Published online before print December 12, 2003
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,1
* First Department of Internal Medicine, Nagoya University School of Medicine, Japan; and
Section of Microbiology, Division of Biological Sciences, University of California at Davis
1 Correspondence: Section of Microbiology, Division of Biological Sciences, University of California at Davis, One Shields Avenue, Davis, CA 95616. E-mail: fhayakawa{at}ucdavis.edu
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Key Words: p300 • GCN5 • histone acetyl transferase • transcription factor • DNA-binding activity
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amino groups of lysine residues, thereby loosening contacts between the histones and negatively charged DNA. Increased histone acetylation also promotes chromatin decompaction [3
]. Both of these effects can increase transactivator binding to nucleosomal DNA, facilitating transcriptional activation. Proteins that possess such histone acetyltransferase (HAT) activity include p300, cyclic AMP response element-binding protein-binding protein (CBP), GCN5, p300/CBP-associated factor (P/CAF), TATA element-binding protein-associated factor (TAF)II250, steroid receptor coactivator-1, and activator of thyroid and retinoic acid receptors (ACTR) [4
5
6
7
8
9
10
11
]. These HATs have different lysine specificities, and some of them form complexes with each other in acetylating histones [6
, 9
, 11
]. It is interesting that recent studies demonstrate that these acetyltransferases have substrates in addition to nucleosomal histones, such as tumor suppressor protein p53 for p300 and P/CAF [12
, 13
], the erythroid Kruppel-like factor (EKLF) for p300 and CBP [14
], the Kruppel-like zinc finger transcription factor specificity protein 1 (Sp1) for p300 [15
], the basal transcription factors TFIIE, TFIIF for p300, CBP, P/CAF, and TAFII250 [16
], cell-cycle regulator E2F1 for P/CAF, p300, and CBP [17
], proto-oncogene product c-Myb for p300, CBP, and GCN5 [18
, 19
], the orphan nuclear receptor hepatocyte nuclear factor-4 (HNF-4) for CBP [20
], and hematopoietic lineage-specific factor GATA-1 and GATA-3 for p300 and CBP [21
22
23
]. Acetylation has been shown to regulate the function of these factors at multiple levels such as DNA-binding activity, protein interaction, nuclear localization, protein stability, and transactivation activity. These findings provide additional insights into the regulation of transcription factor by post-translational modification. However, the physiological and biological significance of acetylating, nonhistone proteins mostly remains an open question. Members of the GATA family proteins play critical roles in cellular proliferation and differentiation of various cell lineages [24 , 25 ]. Three GATA family members (GATA-1/2/3) have been identified as important regulators of gene expression in hematopoietic cells. Gene targeting experiments have demonstrated an essential role for GATA-1 in erythroid and megakaryocytic lineage differentiation [26 ], GATA-2 in the survival and growth of multipotential progenitors and mast cell development [27 ], and GATA-3 in T cell development and differentiation [28 ]. These factors bind to a DNA consensus sequence (T/A)GATA(A/G) using a highly conserved DNA-binding domain composed of amino- and carboxy-terminal zinc fingers [29 ]. Much attention has been focused on how these different factors, which seemingly bind to similar (or identical) cis elements, carry out their distinct, biological functions. Part of the answer may be attributed to their different expression profiles: GATA-1 is highly expressed in developing erythroid cells, mast cells, and megakaryocytes [30 , 31 ]. GATA-2 is also expressed in early erythroid cells, mast cells, and megakaryocytes [32 33 34 35 ], but particularly high levels of expression have been observed in enriched populations of pluripotent hematopoietic stem cells [36 ]. The expression of GATA-3 within hematopoiesis is confined to definitive erythroid cells and T lymphocytes [24 , 37 ]. Functional experiments, in which part of the deficiency caused by loss of function of a given GATA family member is rescued by enforced expression of a different family member, support the notion that GATA factors are partially interchangeable [38 , 39 ]. However, the rescue is yet to be completely effective, thereby implying the existence of intrinsic differences in the functional potentials of the different GATA factors. That there are such intrinsic differences in the biological properties of different GATA factors has also been argued on the basis of ectopic expression experiments conducted in erythroid cells [40 ] and multipotent progenitor cells [41 , 42 ]. Such differences may relate to subtle differences in binding-site affinities or preferences. Some evidence for this has been obtained in vitro [43 44 45 ], but how these findings relate to the in vivo situation will require a fuller understanding of different bona fide GATA target genes.
Modifications of GATA proteins, through phosphorylation [46 , 47 ] and acetylation [21 22 23 ], provide additional control points for the regulation of GATA factor functions. GATA-1 acetylation occurs at two highly conserved, lysine-rich motifs near the zinc finger domains by p300/CBP. Acetylation of GATA-1 enhances transactivation capacity and is required for its biological function, the induction of erythroid differentiation, although its effect on DNA-binding activity is still unconfirmed [21 , 22 ]. Conversely, nothing is known about acetylation of GATA-2. In the present study, we show that GATA-2 exists as an acetylated protein in vivo that is also acetylated in vitro by p300 and GCN5. We have identified multiple acetylation sites in the native GATA-2, which include sites outside the zinc finger domain. We have successfully shown that GATA-2 protein acetylated by p300 in vitro exhibited an increase in DNA-binding activity. We also showed a transcriptional synergism of GATA-2 with p300 in transfected cells that was impaired by mutation of the acetylated lysines. More importantly, those acetylation-defective mutations of GATA-2 abolished the growth-inhibiting effect of wild-type GATA-2 on interleukin (IL)-3-dependent myeloid cells. We conclude that acetylation appears to significantly affect the biological function of GATA-2.
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Antibodies
Antiflag M2 monoclonal antibody, antiglutathione S-transferase (GST) antibody, and anti-His express antibody were purchased from Kodak-IBI (Rochester, NY), Pharmacia (Uppsala, Sweden), and Invitrogen (Carlsbad, CA), respectively. Rabbit polyclonal anti-GATA-2 antibody (H-116) and mouse monoclonal anti-GATA-2 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids
GST-GATA-2/pGEX for expression of GST-fused, full-length GATA-2 was as described [48
]. GST-GATA-2(1-197)/pGEX, GST-GATA-2(198-393)/pGEX, and GST-GATA-2(393-480)/pGEX for GST-fused partial fragment of GATA-2 [amino acids (aa) 1–197, aa 198–393, and aa 393–480, respectively] were constructed using digestion and ligation at convenient restriction sites. Lysine-to-alanine substitution on these GST constructs was performed using overlapping polymerase chain reaction (PCR). Sequence analysis confirmed the absence of PCR errors using ABI 310 autosequencer (Perkin Elmer, Wellesley, MA). GST-p300-HAT/pGEX and GST-GCN5/pGEX for expression of GST-fused HAT domain of p300 (aa 1053–1810) and full-length GCN5 (aa 1–476), respectively, were made by PCR cloning as described previously [18
]. His-p300-HAT/pCDNA was constructed by digestion of corresponding GST constructs and insertion into the His/pCDNA expression vector (Invitrogen).
Flag-GATA-2/promoter cytomegalovirus (pCMV) and His-GATA-2/pCDNA for expression of Flag-tagged and His-tagged, full-length GATA-2 and the constructs for its lysine-to-alanine substituted mutants were constructed by digestion of corresponding GST-GATA-2 constructs and insertion into the Flag/pCMV expression vector (Kodak-IBI) and His/pCDNA expression vector, respectively. EFG2ERP, a kind gift from Stuart H. Orkin (Harvard Medical School, Cambridge, MA), expresses GATA-2/estrogen receptor (ER), an in-frame fusion protein of GATA-2, and the ligand-binding domain of estrogen (aa 282–595). The full-length cDNAs of GATA-2/ER were cloned into a His/pCDNA expression vector to make His-GATA-2/ER/pCDNA for expression of His-tagged GATA-2/ER. The digestion of corresponding mutant Flag-GATA-2 constructs and recombination into His-GATA-2/ER/pCDNA constructed mutant constructs with lysine-to-alanine mutations.
In vitro acetylation assays
GST and GST fusion proteins were expressed in Escherichia coli strain BL21 and purified with glutathione beads (Pharmacia), according to the manufacturer’s instructions. Acetyltransferase assays were performed as described [18
] with a slight modification. Briefly, 1 µg wild-type or mutant GST-GATA-2 was incubated at 37°C for 1 h with 100 ng GST or GST-fused acetyltransferases and 1 µl [14C]acetyl-CoA (CoA; 50 mCi/mmol; Amersham, Little Chalfont, UK) in 30 µl acetyltransferase reaction buffer [50 mM Tris-HCl (pH 8.0), 5% glycerol, 1 mM dithiothreitol, and 10 mM sodium butyrate]. The reaction mixture was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by image analyzing using a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).
In vivo acetylation assays
293T cells (5x105) were transfected with the indicated expression plasmids by lipofectamine 2000 (Gibco BRL, Grand Island, NY) according to the manufacturer’s instructions. Total amounts of plasmids used for transfection were equalized by the addition of corresponding empty vectors. Forty-eight hours after transfection, the medium was replaced with DMEM supplemented with 10% FCS in the presence of 0.05 mCi/ml sodium [14C]acetate (Amersham) for 4 h. Cells were then lysed and immunoprecipitated with M2 antibody-conjugated agarose followed by SDS-PAGE and subsequent image analyzing. KG1 cells (3x107) were also pulse-labeled with RPMI 1640, supplemented with 10% FCS in the presence of 0.05 mCi/ml sodium [14C]acetate for 4 h. Nuclear extracts obtained from the cells were subjected to immunoprecipitation with anti-GATA-2 antibody followed by SDS-PAGE and image analyzing.
DNA-binding reactions
Electrophoretic mobility shift assay using 5'-AGTCCATCTGATAAGACTTATCTGCTGCCC-3' as probe was performed as described previously [48
]. Anti-GST antibody was used for supershift assay.
Luciferase assays
A luciferase reporter plasmid 8x GATA-RE P36 that contains eight GATA consensus motifs was a kind gift from Michael G. Rosenfeld (University of California at San Diego). 293T cells (1x105) were transfected with the indicated expression plasmids using lipofectamine 2000. Total amounts of plasmids used for transfection were equalized by the addition of corresponding empty vectors. Luciferase activities were assayed using a luciferase assay system (Promega, Madison, WI), according to the manufacturer’s instructions. Transfection efficiency was normalized on the basis of ß-galactosidase (ß-gal) activity expressed from cotransfected pCMV/ß-gal plasmids (Promega). The relative luciferase activities presented reflect triplicate values from a representation of no less than three independent experiments.
Coprecipitation assays
293T cells (1x105) were cotransfected with Flag-tagged GATA-2 constructs and His-p300-HAT/pCDNA. His-p300-HAT in cell lysates was precipitated with BD Talon resin (Clontech, Palo Alto, CA) as per the manufacturer’s instruction. Precipitates were subjected to SDS-PAGE followed by immunoblot with anti-Flag antibody.
Generation of cells expressing GATA-2/ER
The His-GATA-2/ER/pCDNA and the mutant constructs disrupted at the acetylated lysines were electropolated into 32D cells with gene pulser (Bio-Rad, Hercules, CA) at 300 V and 960 µF. Single-cell clones were isolated by limiting dilution with selection by 800 µg/mL geneticin (Sigma Chemical Co., St. Louis, MO) and were maintained for further analysis.
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50 kDa was immunoprecipitated with anti-GATA-2 antibody but not with control immunoglobulin G (IgG; Fig. 1
, upper panel); this protein was detected with a second anti-GATA-2 antibody (Fig. 1
, lower panel). This indicates that GATA-2 exists as an acetylated protein in human myeloid progenitor cells.
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Figure 1. GATA-2 is acetylated in hematopoietic cells. KG1 cells (3x107) were pulse-labeled with [14C]acetate for 4 h. Nuclear extracts of these cells were immunoprecipitated with rabbit anti-GATA-2 antibody (anti-GATA-2 R Ab) or control rabbit IgG. Ninety percent of each immunoprecipitate was subjected to SDS-PAGE and autoradiography (upper panel), and 10% of that was subjected to Western blot (WB) analysis using mouse anti-GATA-2 antibody (anti-GATA-2 M Ab; lower panel). The arrows indicate the position of GATA-2 in both panels. The positions of molecular mass markers are shown.
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Figure 2. Acetylation of GATA-2 in vitro. (A) In vitro acetylation of GATA-2. An equal amount (2 µg) of the purified GST-GATA-2 was incubated with CoA and recombinant GST or GST-fused HATs as indicated. Reaction mixtures were separated by SDS-PAGE, fixed, and stained with Coomassie brilliant blue (CBB). [14C]-Incorporation was visualized with an imaging analyzer. The arrows indicate acetylated GATA-2. (B) Proportion of acetylation in GATA-2. Comparable amounts of the different subdomains of GATA-2 were applied to the in vitro acetylation analysis followed by SDS-PAGE, CBB staining, and autoradiography as described above. Asterisks show the nondegraded forms of GATA-2 fused to GST. An image analyzer determined [14C]-incorporation per molecule. The percent [14C]-incorporation per molecule of each GATA-2 subdomain was then calculated and plotted in a histogram. The solid, shaded, and open bar charts represent G2(1-197)Wild, G2(198-393)Wild, and G2(393-480)Wild, respectively.
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Figure 3. Identification of GATA-2 acetylation sites. (A) The amino acid sequence of human GATA-2 showing the lysine-to-alanine mutation sites. Boxed lysines (K) were substituted to alanine (A) as indicated to generate mutation M1–M11. (B) Schematic representation of three subdomains of GATA-2 and their mutants. Asterisks indicate the positions of lysine-to-alanine mutations. Solid and shaded boxes represent amino- and carboxy-terminal zinc fingers, respectively. Numbers correspond to amino acids. (C) In vitro acetylation of wild-type and mutant GATA-2 subdomains. An equal amount (2 µg) of wild-type or mutant GATA-2 fragments fused to GST was acetylated in vitro by GST-p300-HAT and was subjected to SDS-PAGE, CBB staining, and autoradiography as described in Figure 2
. [14C]-Incorporation of each GATA-2 subdomain was measured with an imaging analyzer. Three independent analyses were performed, and the average [14C]-incorporation was plotted on a bar chart as a percentage relative to wild-type GATA-2 along with standard deviations. (D) Conservation of the GATA-2 acetylation sites beyond species and schematic representation of wild-type and mutant GATA-2. Alignments of amino acid sequences of Xenopus (x), chicken (c), and mouse (m) GATA-2 corresponding to the acetylation sites of human (h) GATA-2 (K1, K5, K7, K9, K10, and K11) are shown in the upper panel. Asterisks, solid and shaded boxes, and numbers are the same as in (B). (E) In vitro acetylation of full-length GATA-2 and the mutants. An equal amount (2 µg) of wild-type or mutant full-length GATA-2 protein fused to GST was subjected to in vitro acetylation analysis by p300-HAT as indicated in (C).
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Lysines acetylated in GATA-2 in vivo largely parallel those acetylated in vitro by p300
We examined the effect of p300 coexpression on GATA-2 acetylation in vivo by the use of 293T cells transfected with Flag-tagged, wild-type GATA-2. Cells were labeled with [14C]acetate. Then, GATA-2 in the cell lysate was immunoprecipitated with anti-Flag M2 antibody and subjected to SDS-PAGE. The autoradiograph showed that GATA-2 was acetylated endogenously and that coexpression of p300-HAT enhanced this GATA-2 acetylation approximately by two- to threefold; this was consistent, as revealed by repeated experiments. Representative data are shown (Fig. 4
, lanes 1 and 2). Next, to determine the sites of endogenous acetylation in vivo, we investigated the acetylation status of Flag-tagged, wild-type, and mutant GATA-2 transfected to 293T cells as described above. The levels of acetylation in G2M1, G2M5·7·9, and G2M10·11 were reduced to 50%, 27%, and 28% of the wild-type level, respectively. G2M all showed no detectable acetylation (Fig. 4
, lanes 1, 3–6). Equal efficiency of immunoprecipitation among all GATA-2 constructs was confirmed by WB analysis with anti-GATA-2 antibody (middle panel). These results indicate that p300 can induce acetylation of GATA-2 in vivo and that acetylation of GATA-2 in intact cells occurs at sites similar to those induced by p300 in vitro.
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Figure 4. In vivo acetylation of GATA-2. 293T cells (5x105) were transfected with the Flag-tagged GATA-2 constructs with (+) or without (–) cotransfection of p300-HAT expression vector. Cells were pulse-labeled with [14C]acetate for 4 h. Whole-cell lysates of the cells were immunoprecipitated with agarose-conjugated anti-M2 antibody. Ninety percent of the each immunoprecipitate was subjected to SDS-PAGE and autoradiography (top panel). Ten percent of that was subjected to WB analysis using anti-GATA-2 antibody (middle panel). Relative intensity of [14C]-incorporation to G2Wild was calculated and plotted (bottom panel). Lane numbers are shown at the bottom.
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Figure 5. DNA-binding activity is elevated by acetylation. (A) Equal amounts of GST-GATA-2 (400 ng) were incubated with a radiolabeled oligonucleotide probe containing GATA consensus sequence in the presence (+) or absence (–) of a 200-fold molar excess of unlabeled oligonucleotide (competitor), normal goat IgG (control antibody), or anti ( )-GST antibody as indicated. GATA-2 DNA complex (indicated by an arrow) was visualized with an imaging analyzer. (B) Indicated amount of wild-type GST-GATA-2 was first subjected to in vitro acetylation assay by p300-HAT (50 ng) with (+Acetyl-CoA) or without (–Acetyl-CoA) 2 mM acetyl-CoA and was then applied to EMSA. The intensity of DNA-binding activity was visualized and measured with an imaging analyzer. Three independent experiments were performed. Representative images and the average of the fold increase in the activities of (+)acetyl-CoA samples relative to the corresponding values of (–)acetyl-CoA samples are shown. An arrow indicates the GATA-2-DNA complex. The error bars indicate standard deviation. (C) An equal amount (400 ng) of wild-type and mutant GST-GATA-2 was subjected to EMSA. Two independent experiments were performed. Representative image and the average of the relative DNA-binding activity to G2Wild with standard deviation are shown. An arrow indicates the GATA-2-DNA complex. (D) An equal amount (200 ng) of wild-type and mutant GST-GATA-2 was first subjected to in vitro acetylation by p300-HAT with (+) or without (–) 2 mM acetyl-CoA as indicated and then to EMSA. Three independent experiments were performed. Representative image and the average of the fold increase in the activities of acetyl-CoA (+) samples relative to the those of acetyl-CoA (–) samples are shown with standard deviation.
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Figure 6. Enhanced transactivating potential of GATA-2 by acetylation. (A) 293T cells (1x105) were transfected with GATA reporter gene in the presence (+) or absence (–) of wild-type GATA-2 expression vector and His-p300-HAT expression vector as indicated. Cell lysates were prepared 48 h after transfection and were assayed for luciferase activity. The relative luciferase activities presented reflect triplicate values from a representation of no less than three independent experiments. (B) 293T cells were transfected with GATA reporter gene and empty vector (–) or GATA-2 expression vectors as indicated and were subjected to luciferase assay. The relative luciferase activities presented reflect triplicate values from a representation of no less than three independent experiments. (C) GATA reporter gene and empty vector (–) or GATA-2 expression vectors as indicated were transfected to 293T cells with or without p300-HAT expression vector, and cells were subjected to luciferase assay. Three independent experiments were performed. The average of the fold increase in the activity derived from each GATA-2 construct in the presence of p300-HAT relative to that from respective GATA-2 constructs in the absence of p300-HAT is shown with standard deviation. (D) 293T cells (1x105) were transfected with empty vector (–) or Flag-tagged GATA-2 constructs in the presence (+) or absence (–) of cotransfection of His-tagged p300-HAT expression vector. His-tagged p300-HAT was purified with BD Talon resin and subjected to SDS-PAGE. GATA-2 bound to p300-HAT was detected by immunoblot with anti-Flag antibody. Two independent experiments were performed. Representative image is shown. Arrows indicate GATA-2 bound to p300.
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Acetylation can affect the growth-inhibiting effect of GATA-2
An in-frame fusion of GATA-2 and the ligand-binding domain of ER generate an estrogen-inducible form of GATA-2 (GATA-2/ER) [40
]. The mechanism of this inducible transactivation has been attributed to a ligand-dependent unmasking of the transactivation domain(s) of GATA-2/ER. We therefore fused ER to the C-terminal of His-tagged GATA-2 and the mutants G2M1, G2M5·7·9, G2M10·11, and G2M all; these are designated as G2Wild/ER, G2M1/ER, G2M5·7·9/ER, G2M10·11/ER, and G2M all/ER, respectively. To reveal the biological significance of GATA-2 acetylation, we transfected these ER-fusion constructs into 32D cells, IL-3-dependent early myeloid progenitor cells, by electroporation. Single clones of stable transfectants were established by selection with 800 µg/ml geneticin in the presence of IL-3. WB analyzed the expression of GATA-2/ER of five clones from each construct. One representative clone from each mutant with the strongest expression of GATA-2/ER was designated as G2Wild/ER 32D, G2M1/ER 32D, G2M5·7·9/ER 32D, G2M10·11/ER 32D, and G2M all/ER 32D, respectively, and subjected to further analysis. The GATA-2/ER expression of these clones is shown (Fig. 7A
). In murine IL-3-dependent, multipotential, hematopoietic progenitor, factor-dependent, cell-Patersen (FDCP)-mix cells, the up-regulation of GATA-2/ER by estrogen resulted in arrested growth and an enhanced differentiation down the granulomonocytic pathway that occurred despite the presence of an IL-3-mediated self-renewal signal [41
]. Consistent with this, G2Wild/ER 32D grew slowly under 0.2 µM ß-estradiol. The viable cell number after 4 days in ß-estradiol was only 24% of that without ß-estradiol, and ß-estradiol had no effect on the growth of parental 32D cells (Fig. 7B
and 7C) . The viability of the cells was consistently over 90% regardless of the addition of ß-estradiol (data not shown). No significant, morphological change was obtained in the cells stained by May-Giemsa (data not shown). Having established that GATA-2 has a growth-inhibiting effect on 32D cells, our mutant GATA-2/ER transfectants were subjected to the same assay to test the role of acetylation in the biological function of GATA-2. The growth-inhibiting effect of GATA-2/ER was drastically impaired by the mutation of the acetylated lysines. Specifically, the cell numbers of G2M1/ER 32D, G2M5·7·9/ER 32D, G2M10·11/ER 32D, and G2Mall/ER 32D cultured with ß-estradiol relative to those without ß-estradiol were 85%, 105%, 73%, and 100%, respectively (Fig. 7C)
. The growth curves of these transfectants cultured in the presence or absence of ß-estradiol were almost identical to those of parental 32D cells with or without ß-estradiol and G2Wild/ER 32D without ß-estradiol (data not shown). These results indicate that GATA-2 has a growth-inhibiting effect on 32D cells and that acetylation appears to enhance this biological function of GATA-2.
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Figure 7. Growth inhibitory effect of GATA-2 is counteracted by the introduction of mutations at acetylated lysines. (A) Nuclear extract (50 µg) obtained from the indicated 32D transfectants was subjected to WB analysis using anti-His antibody. The arrow indicated the position of the 85-kD GATA-2/ER. (B) Parental 32D cells and G2Wild/ER 32D cells were cultured at 2 x 104/ml in IL-3-containing medium with or without 0.2 µM ß-estradiol. Viable cells were counted at the indicated times using trypan blue. The assay was performed three times, and the average was calculated. The growth curve was calculated from the ratio of viable cells at each time point against the cells at time 0 (2x104/mL). (C) Cells, as indicated (A), were cultured at 2 x 104/ml in IL-3-containing medium with or without 0.2 µM ß-estradiol. After 4 days of culture, the cells were harvested and counted after trypan blue staining. The number of cells obtained in the presence of ß-estradiol (+E) is expressed as a percentage of the number of respective cells in the absence of ß-estradiol (–E). The results are the average of three independent experiments.
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We have demonstrated that GATA-2 transactivating potential can be modulated by p300 coexpression. Our data suggest that p300 may regulate GATA-2 transactivation through regulation of its DNA binding by acetylation. The mechanism(s), however, has not been fully resolved. One putative mechanism by which p300 may regulate the activity of transcription factors is acetylation of the adjacent histones, leading to an increase in accessibility of the chromatin for the transcriptional machinery or additional cofactors. Acetylation-defective mutants of GATA-2, which retain binding ability to p300 in vivo, did not respond to p300 in the present reporter assay (Fig. 6C and 6D) , suggesting that histone acetylation by p300 is not the main determinant of up-regulation of GATA-2 transactivation in our system. Several other proteins, however, are speculated to associate with GATA-2 and affect its transactivation such as friend of GATA, promyelocytic leukemia protein, PU.1, activation protein-1 (AP1), and Pit1 [48 , 50 51 52 ]. Acetylation might regulate an association of GATA-2 with these candidate accessory proteins (or unknown proteins), resulting in the altered transcriptional activity of GATA-2 we observe. Acetylation of lysine residues neutralizes their positive charge and changes the size of the residues. Such changes conceivably can confer alterations in protein function comparable with those that occur upon phosphorylation. We have reported such an example in the case of c-Myb acetylation, where acetylation may lead to neutralizing of the positive charge, resulting in altered DNA-binding activity [18 ]. Alternatively, acetylation could directly influence protein-DNA interactions or protein-protein interactions, as has been suggested for the binding of histone tails to DNA [1 , 2 ] or to the yeast transcriptional repressor Tup1 [53 ]. It is conceivable that acetylation leads to a conformational change in GATA-2, thereby increasing activity through exposure of the DNA-binding domain and putative activation domain. Furthermore, the docking sites for still undefined coactivators might be exposed by the acetylation. It might be argued that in vitro conditions might not reflect the actual changes in GATA-2 function triggered by acetylation. However, the fact that the lysines acetylated in vitro by our assay are highly conserved among different species together with our demonstration of the coincidence of acetylation sites determined by in vitro to those in vivo strengthen the likelihood that these acetylation domains play an important role in vivo as well as in vitro. The specific roles of these different acetylation sites in the crucial in vivo cell functions of GATA-2 are still unknown, although individual mutations of each individual acetylation motif in GATA-2, i.e., from K1 to K11, diminished its function in vivo to some extent.
As crucial GATA-2 functions have been primarily identified in immature hematopoietic stem cells, the methodology for examining GATA-2 functions in vivo is somewhat limited. One possible solution is to use the GATA-2/ER system as applied in the present study. The in vivo functional analysis clearly indicates that every site of lysine acetylation tested is involved to some degree in GATA-2-directed growth inhibition. The exact mechanism remains to be elucidated by which acetylation of GATA-2/ER exerts its function in ER-dependent cell growth inhibition in IL-3-dependent hematopoietic progenitor cells. One possible mechanism of growth inhibition by GATA-2 is the transactivation of a GATA-2 target gene, which in turn mediates growth inhibition; the specific target gene of GATA-2 involved in this phenomenon remains to be identified. It is interesting that although G2M10·11 showed no detectable transactivation activity, it still inhibited the growth of 32D cells, reducing them to 73%. This suggests that GATA-2 might have additional mechanisms for growth inhibition beyond transactivation of the target gene studied here. GATA-2 has been shown to associate with other transcription factors such as AP1 and PU.1 and to regulate their transactivation activity positively or negatively [50 , 51 ]. One of the mechanisms of GATA-2 for growth inhibition may be a functional modulation of these or other transcription factors related to cell growth. A detailed examination of the expected alteration of DNA-binding activity and transcriptional activity caused by acetylation-defective mutations in the GATA-2/ER system may further confirm our claims. Recently, it has been shown that the functions of GATA-2/ER in embryonic hematopoiesis are somewhat different from those of GATA-2 [54 ]. In committed progenitor cells, however, GATA-2 and GATA-2/ER are reported to have inhibitory effects to hematopoiesis, which is consistent with our finding in 32D cells [41 , 42 ]. In addition, another group has found the growth-inhibitory effects of GATA-2/ER in a very similar system as ours [55 ].
Thus, taken together, our data suggest that acetylation regulates the GATA-2 function at multiple levels. Acetylation has been generally shown to regulate DNA-binding activity, protein interaction, nuclear localization, protein stability, and transactivation activity of the transcription factor. The putative mechanism of these regulations is a conformational change as a result of neutralization of the positive charge of lysine residue by acetylation. In this study, we revealed that acetylation regulates the DNA-binding potential and transactivating potential of GATA-2 and consequently, at least some of its biological functions. More extensive analyses will be required to elucidate additional functions of GATA-2 that may be regulated by acetylation. These may include structural analysis, detection of a functional partner whose association with GATA-2 is regulated by acetylation, and a differentiation assay using GATA-2–/– hematopoietic stem cells rescued by GATA-2 with mutations of acetylated lysines. The generation of specific high-affinity antibody that recognizes the acetylated form of endogenous GATA-2 protein will be useful in addressing this issue. What our results demonstrate is that acetylation represents a novel mechanism by which the activity of GATA-2 is regulated.
Received June 24, 2003; revised November 7, 2003; accepted November 18, 2003.
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gene enhancer Mol. Cell. Biol. 11,2778-2784 oncoprotein Mol. Cell. Biol. 20,6276-6286This article has been cited by other articles:
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 F. Hayakawa, A. Abe, I. Kitabayashi, P. P. Pandolfi, and T. Naoe Acetylation of PML Is Involved in Histone Deacetylase Inhibitor-mediated Apoptosis J. Biol. Chem., September 5, 2008; 283(36): 24420 - 24425. [Abstract] [Full Text] [PDF]
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T. Takaya, T. Kawamura, T. Morimoto, K. Ono, T. Kita, A. Shimatsu, and K. Hasegawa Identification of p300-targeted Acetylated Residues in GATA4 during Hypertrophic Responses in Cardiac Myocytes J. Biol. Chem., April 11, 2008; 283(15): 9828 - 9835. [Abstract] [Full Text] [PDF]
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 R. S. Viger, S. M. Guittot, M. Anttonen, D. B. Wilson, and M. Heikinheimo Role of the GATA Family of Transcription Factors in Endocrine Development, Function, and Disease Mol. Endocrinol., April 1, 2008; 22(4): 781 - 798. [Abstract] [Full Text] [PDF]
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A. Matsushita, S. Sasaki, Y. Kashiwabara, K. Nagayama, K. Ohba, H. Iwaki, H. Misawa, K. Ishizuka, and H. Nakamura Essential Role of GATA2 in the Negative Regulation of Thyrotropin {beta} Gene by Thyroid Hormone and Its Receptors Mol. Endocrinol., April 1, 2007; 21(4): 865 - 884. [Abstract] [Full Text] [PDF]
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 J. M. Lamonica, C. R. Vakoc, and G. A. Blobel Acetylation of GATA-1 is required for chromatin occupancy Blood, December 1, 2006; 108(12): 3736 - 3738. [Abstract] [Full Text] [PDF]
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 B. O. Evert, J. Araujo, A. M. Vieira-Saecker, R. A. I. de Vos, S. Harendza, T. Klockgether, and U. Wullner Ataxin-3 Represses Transcription via Chromatin Binding, Interaction with Histone Deacetylase 3, and Histone Deacetylation. J. Neurosci., November 1, 2006; 26(44): 11474 - 11486. [Abstract] [Full Text] [PDF]
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K. Kitajima, M. Tanaka, J. Zheng, H. Yen, A. Sato, D. Sugiyama, H. Umehara, E. Sakai, and T. Nakano Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2 Blood, March 1, 2006; 107(5): 1857 - 1863. [Abstract] [Full Text] [PDF]
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J. Cleary, K. V. Sitwala, M. S. Khodadoust, R. P. S. Kwok, N. Mor-Vaknin, M. Cebrat, P. A. Cole, and D. M. Markovitz p300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters J. Biol. Chem., September 9, 2005; 280(36): 31760 - 31767. [Abstract] [Full Text] [PDF]
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L.-O. Tykocinski, P. Hajkova, H.-D. Chang, T. Stamm, O. SOzeri, M. LOhning, J. Hu-Li, U. Niesner, S. Kreher, B. Friedrich, et al. A Critical Control Element for Interleukin-4 Memory Expression in T Helper Lymphocytes J. Biol. Chem., August 5, 2005; 280(31): 28177 - 28185. [Abstract] [Full Text] [PDF]
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 N. Minegishi, N. Suzuki, Y. Kawatani, R. Shimizu, and M. Yamamoto Rapid turnover of GATA-2 via ubiquitin-proteasome protein degradation pathway Genes Cells, July 1, 2005; 10(7): 693 - 704. [Abstract] [Full Text] [PDF]
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