SSE complex (lane 14). These findings indicate that acetylation of NF-E4 does not alter its DNA binding properties.
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HAT or the empty vector as a control (Fig. 5A). Cellular extracts were prepared at various time points after cycloheximide treatment and subjected to SDS-PAGE and immunoblotted with antibodies to NF-E4 or tubulin as a control. The expression of PCAF and PCAF
HAT was shown to be comparable in the two cell lines by immunoblotting extracts with
-PCAF (top right panel). As shown in data from a representative experiment (left panel) and graphically from all experiments (bottom right panel), an increase in endogenous NF-E4 stability was observed in the cells transfected with wild-type PCAF compared with vector alone. In contrast, the half-life of NF-E4 in K562 cells transfected with PCAF
HAT was significantly reduced, suggesting that the stability of NF-E4 is dependent on PCAF-mediated acetylation of Lys-43.
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The enhanced stability of both acetylated NF-E4 and the acetylation-deficient K43R mutant was reminiscent of the findings reported with the transcription factors Smad7 and the SREBP family (33, 34). In these proteins, the sites of acetylation that enhance protein stabilization are also the lysine residues targeted by ubiquitination. Consequently, either acetylation or mutation of these lysine residues increases protein stability. To determine whether acetylation of NF-E4 altered the susceptibility of the protein to ubiquitination, we utilized recombinant NF-E4 in both acetylated and nonacetylated forms as substrates in reconstituted in vitro ubiquitination assays (Fig. 5C, top panel). Nonacetylated NF-E4 was ubiquitinated in the presence of rabbit reticulocyte lysate (RRL) (lane 3) and migrated as a high molecular weight polyubiquitinated species (NF-E4-Ubn). Acetylation of NF-E4 markedly reduced its ubiquitination (compare lanes 2 and 3). No ubiquitinated protein was observed in the absence of the rabbit reticulocyte lysate (lane 1). The amount of HA-NF-E4 used in each experiment was equivalent as demonstrated by immunoblotting with anti-HA antisera (lower panel), and acetylation was confirmed in the presence of PCAF and Ac-CoA by immunoblotting with anti-AcK antisera (middle panel). This finding suggests that acetylation of Lys-43 blocks the ubiquitination of NF-E4. To examine the effect of mutation of Lys-43 on the ubiquitination of NF-E4 in a cellular context, we utilized the 293T cells stably transfected with retroviral vectors containing the HA-tagged wild-type (HA-NF-E4) or K43R mutant NF-E4 (HA-K43R) or the empty vector control (Fig. 5D). The levels of expression of the tagged NF-E4 forms were similar in both lines, with a small increase in the K43R protein level reflecting its increased stability (shown in
-HA, third panel). These lines were then transfected with a mammalian expression vector containing FLAG-tagged ubiquitin (+Flag-Ub, second panel) or the empty vector (w/o Flag-Ub, top panel). The levels of expression of the FLAG-Ub were equivalent in all three lines (bottom panel). Cellular extracts were prepared and immunoprecipitated with anti-HA antibody prior to immunoblotting with either antiubiquitin or anti-FLAG antibody. In the absence of FLAG-Ub expression (w/o Flag-Ub, top panel), the
-HA-immunoprecipitate from all three lines contained no ubiquitinated NF-E4 species, with only a nonspecific band (asterisk) visualized in all three lanes. We attributed this to low levels of endogenous ubiquitin expression in these lines. When FLAG-Ub was expressed (+Flag-Ub, second panel), high molecular weight polyubiquitinated NF-E4 species (NF-E4-Ubn) were seen in the immunoprecipitates from the wild type NF-E4-expressing cells. These polyubiquitinated proteins were not seen in cells containing the empty vector, and more significantly, they were not seen in the K43R-expressing cells. These results suggest that lysine 43 is also targeted by ubiquitination in vivo.
Effect of K43R Mutation on NF-E4 Transcriptional ActivityWe predicted that functional effects of stabilization of NF-E4 would be reflected by an increase in the activity of the
-promoter. To test this, we utilized the stable K562 cells in which expression of wild type or K43R mutant NF-E4 had been enforced and a vector-transduced line as a control. These lines were then transiently transfected with a plasmid containing the
-promoter truncated to 53 relative to the CAP site linked to a luciferase reporter gene (32). This construct contains the SSE and the TATA box but none of the other known upstream regulatory elements and is therefore specifically responsive to NF-E4. A
-galactosidase-expressing plasmid was also used to monitor transfection efficiency. As shown in Fig. 6A, a greater than 2-fold increase in
-promoter activity was observed in cells expressing the wild-type NF-E4. Surprisingly, the increase in
-promoter activity observed in the K43R-expressing cells was significantly less than in the wild-type cells despite the higher level of expression of the mutant protein (Fig. 6B). This finding suggested that stabilization of the NF-E4 protein was insufficient in isolation to enhance the protein's activation potential and that acetylation of the protein must play an additional role in NF-E4-mediated
-gene activation.
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-promoter activity in a transient transfection assay. K562 cells were co-transfected with the
-promoter-luciferase reporter and the
-galactosidase-expressing plasmid and divided into three flasks, two of which were induced with TSA in varying concentrations. As shown in Fig. 6C, a dose-dependent increase in
-promoter activity was seen with TSA induction. This finding was consistent with the enhancement of endogenous
-gene expression seen with this compound (35, 36).
Diminished Binding of Acetylated NF-E4 to HDAC1Altered protein-protein interactions are a well established consequence of transcription factor acetylation (19). The affinity of EKLF for the SWI/SNF chromatin-remodeling complex is enhanced by this modification (29), prompting us to consider whether acetylation of NF-E4 could influence its protein interactions to enhance its activation potential. We had previously demonstrated that NF-E4 and the histone deacetylase HDAC1 were co-immunoprecipitated from extract derived from K562 cells using anti-NF-E4 antibody (Fig. 7A). HDAC1 is a member of a family of deacetylases involved in transcriptional repression and gene silencing (reviewed in Ref. 37). To determine whether acetylation of NF-E4 altered its affinity for HDAC1, we utilized recombinant GST-HA-NF-E4 in both acetylated and nonacetylated forms and a GST alone control in pull-down experiments with K562 cell extract (Fig. 7B). Acetylation of the recombinant protein was confirmed by immunoblotting with anti-AcK antibody (bottom panel). Analysis of the eluate from the three GST matrices with anti-HDAC1 antibody (
-HDAC1) demonstrated that nonacetylated NF-E4 bound significantly greater amounts of HDAC1 derived from K562 extract than acetylated NF-E4. In contrast, CP2 from K562 cell extract was retained equally on both acetylated and nonacetylated NF-E4 as detected by CP2 antibody (
-CP2). GST alone bound neither protein. These findings suggest that acetylation of NF-E4 plays a dual role in enhancing the activation potential of the factor via increased protein stability and diminished association with the transcriptional repressor, HDAC1.
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| DISCUSSION |
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-globin locus through activation of the
-genes and competitive silencing of
-gene expression (30). The mechanisms underlying these activities are gradually emerging, and appear to hinge on both critical protein/protein interactions and post-translational modification of the factor. In the fetal erythroid environment, NF-E4 is a component of a multiprotein complex that contains the transcriptional activator CP2 (30, 31). We now provide evidence that the acetyltransferase PCAF also complexes with NF-E4 in this environment and covalently modifies the protein. This modification has a number of sequelae, including increased stability of the protein through resistance to ubiquitination and alteration of protein partners to favor interactions that promote transcriptional activation.
The PCAF-interacting domain of NF-E4 is contained within the amino-terminal region of the NF-E4 protein. Although the full-length NF-E4 protein contains a number of lysine residues, it appears that in the context of PCAF, only the single residue at position 43 is acetylated. We found no evidence that CBP directly modifies the NF-E4 protein. In this regard, NF-E4 is unlike several of the other erythroid transcription factors that bind to the globin locus, including GATA-1, EKLF, and p45 NF-E2, all of which are acetylated by CBP (1012, 38). Interestingly, these factors have all been directly implicated in recruitment of acetyltransferases to the adult
-globin promoter (29, 39, 40). In contrast, the fetal globin regulatory factors, NF-E4 and FKLF-2, are both acetylated and functionally modified by PCAF, raising the possibility that stage-specific recruitment of acetyltransferases could occur at the globin locus (41). This could provide a potential explanation for the paradox of the highly stage-specific effects of factors such as EKLF and NF-E4 that are observed despite expression of these factors at all stages of erythroid development.
The functional consequences of acetylation of several of the globin locus binding factors have been examined previously. Modification of the MafG component of NF-E2 augments the DNA binding activity of this complex (38). Enhanced DNA binding has also been reported for acetylated GATA-1 (10), although there is some conjecture about this conclusion (11). We observed no effect of acetylation on the DNA-binding properties of NF-E4, a finding that parallels the lack of effect of acetylation on EKLF binding to its cognate site (29). The effects of acetylation on EKLF involve enhanced interaction between the factor and the SWI/SNF chromatin-remodeling complex (29). This results in maximization of the activation potential of EKLF at the
-promoter. The acetylation of NF-E4 also appears to maximize its activation potential through increased levels of the protein and a diminished association with the transcriptional repressor HDAC1. HDAC1 is a member of a family of proteins involved in mammalian gene silencing through deacetylation of core histones (37). Several HDAC-dependent co-repressor complexes have been identified in mammalian cells including mSin3A (4247) and NuRD/Mi2 (48, 49). Although the nature and role of such complexes are yet to be established at the
-promoter, our identification of HDAC1 and more recently another NuRD component, SHARP (50)2 as protein partners of NF-E4 suggest the potential for an NF-E4-dependent repressor complex to assemble on the promoter. As such, it appears that NF-E4 may play a dual role in fetal globin gene regulation depending on the developmental context.
Modification of a number of transcription factors has been shown to influence their interaction with HDAC1. The phosphorylation status of NF-
B determines whether it associates with CBP/p300 or HDAC1 (51). The acetylation status of MyoD and YY1 also influences the ability of these factors to bind HDAC1 (52, 53). In both cases, acetylation increases the interaction with the deacetylase, in contrast to the reduced association of acetylated NF-E4 with HDAC1 that we observed. Another hematopoietic transcription factor, SCL, has also been shown to have an impaired interaction with the mSin3A repressor complex including HDAC1 when acetylated (13). This may provide a novel mechanism to expand the potential roles of a factor at a single complex locus.
The enhanced stability of NF-E4 we observed in the context of acetylation has been reported for a number of transcription factors, including E2F1, members of the SREBP family, and Smad7 (14, 33, 34). In the latter two examples, it was demonstrated that specific lysine residues were targeted by ubiquitination and that acetylation of these residues prevented the subsequent ubiquitination of the protein. Our data indicate that a similar mechanism governs NF-E4 stability. The potential link between acetylation status of proteins and their ubiquitination has been strengthened by the identification of ubiquitin pathway components in the HDAC6 complex (54). NF-E4 degradation may be dependent on its deacetylation and ubiquitination, and it is tempting to speculate that this may be mediated by NF-E4 serving as a substrate for HDAC1. Our future studies will explore this possibility.
| FOOTNOTES |
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¶ To whom correspondence should be addressed: Rotary Bone Marrow Research Laboratory, c/o Royal Melbourne Hospital Post Office, Grattan Street, Parkville, Victoria 3050, Australia. Tel.: 61-3-93428641; Fax: 61-3-93428634; E-mail: jane{at}wehi.edu.au.
1 The abbreviations used are: HAT, histone acetyltransferase; EKLF, erythroid Kruppel-like factor; SSP, stage selector protein complex; SSE, stage selector element; GFP, green fluorescent protein; GST, glutathione S-transferase; TSA, trichostatin A; HA, hemagglutinin; Ub, ubiquitin; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein. ![]()
2 S. M. Jane and J. M. Cunningham, unpublished observations. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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