Stimulation of NF-E2 DNA Binding by CREB-binding Protein (CBP)-mediated Acetylation*

Hsiao-Ling HungDagger , Alexander Y. KimDagger , Wei HongDagger , Carrie RakowskiDagger , and Gerd A. BlobelDagger §

From the Dagger  Division of Hematology, Children's Hospital of Philadelphia and the § University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, August 28, 2000, and in revised form, January 11, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The basic-zipper (bZip)1 transcription factor NF-E2 plays a critical role in erythroid and megakaryocytic gene expression (for review see Ref. 1). NF-E2 binds to an extended AP-1-like element, TGCTGA(G/C)TCA, which is found in the locus control regions (LCRs) of the alpha - and beta -globin genes and in the promoters of several heme biosynthetic enzyme genes (for review see Refs. 2 and 3)). NF-E2 binding sites in the DNase I hypersensitive site 2 (HS2) of the beta -globin LCR are essential for its enhancer activity (4-6).

NF-E2 is a heterodimer consisting of a hematopoietic-specific subunit p45, which is a member of the cap and collar (CNC) family, and a more widely expressed small subunit, which is a member of the small Maf protein family (MafG, MafK, and MafF) (for review see Refs. 2 and 3)). MafG and MafK are the predominant small Maf molecules in erythroid cells and megakaryocytes (7). p45 and a small Maf protein dimerize through their leucine zipper domains to generate a composite DNA binding domain that consists of the basic regions of both molecules. Other members of the CNC family, including Nrf1 (8), Nrf2 (9), Nrf3 (10), Bach1, and Bach2 (11) can also dimerize with small Maf proteins. Despite the high levels of p45 expression in erythroid cells, mice that are null for p45 displayed a surprisingly mild defect in globin gene expression, suggesting that other members of the CNC protein family can substitute for p45 function in vivo (12).

The N terminus of p45 contains an activation domain that is important for the biological activity of p45 (13, 14). Several molecules interact with this domain and are candidate mediators of p45 activity. These include TAFII130 (a component of the TFIID complex) (15), cAMP-response element-binding protein (CREB)-binding protein (CBP) (16), and several ubiquitin ligases (17, 18). The small Maf proteins lack a typical activation domain and are believed to activate transcription as heterodimers with members of the CNC family of proteins. Small Maf proteins can also form homodimers and repress transcription (19).

CBP and its close relative p300 serve as coactivators for a large and diverse set of nuclear factors (20, 21). CBP and p300 possess intrinsic histone acetyltransferase (AT) activity (21-23). Histone acetylation is associated with a relaxed chromatin configuration, suggesting that coactivators act in part through modifying chromatin structure. Consistent with this idea, at the chicken beta -globin gene locus, the area of general DNaseI sensitivity coincides well with the region of elevated histone acetylation (24). Interestingly, hyper-acetylation of histones H3 and H4 was observed at the human beta -globin LCR and at the transcribed beta -globin gene when compared with the acetylation status of the inactive beta -like globin genes (25, 26). Together, these findings suggest that erythroid transcription factors might recruit histone-modifying enzymes such as CBP/p300 to the LCR and globin gene locus, thereby altering chromatin structure (27). This idea is supported by our observation that E1A-mediated inactivation of CBP/p300 in erythroid cells leads to a block in cell differentiation and globin gene induction (28). CBP binds to several hematopoietic-restricted transcription factors involved in globin gene expression and enhances their activity, including GATA-1 (28) and the erythroid Krüppel-like factor EKLF (29).

A new layer of complexity in the function of acetyltransferases emerged with the discovery that CBP/p300 also acetylates a variety of transcription factors. Acetylation can alter transcriptional activity through several mechanisms. For example, acetylation of the tumor suppressor protein p53 strongly increases its affinity for DNA (30-32). Both GATA-1 and EKLF are also acetylated by CBP (29, 33, 34). Mutations in the acetylation sites in GATA-1 compromise its function in erythroid cells, suggesting that GATA-1 acetylation is biologically relevant (34).

Recent studies implicated CBP/p300 in the regulation of NF-E2 activity by showing that E1A, which inhibits CBP/p300 function, reduced the enhancer activity of HS2 and that NF-E2 was an important target of E1A-mediated inhibition (35). In addition, glutathione S-transferase (GST) pull-down experiments showed that p45 binds CBP in vitro (16).

Here we report that both subunits of NF-E2 interact with CBP in vitro. In addition, NF-E2 can recruit CBP to a DNA template containing NF-E2 binding sites. Immunoprecipitation experiments demonstrate in vivo association between MafG and CBP in erythroid cells. CBP acetylates MafG predominantly in the basic region, thereby stimulating DNA binding of NF-E2. Mutations at the major acetylation sites reduce DNA binding and transcriptional activation by NF-E2. Thus, recruitment of CBP by NF-E2 might serve two functions, regulation of chromatin structure and transcription factor activity.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Full-length and truncated variants of GST murine p45 and GST murine MafG were generated by polymerase chain reaction and subcloned in the BamHI and the EcoRI sites of pGEX2TK (Amersham Pharmacia Biotech). GST-tethered-NF-E2 contains murine p45 and human MafG physically tethered by a flexible peptide linker as described (36). pXM-Tethered NF-E2 was described previously (36). Mutagenesis replacing the four lysine residues in the basic region of MafG (see Fig. 4) was performed using the QuikChangeTM method as instructed by the manufacturer (Stratagene), and the resulting construct was verified by sequencing.

GST pull-down experiments in Fig. 1 were performed as previously described (34) using 5 µg of GST protein and 10 µl of programmed reticulocyte lysates to map the interaction domains of NF-E2 and CBP. Experiments in Fig. 6A were modified to determine the effect of acetylation on p45-MafG heterodimerization. Two µg of GST-MafG were acetylated by 0.3 µg of GST-CBPH-AT in the presence of cold acetyl coenzyme A (0.6 mM) at 30 °C for 90 min and incubated with 35S-labeled p45 in the presence or absence of 1 µg of double-stranded oligonucleotide containing the NF-E2 binding site from the porphobilinogen deaminase (PBGD) promoter (5'-GATCCTGGGGAACCTGTGCTGAGTCACTGGAGG-3') (37). GST-MafG quickly associates with p45 in the presence of the NF-E2 binding site, and equilibrium was reached within 30 min.

Coimmunoprecipitation of CBP and MafG-- EF1alpha -neo-HA-MafG was transfected into murine erythroid leukemia cells (MEL) by DMRIE-C (Life Technologies, Inc.) and selected in the presence of G418. Western blots using anti-HA antibodies (12CA5, Roche Molecular Biochemicals) were used to identify HA-MafG-expressing clones. A line expressing high levels of HA-MafG was expanded and used in the experiments. Coimmunoprecipitation of CBP and HA-MafG was carried out essentially as described using anti-CBP (A-22, Santa Cruz Biotechnology) as the precipitating antibody (28).

Recruitment of CBP to HS2-- A HindIIII-XbaI fragment (374 base pairs) containing HS2 from the human beta -globin LCR (a gift from R. Hardison) (38) was biotinylated and coupled to M-280 streptavidin magnetic beads (Dynal) according to the manufacturer's instructions. The tandem NF-E2 sites from 8661 to 8677 (GenBankTM HUMHBB) was replaced with a SalI site in HS2Delta NF-E2 fragment (35). One pmol of HS2 coupled to 100 µg of Dynal beads was resuspended in 100 µl of 1× DNA-protein binding buffer (10 mM Tris (pH 7.5), 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5.5 mM dithiothreitol, 5% glycerol, 0.03% Igepal, 5 mg/ml bovine serum albumin, protease inhibitor mixtures) and incubated with 5 pmol of GST-p45 plus 5 pmol of GST-MafG at room temperature for 20 min. To remove unbound recombinant proteins, beads were washed twice with 1 ml of DNA-protein binding buffer and resuspended in 1 ml of DNA-protein binding buffer supplemented with 2.5 µg of poly(dI-dC). One hundred µl of MEL cell nuclear extracts corresponding to 107 cells were added to the beads and incubated for 1 h at 4 °C. Beads were washed twice in 1 ml of 0.5× DNA-protein binding buffer supplemented with 100 mM NaCl and analyzed on SDS-PAGE followed by Western blotting for CBP.

Acetyltransferase assays were performed as described (34). All substrates and enzymes were expressed as recombinant GST fusion proteins in Escherichia coli (DH5alpha ) except for the full-length GST-MafG and the full-length His-tagged CBP, which were produced in baculovirus. GST proteins were prepared as described (30). Reactions were carried out with 50 pmol of substrate and 5 pmol of enzyme in the presence of 0.06 µCi of 14C-labeled acetyl coenzyme A (55 mCi/mmol, PerkinElmer Life Sciences) at 30 °C for 90 min. Tethered NF-E2 protein used in gel shift experiments was acetylated in the presence of 0.6 mM unlabeled acetyl coenzyme A.

Anti-acetyl Lysine (AK) Immunoprecipitations-- Anti-acetyl lysine antibodies (New England Biolabs) were used to immunoprecipitate acetylated MafG from MEL cells expressing HA-MafG, transfected NIH3T3 cells, and transfected COS-7 cells. Preliminary experiments showed that these antibodies recognize recombinant MafG acetylated by CBP. For Fig. 5B, 3 µg each of EF1alpha -HA-MafG (hemagglutinin-tagged MafG), pCMV5CBP, and EF1alpha -E1A (28) were transfected into 50% confluent NIH3T3 cells in 10-cm dishes using LipofectAMINE (Life Technologies, Inc.). For Fig. 5C, 8 µg of pXM-tethered NF-E2 was transfected into 40% confluent COS-7 cells in 10-cm dishes using LipofectAMINE. COS cells express high levels of endogenous CBP and, therefore, were chosen to evaluate the in vivo acetylation of wild type and mutant NF-E2. High salt whole cell lysates were prepared 48 h after transfections and immunoprecipitated using anti-AK antibodies (0.5 µg/sample) as described (34). Rabbit IgG was used in the control precipitation. Immunoprecipitates were analyzed by Western blotting using anti-HA antibodies to detect HA-MafG and anti-p45 (a gift from E. Bresnick) to detect tethered NF-E2.

Gel mobility shift assays were performed as described previously (28, 34). The oligonucleotide probes used in gel shift assays contain the NF-E2 sites from the PBGD promoter (5'-GATCCTGGGGAACCTGTGCTGAGTCACTGGAGG-3') (37) and human HS2 (5'-GCAGTGCTGAGTCATGCTGAGTCATGCTG-3') (19). Tethered NF-E2 protein (1.2 µg) was acetylated by full-length His-tagged CBP (100 ng) before use in the gel shift reactions. Incubation of DNA complex proceeds for 15 min on ice before loading on a 5% nondenaturing acrylamide gel in 0.5× Tris borate EDTA.

Reporter Gene Assays-- A PBGD-GH reporter containing -310 to +78 of the PBGD promoter (39) linked to the human growth hormone gene was used to assay NF-E2 activity. 30% confluent NIH3T3 cells were transfected using LipofectAMINE with 0.2 µg of PBGD-GH and with increasing amounts (0.4, 0.8, and 1.6 µg) of pXM plasmids expressing wild type or acetylation-defective (4A) NF-E2. The amounts of transfected plasmid were kept constant by adding empty pXM. To account for variability in transfection efficiency, a control plasmid (0.1 µg) expressing the firefly luciferase gene SV40-GL3 (Promega) was included in the transfections. Growth hormone levels were determined using a radio-immunoassay (Nichols Diagnostic). Whole cell lysates were prepared to determinate luciferase activity (Promega) and to monitor NF-E2 expression by Western blot.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Both Subunits of NF-E2 Interact with CBP-- Previous work demonstrated a functional link between NF-E2 and CBP (35). In addition, it has been shown that p45 interacts with CBP in vitro (16). To examine whether MafG also associates with CBP, GST pull-down experiments were performed. Both full-length p45 and MafG proteins fused to GST interact with in vitro translated CBP (Fig. 1A). We next mapped the domains in p45 and MafG that mediate the association with CBP. The results show that the N terminus (aa 1-144) of p45 is necessary and sufficient for CBP binding (Fig. 1B). The domain in MafG that contacts CBP was mapped to the bZip domain (aa 51-162) (Fig. 1C). Deletion of the basic region (construct 77-162) resulted in complete loss of CBP binding. These results are summarized in Fig. 1D. To map the domain of CBP that mediates binding to MafG and p45, various CBP fragments were generated by in vitro translation. The results in Fig. 1E show that a C-terminal fragment of CBP containing the CH3 domain (aa 1626-2260) (20) binds strongly to both p45 and MafG (Fig. 1E). Consistent with a previous report (16), a fragment of CBP containing the CREB binding domain (aa 117-737) also bound to p45, although with much lower affinity. In summary, both subunits of NF-E2 bind to CBP, suggesting that the NF-E2-coactivator complex might be stabilized by multiple protein contacts.



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Fig. 1.   In vitro binding of NF-E2 to CBP. A, both p45 and MafG associate with CBP in vitro. GST, GST-p45, and GST-MafG (5 µg of each) were assayed for binding to in vitro translated, [35S]methionine-labeled CBP. Input: 10% of in vitro translated material. B, mapping of CBP binding sites in p45. C, mapping of CBP binding sites in MafG. D, summary of mapping experiments. EHR, extended homology region. E, mapping of CBP domains that bind p45 and MafG.

We next examined whether NF-E2 and CBP associate in vivo. Due to the lack of an appropriate MafG antibody, we generated MEL cells stably expressing HA-tagged MafG. There was no detectable difference in the growth, differentiation, and hemoglobinization of HA-MafG-expressing cells when compared with parental MEL cells (data not shown). This indicates that HA-MafG expression did not occur at levels sufficient to perturb cellular functions, as might have been expected from studies in which small Maf proteins were over-expressed in MEL cells (40). Nuclear extracts from these cells were immunoprecipitated with anti-CBP antibodies followed by Western analysis using anti-HA antibodies. HA-MafG was detected in immunoprecipitates with anti-CBP antibodies but not with control antibodies (Fig. 2A). These results suggest that NF-E2 associates with CBP in erythroid cells.



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Fig. 2.   NF-E2 interacts with CBP in vivo and recruits CBP to a NF-E2 binding site-containing DNA template. A, nuclear extracts from MEL cells expressing HA-MafG were immunoprecipitated (I.P.) with anti-CBP antibodies or nonimmune (n.i.) rabbit IgG followed by anti-HA Western blotting. B, a DNA fragment containing HS2 from the human beta -globin LCR was immobilized on magnetic beads and incubated with recombinant GST-p45 and GST-MafG followed by incubation with MEL cell nuclear extracts (NE). After extensive washing, the presence of CBP on the HS2 template was determined by Western blot. Input: 10% of input MEL nuclear extracts. WT, wild type.

We next determined whether NF-E2 can recruit CBP to an enhancer containing NF-E2 binding sites. A DNA fragment containing HS2 from the human beta -globin LCR was used as DNA template since HS2 contains two functionally important NF-E2 binding sites that are positioned in a tandem configuration (5). After biotinylation and immobilization on streptavidin-coupled magnetic beads, the DNA template was incubated with recombinant GST-MafG and GST-p45 proteins. After several washes to remove free protein, DNA-bound NF-E2 was incubated with nuclear extracts from MEL cells, washed, and analyzed for the presence of CBP by Western blotting. In the presence of NF-E2, CBP derived from MEL nuclear extracts bound to the NF-E2-DNA complex (Fig. 2B). In the absence of NF-E2 protein (GST alone) or when a HS2 fragment that lacked functional NF-E2 binding sites (HS2Delta NF-E2) was used, little or no CBP was retained (Fig. 2B), indicating that recruitment of CBP is mediated by DNA-bound NF-E2. These results suggest that NF-E2 might contribute to the strong enhancer activity of HS2 by recruiting CBP, consistent with experiments that functionally linked NF-E2 elements of HS2 with CBP action (35).

Acetylation of MafG by CBP-- Since several CBP-associated nuclear factors are regulated by protein acetylation (21), we examined whether either subunit of NF-E2 is a substrate for CBP. In vitro acetylation assays were performed using recombinant purified GST fusion proteins containing the acetyltransferase domain of CBP (CBP-AT), p45, and MafG in the presence of [14C]acetyl coenzyme A. Products were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The results show that CBP-AT acetylates full-length MafG (Fig. 3A) but not p45 (Fig. 3B). Deletion analysis revealed that CBP-AT strongly acetylates MafG at the bZip domain (aa 51-162) but only very weakly at the N terminus (aa 1-50) (Fig. 3B). Deletion of the basic region (aa 51-77) significantly reduced acetylation, suggesting that it is the predominant CBP-acetylated site (Fig. 4A). The basic region contains 4 lysine residues at positions 53, 60, 71, and 76 (Fig. 4B). Deletion of lysines 53 and 60 (construct 61-162) results in decreased acetylation, whereas loss of all four lysines further reduces acetylation (construct 77-162), suggesting that all 4 lysines are targets for acetylation (Fig. 4A). Of note, we observed residual acetylation of a construct that contained the leucine zipper but lacked the basic region (77).



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Fig. 3.   MafG is acetylated by CBP. GST-p45 and GST-MafG were acetylated by GST-CBP-AT in the presence of 14C-labeled acetyl coenzyme A and resolved on SDS-polyacrylamide gel electrophoresis followed by autoradiography. GST-GATA-1 served as a positive control (34).



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Fig. 4.   Acetylation of MafG by CBP occurs primarily at the basic region. A, upper panel, acetylation of MafG truncation constructs. Lower panel, Coomassie staining. B, acetylation by full-length CBP of tethered wild type (WT) NF-E2 and NF-E2-4A.

Since both small Maf proteins and p45 can form homodimers, we examined whether lysines 53, 60, 71, and 76 of MafG are the major acetylation sites in the context of heterodimeric NF-E2. For this purpose we used a tethered form of NF-E2 that contains p45 linked to MafG by a flexible peptide. Importantly, tethered NF-E2 binds DNA with high efficiency and is fully active in restoring NF-E2 function in p45 null erythroid cells, demonstrating that the linker peptide does not adversely affect NF-E2 function (13, 36). The advantage of using tethered NF-E2 over both subunits prepared separately is the absence of homodimeric forms of MafG and p45, respectively (see also below). To examine the acetylation of lysines 53, 60, 71, and 76 of MafG, we generated a form of tethered NF-E2 in which all 4 lysines were substituted with alanines (NF-E2-4A). GST-CBP-AT acetylated tethered NF-E2 somewhat less efficiently than MafG (data not shown). However, when full-length baculovirus-expressed CBP was used, tethered NF-E2 was acetylated with high efficiency (Fig. 4B). In contrast, acetylation of NF-E2-4A was substantially reduced when compared with wild type NF-E2. This indicates that the four lysines in the basic region of MafG are the major acetylation sites of the NF-E2 heterodimer. Since the basic region is directly involved in DNA binding, these results raised the possibility that acetylation of MafG might modulate its ability to bind DNA (see below).

MafG Is Acetylated in Vivo-- The use of anti-acetyl lysine (anti-AK) antibodies allowed monitoring of protein acetylation in vivo. Broad-specificity anti-AK antibodies reacted well with MafG acetylated by CBP in vitro but not with nonacetylated MafG (data not shown). Using these antibodies, we determined whether acetylation of MafG occurs in vivo. Extracts of MEL cells stably expressing HA-MafG were immunoprecipitated by anti-AK antibodies followed by Western blotting with anti-HA antibodies. The results show that anti-AK, but not nonimmune rabbit IgG, precipitated significant amounts of HA-MafG (Fig. 5A), indicating that MafG is acetylated in erythroid cells. To determine whether MafG acetylation is regulated by CBP, NIH 3T3 cells were transfected with HA-MafG alone or together with CBP. As shown in Fig. 5B, MafG was acetylated, consistent with the results obtained in MEL cells. Coexpression of CBP enhanced acetylation, which in turn was inhibited when an E1A-expressing plasmid was included in the transfections (Fig. 5B). Control Western analysis showed that coexpression of E1A and CBP did not alter MafG protein levels. These results demonstrate that CBP stimulates MafG acetylation in an E1A-sensitive manner in intact cells, similar to what we observed for GATA-1 (34). To determine whether acetylation of MafG occurs at the same sites in vitro and in vivo when bound to p45, wild-type tethered NF-E2 and NF-E2-4A were transfected into COS cells, which contain high levels of endogenous acetyltransferase activity (34), and analyzed by anti-AK immunoprecipitation. Anti-AK precipitated substantial amounts of wild type NF-E2 but not NF-E2-4A (Fig. 5C), suggesting that the four lysine residues in the basic region of MafG are the major acetylation sites in vivo.



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Fig. 5.   In vivo acetylation of MafG. A, acetylation of MafG occurs in erythroid cells. Whole cell lysates from MEL cells expressing HA-MafG were immunoprecipitated (I.P.) with anti-AK antibodies followed by Western blotting using anti-HA antibodies. n.i., nonimmune rabbit IgG. As control, 10% of the lysates were analyzed directly (input). B, in vivo acetylation of MafG is enhanced by over-expression of CBP, which in turn is inhibited by coexpressed E1A. HA-MafG, CBP, and E1A constructs were transiently transfected in NIH 3T3 cells. Whole cell lysates were immunoprecipitated with anti-AK antibodies (Ab) as in A. C, acetylation of MafG occurs at the same sites in vivo and in vitro. Whole cell lysates from COS cells transfected with wild type (WT) NF-E2 or NF-E2-4A were immunoprecipitated (I.P.) with anti-AK antibodies (Ab) followed by Western analysis with anti-p45 antibodies.

Acetylation by CBP Stimulates DNA Binding of NF-E2-- Since CBP-mediated acetylation of MafG occurs in the bZip region, we examined whether acetylation affects the interaction between the p45 and MafG subunits or their ability to bind DNA. To test whether acetylation affects heterodimerization of MafG and p45, GST pull-down experiments were carried out using acetylated or mock-acetylated GST-MafG and in vitro translated, 35S-labeled p45. Acetylated and nonacetylated GST-MafG bound equal amounts of p45 (Fig. 6A, left panel). However, since in both cases p45 binding was very inefficient, we examined whether heterodimer formation might be facilitated or stabilized in the presence of DNA. As shown in Fig. 6A (right panel), inclusion in the binding reaction of an oligonucleotide containing a single NF-E2 binding site substantially increased binding between GST-MafG and p45. p45 associated very rapidly with GST-MafG, reaching a maximum within 30 min. Acetylation of GST-MafG did not alter the association with p45, even at a time point before saturation was reached (10 min, Fig. 6A, right panel). Together, these results suggest that acetylation does not significantly affect association between MafG and p45.



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Fig. 6.   Dimerization and DNA binding of NF-E2. A, dimerization between acetylated or mock-acetylated GST-MafG and in vitro translated p45 in the presence (right panel) or absence (left panel) of an oligonucleotide containing the NF-E2 element derived from the PBGD gene. Two µg of acetylated or mock-acetylated GST-MafG were incubated with in vitro translated, 35S-labeled p45 for 1 h (without oligonucleotide) or 10 min (with oligonucleotide). Input: 10% of the in vitro translation reaction. B, DNA binding of in vitro acetylated NF-E2. Tethered NF-E2 was acetylated by His-tagged full-length CBP, and increasing amounts of protein (3, 6, 12 ng) were used in gel shift assays. Mock acetylation reactions lacked acetyl coenzyme A. The oligonucleotides used in the gel shift reactions contained the NF-E2 sites of the PBGD promoter (left panel) and HS2 from the human beta -globin LCR (right panel), respectively. The experiments were done in duplicate with two independent acetylation reactions, one of which is shown. The averages of fold-increases in DNA binding upon acetylation of NF-E2 were 2.8-, 3.4-, and 2.5-fold (when 3, 6, and 12 ng of NF-E2 were used) for the PBGD probe and 1.9-, 1.9-, and 2.1-fold for the HS2 probe. C, DNA binding of NF-E2 constructs expressed in COS cells. Wild type (WT) NF-E2 and NF-E2-4A were expressed in COS cells, and nuclear extracts were used in gel shift experiments using the oligonucleotide containing the PBGD-derived NF-E2 element. Western blotting confirmed that wild type NF-E2 and NF-E2-4A proteins were expressed at comparable levels.

Since the predominant acetylation sites reside in the basic region of MafG, we tested whether acetylation by CBP affects DNA binding of NF-E2. Initial gel shift experiments with recombinant GST-p45 and GST-MafG showed that GST-p45 bound DNA relatively well as homodimer and that the presence of GST-MafG led to an only moderate increase in DNA binding (data not shown). Furthermore, p45 homodimers migrated with a mobility very similar to that of p45-MafG heterodimers in gel shift experiments. These results contrast with previous reports where maltose binding protein (MBP) fusion proteins of p45 and MafG bound DNA significantly better as heterodimers than as homodimers (19, 41). We suspect that the difference between these observations might be related to the use of different tags (GST versus MBP) or to differences in protein preparation. In mammalian nuclear extracts, p45 is found in a complex with small Maf proteins (42, 43), and the p45-Maf heterodimer is the predominant DNA-bound form of NF-E2 (13, 42). To test DNA binding of the NF-E2 heterodimer in the absence of confounding homodimeric complexes, we used the tethered form of NF-E2, which binds DNA as an obligate heterodimer. As shown in Fig. 6B (left panel), acetylation by CBP significantly increased DNA binding of NF-E2 at various protein concentrations. Phosphorimaging analysis revealed that acetylated NF-E2 binds to the NF-E2 binding site derived from the PBGD promoter with up to 3.4-fold higher efficiency when compared with nonacetylated NF-E2. We also observed stimulation of DNA binding when the NF-E2 binding site from HS2 of the human beta -globin LCR was used (Fig. 6B, right panel).

If acetylation of the basic region in MafG enhances DNA binding, mutations of the acetylated residues would be expected to reduce DNA binding of mammalian-expressed NF-E2. To test this possibility, NF-E2 constructs were expressed in COS cells where wild type NF-E2 but not NF-E2-4A is acetylated with high efficiency (Fig. 5C). When nuclear extracts were examined by gel shift analysis, strong DNA binding was observed with wild type NF-E2, whereas NF-E2-4A failed to bind DNA very efficiently (Fig. 6C). Western analysis confirmed the presence of equal amounts of proteins in the reactions. These results indicate that the acetylated residues are important for DNA binding of mammalian-expressed NF-E2 and establish a correlation between acetylation and DNA binding of NF-E2 (Figs. 5C and 6C).

Intact Acetylation Sites Are Required for Transcriptional Activation by NF-E2-- To determine whether the major acetylation sites are important for the function of NF-E2, transient transfection assays were performed in NIH 3T3 cells using the NF-E2-dependent promoter of the PBGD gene fused to the human growth hormone gene as a reporter. As shown in Fig. 7, NF-E2-4A displayed significantly lower activity when compared with wild type NF-E2. Control Western analysis showed that NF-E2-4A was consistently expressed at about 2-fold higher levels than wild type NF-E2 (Fig. 7, lower panel), making the observed reduction in activity even more significant. These results indicate that the acetylation sites of NF-E2 are important for NF-E2 as a transcriptional activator.



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Fig. 7.   Acetylation-deficient NF-E2 has reduced transcriptional activity. Increasing amounts of plasmid expressing wild-type (WT) NF-E2 and NF-E2-4A were cotransfected into NIH3T3 cells with a PBGD-GH reporter. The transcriptional activity of NF-E2 was determined by measuring the levels of secreted growth hormone. Results represent the averages of three independent experiments. Whole cell lysates were used in Western blot to determine the levels of NF-E2.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we demonstrate that both subunits of NF-E2 interact with CBP. We further show that MafG and CBP associate in erythroid cells. CBP and acetylates MafG in vitro and in vivo mainly in the basic region, thereby increasing DNA binding and transcriptional activation of NF-E2.

Our observation that both subunits of NF-E2 bind to CBP suggests that the complex might be stabilized by multiple protein contacts in vivo. It is possible that by binding to both subunits, CBP might stimulate heterodimer formation, leading to enhanced DNA binding independent of protein acetylation. Recruitment of CBP to the beta -globin LCR might be aided further by the presence of additional erythroid transcription factors that interact with CBP. Both GATA-1 and EKLF fulfill these criteria since GATA-1 and EKLF binding sites are important elements at the LCR, and both factors have been shown to bind CBP (28, 29). GATA-1 and EKLF are also acetylated by CBP (29, 33, 34), indicating that a common theme underlies the regulation of structurally diverse nuclear factors involved in globin gene expression.

In agreement with a requirement of CBP for LCR function, interference with CBP/p300 activity leads to a complete block in globin gene expression and erythroid differentiation (28). In the context of HS2, NF-E2 binding sites are the predominant E1A-sensitive cis-acting elements, implicating a functional link between NF-E2 and CBP (35). Therefore, it is possible that the effects of E1A on globin gene expression are the combined result of inhibition of GATA-1, EKLF, and NF-E2 function.

In vitro acetylation of MafG by CBP occurs predominantly at a fragment in the basic region containing four lysine residues. Further deletion analysis showed that each of two lysine pairs (residues 53 and 60, and 71 and 76, respectively) contributes to MafG acetylation. Although all four lysines account for the majority of MafG acetylation when assayed alone or in a complex with p45, the relative contribution of each single lysine to the total acetylation of MafG remains to be determined. All four sites are conserved among all small Maf proteins and across all species examined (44), suggesting that they are functionally important. Minor acetylation was also observed at the N terminus and the leucine zipper. The functional significance of these acetylation sites is unknown. In contrast, in vivo acetylation as determined by anti-AK immunoprecipitation experiments showed virtually no acetylation outside the basic region (Fig. 5C). This suggests that the basic region is the major acetylation site in vivo. However, it is possible that the anti-AK antibodies used may have selectivity toward the acetylated residues in the basic region.

Although the molecular consequences of GATA-1 and EKLF acetylation are not yet established (29, 33, 34), this work shows that acetylation by CBP augments DNA binding of NF-E2. Although acetylation of MafG might increase DNA binding by multiple mechanisms, the observation that acetylation occurs predominantly in the basic region suggests that it might directly increase the affinity of MafG for DNA. Although the residues in MafG that contact DNA have not been determined, the crystal structure of the yeast bZip protein GCN4 complexed with DNA provides some insight into how bZip proteins contact DNA (45). In GCN4, the underlined residues of the NTEAARRSR motif in the basic region contact the central 7 base pairs in the GCN4 binding site. Two of the four major acetylation sites in MafG (aa 60 and aa 71, in bold) directly flank the KNXXYAXXCRYK core motif (Fig. 4B), supporting a possible role for acetylation in the formation of DNA contacts. However, it remains possible that acetylation might stimulate DNA binding by triggering allosteric changes in NF-E2, similar to what has been described for p53 (30). Some acetylation of MafG was also observed in the leucine zipper domain, suggesting that this might stimulate heterodimerization with p45, thereby indirectly increasing DNA binding. However, two observations suggest that this is unlikely. First, in vitro protein binding studies failed to yield any significant differences in dimerization of acetylated and nonacetylated MafG with p45. Second, acetylation in the zipper domain both in vitro and in vivo is minimal when compared with the acetylation observed in the basic region. Although an acetylation-induced increase in DNA binding was observed on two distinct NF-E2 elements derived from the PBGD promoter and human HS2, respectively, it remains possible that acetylation might lead to subtle changes in DNA binding site preferences between variant NF-E2 sites. Comparable increases in DNA binding upon acetylation have been observed in several transcription factors (21). One can envision at least two scenarios regarding the order of events. First, DNA binding of NF-E2 might occur before recruitment of CBP and NF-E2 acetylation. In this case, acetylation might stabilize the NF-E2 complex on DNA. Second, CBP and NF-E2 might be bound to each other in solution before DNA binding. In this event, acetylation might increase the rate of association of NF-E2 with its cognate binding element. Our observation that CBP can bind and acetylate NF-E2 in solution in the absence of DNA is consistent with the latter possibility.

We showed that acetylation-defective NF-E2 (NF-E2-4A) has diminished transcriptional activity in transient reporter gene assays, further suggesting that acetylation of NF-E2 is important for its function. However, NF-E2-4A still retained significant activity, especially at higher amounts of transfected DNA. This suggests that the reduced affinity of NF-E2-4A for DNA can be overcome by increased protein concentrations.

The interaction between NF-E2 and CBP suggests that CBP regulates transcription by modulating chromatin structure as well as transcription factor activity. Although protein acetylation is an attractive mechanism by which CBP acts at the LCR or other erythroid or megakaryocytic genes, additional mechanisms have to be considered as well. For example, since CBP also contacts certain components of the basal transcription machinery (for review see Refs. 20 and 21), it might mediate enhancer activity of the LCR in an acetylation-independent manner, for example by bridging to the globin gene core promoters. Furthermore, NF-E2 has been shown to associate with a chromatin remodeling activity (46, 47), suggesting that CBP-independent activities might contribute to NF-E2 function. Analogously, EKLF associates with E-RC1, an SNF/SWI-related protein complex with ATP-dependent chromatin-remodeling activity (48). It is possible that ATP-dependent remodeling complexes and AT complexes act in different promoter contexts. Alternatively, it is conceivable that ATP-dependent and AT-containing complexes act sequentially at the same genes, similar to what has been described for the yeast HO gene (49, 50).


    ACKNOWLEDGEMENT

We thank Paul Lieberman and Chi-Ju Chen for full-length His-tagged CBP, Volker Blank for cDNAs encoding p45, MafG, and tethered NF-E2, Ross Hardison and Laura Elnitski for the HS2 fragment, Emery Bresnick and Camilla Forsberg for the p45 antibody and HS2Delta NF-E2 DNA, Hui Zhang for the anti-acetyl lysine antibodies, and Mitch Weiss, Merlin Crossley, and Margaret Chou for critical reading of the manuscript.


    FOOTNOTES

* This work was supported by the American Society of Hematology Scholar Award (to G. A. B.) and National Institutes of Health Grants 1R01DK54937 (to G. A. B.) and 1F32DK10027 (to H-L. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Abramson Pediatric Research Center 316A, The Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd. Philadelphia, PA 19104. Tel.: 215-590-3988; Fax: 215-590-4834; E-mail: blobel@email.chop.edu.

Published, JBC Papers in Press, January 11 , 2001, DOI 10.1074/jbc.M007846200


    ABBREVIATIONS

The abbreviations used are: bZip, basic leucine zipper; CNC, cap and collar; CBP, cAMP-response element-binding protein (CREB)-binding protein; LCR, locus control region; HS2, hypersensitive site 2; EKLF, erythroid Krüppel-like factor; AT, acetyltransferase; GST, glutathione S-transferase; HA, hemagglutinin; AK, acetyl lysine; MEL, murine erythroid leukemia; PBGD, porphobilinogen deaminase; aa, amino acids.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Andrews, N. C. (1998) Int. J. Biochem. Cell Biol. 30, 429-432[CrossRef][Medline] [Order article via Infotrieve]
2. Motohashi, H., Shavit, J. A., Igarashi, K., Yamamoto, M., and Engel, J. D. (1997) Nucleic Acids Res. 25, 2953-2959[Abstract/Free Full Text]
3. Blank, V., and Andrews, N. C. (1997) Trends Biochem. Sci. 22, 437-441[CrossRef][Medline] [Order article via Infotrieve]
4. Ney, P. A., Sorrentino, B. P., Lowrey, C. H., and Nienhuis, A. W. (1990) Nucleic Acids Res. 18, 6011-6017[Abstract]
5. Ney, P. A., Sorrentino, B. P., McDonagh, K., and Nienhuis, A. W. (1990) Genes Dev. 4, 993-1006[Abstract]
6. Talbot, D., and Grosveld, F. (1991) EMBO J. 10, 1391-1398[Abstract]
7. Shavit, J. A., Motohashi, H., Onodera, K., Akasaka, J.-E., Yamamoto, M., and Engel, J. D. (1998) Genes Dev. 12, 2164-2174[Abstract/Free Full Text]
8. Chan, J. Y., Han, X. L., and Kan, Y. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11371-11375[Abstract]
9. Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9926-9930[Abstract/Free Full Text]
10. Kobayashi, A., Ito, E., Toki, T., Kogame, K., Takahashi, S., Igarashi, K., Hayashi, N., and Yamamoto, M. (1999) J. Biol. Chem. 274, 6443-6452[Abstract/Free Full Text]
11. Oyake, T., Itoh, K., Motohashi, H., Hayashi, N., Hoshino, H., Nishizawa, M., Yamamoto, M., and Igarashi, K. (1996) Mol. Cell. Biol. 16, 6083-6095[Abstract]
12. Shivdasani, R. A., and Orkin, S. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8690-8694[Abstract]
13. Kotkow, K. J., and Orkin, S. H. (1995) Mol. Cell. Biol. 15, 4640-4647[Abstract]
14. Bean, T. L., and Ney, P. A. (1997) Nucleic Acids Res. 25, 2509-2515[Abstract/Free Full Text]
15. Amrolia, P. J., Ramamurthy, L., Saluja, D., Tanese, N., Jane, S. M., and Cunningham, J. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10051-10056[Abstract/Free Full Text]
16. Cheng, X., Reginato, M. J., Andrews, N. C., and Lazar, M. A. (1997) Mol. Cell. Biol. 17, 1407-1416[Abstract]
17. Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, C. J. (1997) J. Biol. Chem. 272, 24105-24108[Abstract/Free Full Text]
18. Mosser, E. A., Kasanov, J. D., Forsberg, E. C., Kay, B. K., Ney, P. A., and Bresnick, E. H. (1998) Biochemistry 37, 13686-13695[CrossRef][Medline] [Order article via Infotrieve]
19. Igarashi, K., Kataoka, K., Itoh, K., Hayashi, N., Nishizawa, M., and Yamamoto, M. (1994) Nature 367, 568-572[CrossRef][Medline] [Order article via Infotrieve]
20. Shikama, N., Lyon, J., and LaThangue, N. B. (1997) Trends Cell Biol. 7, 230-236[CrossRef]
21. Goodman, R. H., and Smolik, S. (2000) Genes Dev. 14, 1553-1577[Free Full Text]
22. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve]
23. Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve]
24. Hebbes, T. R., Clayton, A. L., Thorne, A. W., and Crane-Robinson, C. (1994) EMBO J. 13, 1823-1830[Abstract]
25. Schubeler, D., Franxastel, C., Cimbora, D. M., Reik, A., Martin, D. I. K., and Groudine, M. (2000) Genes Dev. 14, 940-950[Abstract/Free Full Text]
26. Forsberg, E. C., Downs, K. M., Christensen, H. M., Im, H., Nuzzi, P. A., and H, B. E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 14494-14499[Abstract/Free Full Text]
27. Blobel, G. A. (2000) Blood 95, 745-755[Free Full Text]
28. Blobel, G. A., Nakajima, T., Eckner, R., Montminy, M., and Orkin, S. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2061-2066[Abstract/Free Full Text]
29. Zhang, W., and Bieker, J. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9855-9860[Abstract/Free Full Text]
30. Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve]
31. Sakaguchi, K., Herrera, J. E., Saito, S., Miki, T., Bustin, M., Vassilev, A., Anderson, C. W., and Appella, E. (1998) Genes Dev. 12, 2831-2841[Abstract/Free Full Text]
32. Liu, L., Scolnick, D. M., Trievel, R. C., Zhang, H. B., Marmorstein, R., Halazonetis, T. D., and Berger, S. L. (1999) Mol. Cell. Biol. 19, 1202-1209[Abstract/Free Full Text]
33. Boyes, J., Byfield, P., Nakatani, Y., and Ogryzko, V. (1998) Nature 396, 594-598[CrossRef][Medline] [Order article via Infotrieve]
34. Hung, H.-L., Lau, J., Kim, A. Y., Weiss, M. J., and Blobel, G. A. (1999) Mol. Cell. Biol. 19, 3496-3505[Abstract/Free Full Text]
35. Forsberg, E. C., Johnson, K., Zaboikina, T. N., Mosser, E. A., and Bresnick, E. H. (1999) J. Biol. Chem. 274, 26850-26859[Abstract/Free Full Text]
36. Blank, V., Kim, M. J., and Andrews, N. C. (1997) Blood 89, 3925-3935[Abstract/Free Full Text]
37. Mignotte, V., Eleouet, J. F., Raich, N., and Romeo, P.-H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6548-6551[Abstract]
38. Elnitski, L., Miller, W., and Hardison, R. (1997) J. Biol. Chem. 272, 369-378[Abstract/Free Full Text]
39. Chretien, S., Dubart, A., Beaupain, D., Raich, N., Grandchamp, B., Rosa, J., Goossens, M., and Romeo, P. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6-10[Abstract]
40. Igarashi, K., Itoh, K., Hayashi, N., Nishizawa, M., and Yamamoto, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7445-7449[Abstract]
41. Igarashi, K., Itoh, K., Motohashi, H., Hayashi, N., Matuzaki, Y., Nakauchi, H., Nishizawa, M., and Yamamoto, M. (1995) J. Biol. Chem. 270, 7615-7624[Abstract/Free Full Text]
42. Andrews, N. C., Erdjument-Bromage, H., Davidson, M. B., Tempst, P., and Orkin, S. H. (1993) Nature 362, 722-728[CrossRef][Medline] [Order article via Infotrieve]
43. Ney, P. A., Andrews, N. C., Jane, S. M., Safer, B., Purucker, M., Weremowicz, S., Morton, C. C., Goff, A. C., Orkin, S. H., and Nienhuis, A. W. (1993) Mol. Cell. Biol. 13, 5604-5612[Abstract]
44. Onodera, K., Shavit, J. A., Motohashi, H., Katsuoka, F., Akasaka, J.-E., Engel, J. D., and Yamamoto, Y. (1999) J. Biol. Chem. 274, 21162-21169[Abstract/Free Full Text]
45. Ellenberger, T. E., Brandl, C. J., Struhl, K., and Harrison, S. C. (1992) Cell 71, 1223-1237[Medline] [Order article via Infotrieve]
46. Armstrong, J. A., and Emerson, B. M. (1996) Mol. Cell. Biol. 16, 5634-5644[Abstract]
47. Gong, Q., McDowell, J. C., and Dean, A. (1996) Mol. Cell. Biol. 16, 6055-6064[Abstract]
48. Armstrong, J. A., Bieker, J. J., and Emerson, B. M. (1998) Cell 95, 93-104[Medline] [Order article via Infotrieve]
49. Cosma, M. P., Tanaka, T., and Nasmyth, K. (1999) Cell 97, 299-311[Medline] [Order article via Infotrieve]
50. Krebs, J. E., Kuo, M.-H., Allis, C. D., and Peterson, C. L. (1999) Genes Dev. 13, 1412-1421[Abstract/Free Full Text]


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