Use of Altered Specificity Mutants to Probe a Specific Protein–Protein Interaction in Differentiation

Molecular Cell, Vol. 3, 219–228, February, 1999, Copyright 1999 by Cell Press

Use of Altered Specificity Mutants to Probe a Specific Protein–Protein Interaction in Differentiation: the GATA-1:FOG Complex John D. Crispino,*† Maya B. Lodish,*† Joel P. MacKay,‡ and Stuart H. Orkin*†§ * Division of Hematology-Oncology Children’s Hospital and Dana Farber Cancer Institute † Department of Pediatrics Harvard Medical School and Howard Hughes Medical Institute Boston, Massachusetts 02115 ‡ Department of Biochemistry University of Sydney Sydney, NSW 2006 Australia

Summary GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-12 erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein–protein associations involved in mammalian development. Introduction The development of blood cells of various lineages from hematopoietic stem cells is controlled by cellrestricted transcription factors (Orkin, 1996). Among these, GATA-1 has been the subject of particular interest. Its expression is highly restricted to hematopoietic cells, and specifically to the erythroid, megakaryocytic, eosinophilic, and mast cell lineages (Orkin, 1992). Enforced expression reprograms myeloid cells to megakaryocytes, eosinophils, or erythroid cells (Visvader et al., 1992; Kulessa et al., 1995). Loss of GATA-1 function in mice leads to blocks in both erythroid and megakaryocytic cell maturation and accompanying apoptosis and hyperproliferation, respectively, in precursor cells (Pevny et al., 1991; Weiss et al., 1994; Fujiwara et al., 1996; Shivadasani et al., 1997). Thus, GATA-1 is an essential transcription factor for at least two hematopoietic lineages. Other GATA factors, which are related to GATA-1 in their DNA-binding domains, are critical to the development of hematopoietic and nonhematopoietic cells. GATA-2 is necessary within hematopoietic progenitors § To whom correspondence should be addressed (e-mail: orkin@ rascal.med.harvard.edu).

or stem cells (Tsai et al., 1994), whereas GATA-3 is required for T lymphoid cell development (Ting et al., 1996; Zheng and Flavell, 1997). GATA-4, -5, and -6 are expressed at multiple sites, including the heart and intestinal epithelium (Gao et al., 1998). Loss of GATA-4 leads to a failure of ventral morphogenesis and heart tube formation (Kuo et al., 1997; Molkentin et al., 1997). Of interest is how GATA factors function in transcriptional control. Each contains a DNA-binding domain comprised of two homologous zinc fingers (Evans and Felsenfeld, 1989; Tsai et al., 1989). The carboxyl (C) finger provides DNA contacts critical for recognition of the consensus sequence (T/A)GATA(A/G). The role of the amino (N) finger is more complex. In GATA-1, the N finger is unable to bind DNA on its own. Although the N finger of GATA factors is largely dispensable for binding to simple GATA motifs (Martin and Orkin, 1990), its presence enhances the stability and specificity of binding of the two-finger DNA-binding domain to palindromic sites (Trainor et al., 1996). Such sites are found in selected elements, as in the promoters for the chicken a-D globin gene and the GATA-1 gene of several species. In contrast to transient transcriptional reporter assays in nonhematopoietic cells in which the N finger of GATA-1 is nonessential, a differentiation assay based on the rescue of GATA-12 erythroid cells reveals that the N finger provides a critical function (Weiss et al., 1997). In this assay, as in conversion of myeloid 416B cells to megakaryocytes (Visvader et al., 1995), an N-terminal activation domain defined previously in heterologous cells is dispensable for function. These findings suggested that GATA-1 contributes to differentiation by associating with another hematopoietic-specific nuclear factor, possibly interacting specifically with its N finger. A candidate for this postulated cofactor, FOG (Friend of GATA-1), was isolated by a yeast two-hybrid screen (Tsang et al., 1997). FOG, a complex zinc finger protein, physically associates with the N finger of GATA-1 via at least one of its fingers (Finger 6, F6). FOG is coexpressed with GATA-1 during embryonic development and within erythroid cells and megakaryocytes. GATA-1 and FOG cooperate in promoting both erythroid and megakaryocytic maturation in cellular assays (Tsang et al., 1997). While these findings are compatible with GATA-1 and FOG acting together, evidence also suggests that these two factors have independent roles in hematopoiesis. While loss of GATA-1 leads to a block to megakaryocyte development in midmaturation, targeted mutation of FOG prevents megakaryocyte formation (Shivadasani et al., 1997; Tsang et al., 1998). Therefore, FOG may exert GATA-1-independent roles. Although mice deficient for expression of either gene exhibit a block of erythroid maturation at the proerythroblast stage, FOG2/2 erythroid precursors appear to survive longer than GATA-12 cells, suggesting that GATA-1 may have FOG-independent functions. By ablation experiments, it cannot be determined to what extent the requirement for GATA-1 in erythroid development or megakaryocyte differentiation reflects coordinated action of a GATA-1:FOG complex versus independent, but essential, roles of these factors. Discriminating between these alternatives is a recurring

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Figure 1. Identification of FOG Noninteracting Mutants of GATA-1 (A) Schematic depiction of the split two-hybrid screen. Tet R, Tet repressor; VP16 AD, VP16 activation domain; lexA, lexA DNA-binding domain. (B) Standard two-hybrid analysis to confirm the absence of the protein–protein interaction. GATA-1 mutants isolated in the split two-hybrid screen were cotransformed into yeast strain Y190 with a plasmid encoding a fusion of FOG fingers 5 through 6 with the DNA-binding domain of Gal4. Colonies were streaked onto His2 media containing 10 mM 3-aminotriazole (3-AT). (C) Summary of noninteracting GATA-1 mutants. The amino acids comprising the N-terminal zinc finger are depicted in one line, while the selected mutants are below. (D) Coimmunoprecipitation of FOG with GATA-1 in transfected COS cells. Full-length GATA-1 and a FOG cDNA comprising fingers 5 and 6 fused to the Flag epitope were coexpressed. Nuclear extracts from cells were immunoprecipitated with antibody against the Flag moiety and analyzed by Western blotting using a GATA-1 antibody. The GATA-1 band is indicated by an arrow. The GATA-1 antibody (N6: Santa Cruz Biotechnology) binds near the N terminus and recognized all mutant versions on Western blots. The upper band corresponds to IgG, the heavy chain of the anti-Flag antibody. (E) Structural model of the N finger of GATA-1, shown facing away from DNA. The three novel mutations, highlighted in blue, lie at residues that form one contiguous surface. Residue G208 is shown in red. Residues in green face away from the DNA, whereas those in yellow lie on the DNA side.

problem in assessing the in vivo relevance of individual protein–protein interactions. To address the significance of the GATA-1:FOG complex for GATA-1 function, we have employed altered specificity mutants selected in yeast. Our strategy involved identification of mutants of GATA-1 specifically impaired for interaction with FOG and subsequent isolation of FOG mutants exhibiting enhanced association with these novel GATA-1 mutants. These altered specificity mutants were then tested pairwise for their capacity to promote erythroid differentiation in order to assess the role of the interaction. Our findings demonstrate that the association of FOG with GATA-1 is, indeed, critical to GATA-1’s transcriptional function. This approach provides a paradigm for the assessment of protein–protein interactions in complex developmental pathways. Results Identification of GATA-1 Mutants Impaired for Interaction with FOG A split two-hybrid system in yeast was used to select for mutations in GATA-1 that cripple association with

FOG (Figure 1A; Shih et al., 1996). In this screen, interaction of two proteins drives expression of the Tet repressor, which represses transcription of HIS3. Disruption of interaction leads to derepression of HIS3 transcription and growth on selective media. Since the N finger of GATA-1 is necessary and sufficient for interaction with FOG (Tsang et al., 1997), altered specificity mutants of this domain were sought. PCR mutagenesis was performed to create a library of GATA-1 N finger sequences fused to the activation domain of VP16 and b-galactosidase (lacZ). Clones were cotransfected into yeast with a plasmid expressing fingers 5 and 6 of FOG fused to the DNA-binding domain of lexA. Among 16,000 transformants screened, 139 candidate mutants were obtained. Of these, 98 expressed LacZ, confirming the absence of termination or frame-shift mutations. Nine harbored single substitutions at noncysteine residues, while 20 had replacements of a cysteine of the finger. The remaining clones sustained two or more changes. To validate that mutants were impaired for association with FOG, a forward two-hybrid assay was performed (Figure 1B). Of the nine noncysteine mutants, two, K233E and D218V, interacted sufficiently with FOG to permit

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Figure 2. Several GATA-1 Mutants Bind DNA with Wild-Type Affinity (A) Dissociation gel shift assays. Nuclear extracts of COS cells expressing wild-type and mutant proteins were subjected to gel shift assays. The labeled double-stranded oligonucleotide probe comprising a palindromic GATA site was incubated with each extract for 20 min on ice. Unlabelled competitor was added at t 5 0 min, and samples were loaded onto the gel at the designated time points. (B) Results of one representative dissociation assay. The percentage of probe bound by wild-type or mutant forms of GATA-1 was plotted against time after addition of competitor. (C) Dissociation rate constants of wild-type GATA-1 and several point mutants. Nuclear extracts from untransfected COS cells exhibited a gel shift activity that comigrated with that of GATA-1 but displayed rapid dissociation. The koff for this activity was calculated to be 1.36 6 0.3 3 1023 sec21. Values were averaged from multiple experiments: Wild-type, n 5 7; E203V, V205G, COS extract, n 5 5; C204R, G208V, n 5 4; H222R, n 5 3.

growth on His2 media. The remaining seven clones expressed mutant N finger fusion proteins that failed to sustain growth in cells expressing a FOG finger 5, 6-Gal4 DNA-binding domain fusion. Replacements found to disrupt association with FOG are summarized in Figure 1C. These include E203V, V205G, V205M, G208V, G208E, H222R, and L224P, as well as mutations in all four cysteine residues. Since cysteine residues are required for coordination of zinc and the architecture of the finger, these mutants serve as negative controls in assays to define the activities of N finger mutants. Coimmunoprecipitation confirmed impaired protein– protein interaction indicated by the yeast two-hybrid assay (Figure 1D). Full-length versions of wild-type or mutant GATA-1 cDNAs were coexpressed in COS cells with a Flag-tagged FOG molecule comprising fingers 5 and 6. Following immunoprecipitation with a Flagantibody, samples were subjected to Western blot analysis with anti-GATA-1 antibody. Interaction between FOG and the V205G, H222R, or C204R mutants was undetectable. The E203V mutant displayed reduced, but detectable, binding. Thus, mutants of the N finger of GATA-1 selected in the split two-hybrid system fail to associate with FOG efficiently in mammalian cells.

While the structure of the C-terminal finger of GATA-1 has been described (Omichinski et al., 1993), the structure of the N finger of GATA-1 bound to DNA has only recently been solved (Kowalski et al., 1999). The finger consists of two distorted b hairpins and a single a helix and shows substantial homology to other C4-type zincbinding motifs, including hormone receptor and LIM domains. The four mutated residues identified in the split two-hybrid screen, E203, V205, G208, and H222, lie on a single face of the finger, positioned away from the predicted DNA-binding surface (Figure 1E). The conservation of these residues in all vertebrate GATA factors, as well as in Drosophila pannier (Ramain et al., 1993), suggests that characterization of the determinants of the GATA-1:FOG association will serve as a model for potential interactions between GATA family members and their respective cofactors.

Novel GATA-1 Mutants Exhibit Normal DNA-Binding Properties Our goal in selection of GATA-1 mutants impaired for FOG interaction was to discriminate N finger functions involved in DNA-binding and protein–protein interaction.

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Since prior studies show that the N finger makes contacts with palindromic GATA-1 sites to stabilize binding (Martin and Orkin, 1990; Trainor et al., 1996), we reasoned that mutations that significantly alter its structure would bind less tightly to the palindromic element and dissociate from it rapidly. In contrast, substitutions that selectively alter a protein interaction surface should display normal dissociation kinetics. Following expression of wild-type and mutant GATA-1 proteins in COS cells, nuclear extracts were subjected to gel shift assays. A labeled oligonucleotide comprising a palindromic GATA site was incubated with nuclear extract. Unlabelled competitor oligonucleotide was added, and samples were loaded on the gel at successive times. The E203V, V205G, and H222R mutants bound normally to DNA (Figure 2A). As anticipated, the C240R mutant dissociated rapidly. The G208V mutant also failed to bind normally, as did the G208M mutant (data not shown). This poor binding can be rationalized upon inspection of the structure of the N finger (Kowalski et al., 1999), which shows that G208 adopts a positive backbone φ angle. For residues other than glycine, positive φ angles are energetically unfavorable, as a result of steric limitations imposed by the amino acid sidechain, and it would therefore be expected that these substitutions would disrupt the structure of the N finger to some extent, depending on the particular substitution. The L224P mutant also exhibited unstable DNA binding (data not shown). In this instance, the replacement of leucine with proline is predicted to alter the structure of the finger domain. Figures 2B and 2C present dissociation curves and calculated dissociation rate constants for the GATA1:DNA interactions. Wild-type GATA-1 and the E203V, V205G, and H222R mutants dissociate from DNA with similar kinetics (z2.4 3 1024 sec21), whereas the C204R and G208V mutants dissociate with .5-fold larger rate constants. These data demonstrate that three N finger GATA-1 mutants, E203V, V205G, and H222R, retain normal DNA-binding properties despite poor association with FOG.

FOG Noninteracting GATA-1 Mutants Fail to Induce Differentiation of GATA-12 Erythroid Cells To determine the extent to which the requirement for GATA-1 in erythroid development reflects concerted action of a GATA-1:FOG complex rather than independent action of GATA-1, we examined the activity of FOG noninteracting GATA-1 mutants to rescue terminal erythroid maturation of murine GATA-12 G1E cells (Weiss et al., 1997). G1E cells were infected with a retrovirus harboring wild-type or mutant GATA-1 cDNAs and assayed for erythroid differentiation. Under the conditions of retrovirus production and infection used, a small proportion of G1E cells are transduced. Expression of wild-type GATA-1 induced the appearance of hemoglobin-containing (benzidine positive) cells in z12% of cells (Figures 3A and 3B). In contrast, few, if any, positive cells were induced by the E203V, V205G, or H222R GATA-1 mutants. As expected, the C204R mutant also failed to rescue terminal maturation. Nuclear extracts generated

Figure 3. FOG Noninteracting GATA-1 Mutants Fail to Induce Differentiation of G1E Cells (A) G1E cells were infected with retrovirus expressing either wildtype GATA-1 or mutant versions. G1E cells were stained with benzidine reagent to detect the accumulation of hemoglobin. Darkly staining cells are benzidine positive (marked by white arrows). The figures are representative of three independent experiments. Original magnification, 4003. (B) Quantitative analysis of G1E differentiation in wild-type and mutant transfections. Wild type: 589 6 107 benzidine positive cells per 5000 (n 5 3); H222R 8.2 6 0.2 (n 5 3); V205G 9 6 4 (n 5 3); C204R 0 (n 5 1); E203V 15 (n 5 1); mock infected 0 (n 5 3). (C) Nuclear extracts were prepared from pools of infected G1E cells in (A) and subjected to electrophoretic mobility shift assays. The arrow indicates the position of the GATA-1:DNA complex.

from these cells displayed comparable DNA-binding activities, indicating that the mutant proteins were expressed and stable (Figure 3C; data not shown). To test GATA-1 function under conditions in which wild-type GATA-1 promotes maturation of a higher proportion of cells, we created a conditionally active form of the V205G mutant by fusing the coding region of GATA-1 to the ligand-binding domain of the estrogen receptor (ER). Previously, we demonstrated that the stably expressed wild-type GATA-1/ER fusion protein induces terminal erythroid maturation of G1E cells in an estrogen-dependent manner (Tsang et al., 1997). Three independent, stable cell lines (WT-1, -2, and -3) were created with wild-type GATA-1/ER and assayed by benzidine staining after estrogen induction. Estrogen treatment of WT-1, -2, and -3 resulted in the appearance of mature erythroid cells with a frequency correlating with the level of expressed protein expressed (Figures 4A and 4B). At the lowest level of GATA-1, z2% of the WT-1 cells became benzidine positive upon estrogen

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Figure 4. Stable Cell Lines Expressing a Conditionally Active Version of the V205G GATA-1 Mutant Fail to Induce Differentiation after Estrogen Addition (A) Benzidine staining of stably infected G1E cell lines 48 hr after estrogen addition. Three independent wild-type (top panels) and six independent mutant cell lines (bottom panels) are shown. The figures depicted are representative of two independent experiments. Original magnification, 4003. (B) Nuclear extracts were assayed by a Western blot with antibody to GATA-1.

induction. In WT-3, which expressed the highest level of protein, .50% of cells are terminally differentiated. Six independent clonal lines expressing the V205G/ER fusion protein were examined. Estrogen treatment failed to induce the formation of benzidine positive cells (Figure 4A) despite protein expression greatly exceeding that in WT-3. Thus, FOG noninteracting GATA-1 mutants that retain normal DNA-binding properties fail to promote terminal erythroid maturation. A Compensatory Mutant of FOG Capable of Interacting with Mutant GATA-1 Rescues Terminal Erythroid Maturation Multiple protein–protein interactions of the DNA-binding region of GATA-1 have been reported. Besides an interaction with FOG, the domain associates with Kruppel proteins (Sp1 and EKLF) (Merika and Orkin, 1995), LMO2 (Osada et al., 1995), and CREB-binding protein (CBP)/ p300 (Blobel et al., 1998). The functional effects of these interactions for GATA-1 function are poorly defined. Given the multifunctional nature of the GATA DNA-binding domain and the possible involvement of non-FOG cofactors, known or unknown, effects of our novel mutations on aspects of GATA-1 function unrelated to FOG interaction could not be discounted. Hence, we sought mutations of FOG that would restore interaction with a FOG noninteracting GATA-1 mutant. Coexpression of mutant GATA-1 and FOG proteins with altered interaction specificity would then permit assessment of the contribution of the GATA-1:FOG complex to GATA-1 function in erythroid differentiation. Compensatory FOG mutants were selected in yeast.

As in the split two-hybrid screen, a library of PCR-generated mutants of FOG fingers 5 and 6 was introduced into yeast cells harboring V205G GATA-1. Among 65,000 colonies, three suppresser alleles of FOG (designated 1, 4, and 5) were recovered. These supported growth on His2 media to the same extent as wild-type FOG plus GATA-1 (Figure 5A). These alleles also restored interaction with the H222R mutant but were unable to bind efficiently to E203V (data not shown). FOG mutant-5 was selected for further analysis because it contained a single substitution within finger 6, S706R. The interaction of the FOG compensatory mutant with V205G GATA-1 was confirmed by coimmunoprecipitation (Figure 5B). FOG S706R bound V205G GATA-1 at levels z20% of wild-type, while wild-type FOG exhibited no detectable binding to V205G. S706R FOG interacted with wild-type GATA-1 as efficiently as wild-type FOG, suggesting that the change within FOG finger 6 might not significantly alter its in vivo function. The S706R finger 6 mutation was transferred into fulllength FOG cDNA within a retrovirus containing a zeocin-resistance cassette. Following infection of G1E cells containing the V205G/GATA-1/ER fusion protein (clone 5), cells were selected with zeocin, and estrogen-dependent erythroid differentiation was assessed. S706R FOG promoted extensive estrogen-dependent differentiation (z10% of cells), whereas no differentiation was observed in cells infected with vector alone. Introduction of wild-type FOG provided weak rescue (z2% of cells) (Figure 5C). Cellular maturation either with wild-type or S706R FOG was estrogen-dependent and hence required functional GATA-1. Weak rescue of differentiation

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Figure 6. Northern Blot Analysis of mRNA Expression in V205G/ER versus Wild-Type GATA-1/ER Cells RNA was isolated from wild-type GATA-1/ER cells (clone 3) and from V205G/ER GATA-1 cells (clone 2) at the indicated times after treatment with estrogen. Blots were hybridized with the indicated DNA probes. A control hybridization with actin is shown in the lower right panel.

provides compelling evidence that the failure of V205G GATA-1 to direct normal cellular maturation is due to its reduced affinity for FOG, rather than an impairment in some other, as yet undefined, property of GATA-1. Thus, FOG is, indeed, an essential cofactor for GATA-1 in promoting erythroid development. Figure 5. A Compensatory FOG Mutant Rescues Differentiation of G1E Cells Harboring V205G/ER GATA-1 (A) Yeast clones expressing pairs of wild-type and mutant GATA-1 and FOG proteins were streaked to His2, 20 mM 3-AT media. (B) Coimmunoprecipitation of FOG with GATA-1 in transfected COS cells. Full-length versions of either wild-type or the V205G mutant of GATA-1 were expressed pairwise with wild-type (wt) or mutant-5 FOG (M5) and immunoprecipitated as in Figure 1D. The GATA-1 band is indicated by an arrow, while the upper band corresponds to IgG heavy chain. (C) Benzidine staining of V205G/ER stable cells infected with wildtype FOG, FOG mutant-5, or vector alone. The figures are representative of three independent experiments. Original magnification, 4003.

in cells coexpressing V205G GATA-1 and wild-type FOG was not initially anticipated but is readily attributed to low residual affinity of the GATA-1 mutant for FOG and overproduction of FOG in transduced cells. Markedly enhanced rescue by S706R FOG then reflects the greater affinity of this altered specificity mutant for V205G GATA-1. Terminal erythroid maturation in a second stable cell line (clone 1) was also restored by expression of S706R FOG. Wild-type and S706R FOG proteins were expressed at comparable levels in these experiments (data not shown). That differentiation is rescued by overexpression of either wild-type or S706R FOG

Multiple, but Not All, Target Genes in Erythroid Cells Depend on the GATA-1:FOG Complex for Proper Regulation Having established that terminal erythroid maturation is strictly dependent on the association of GATA-1 and FOG, we asssessed the extent to which this partnership is required for expression of specific targets. We performed Northern blot analysis at different time points after estrogen addition from cells expressing wild-type and V205G/ER fusion proteins. Consistent with the absence of benzidine positive cells with mutant GATA-1 protein, a- and b-globin RNAs are either repressed or poorly induced, respectively, following estrogen treatment (Figure 6). Transcripts for the anion transporter band 3 and two novel, inducible genes, HD2 and DC11 (Weiss, Shirihai, and S. H. O., unpublished data) are also absent or weakly induced in V205G/ER-containing cells. In contrast, transcripts for erythroid Kruppel-like factor (EKLF) (Bieker and Southwood, 1995) and the heme-regulated eIF-a-kinase (HRI) (Crosby et al., 1994) are substantially induced by V205G GATA-1. Moreover, FOG RNA transcripts are normally upregulated by V205G GATA-1. Finally, the expression of two genes whose expression is normally downregulated during GATA-1-induced differentiation (GATA-2 and c-myc) remains unchanged in estrogen-treated V205G G1E cells.

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Thus, rather than influencing the expression of only a small subset of genes ultimately controlled by GATA-1, loss of FOG interaction deregulates multiple targets in erythroid cells. In several instances where transcripts are induced by wild-type GATA-1, induction by a FOG noninteracting GATA-1 variant is impaired. Selected genes, such as FOG itself, EKLF, and HRI, however, are expressed in a relatively FOG-independent manner. Discussion Evidence suggests that lineage selection and differentiation of hematopoietic cells require a complex interplay between multiple regulatory proteins (Sieweke and Graf, 1998). Support for this view includes the demonstration that several cell-restricted transcription factors are individually required for the development of a single lineage, multiple factors bind to cis elements of genes expressed in a lineage-specific fashion, and these factors often physically interact with one another. Despite the numerous pairwise and higher order interactions described, assessment of their contribution to transcription control during differentiation is lacking. This general problem is addressed here by the selection and expression of modified regulatory factors altered in their interaction specificities. Our findings establish that physical association of GATA-1 with FOG is indeed required for erythroid differentiation. Altered Specificity Mutants in the Analysis of Protein Associations Our strategy relied on isolation of mutants selectively altered for a specific protein–protein interaction. While this approach has been used to study gene function in prokaryotes and nonvertebrates, it has not been applied widely in mammalian systems. On occasion, pairwise changes of charged residues of putative partners have been tested for function (Jucovic and Hartley, 1996; Tansey and Herr, 1997; Whipple et al., 1998). We selected mutants of the N finger of GATA-1 impaired for interaction with FOG. Among these, we identified those which retained normal DNA-binding properties. Three residues, E203, V205, and H222, contribute specifically to FOG binding. Substitutions in any one of these greatly reduce interaction. Moreover, mutants containing these changes were inactive in promoting terminal maturation of GATA-12 erythroid cells. We used the yeast two-hybrid system to screen for suppressor alleles of finger 6 of FOG that restored interaction with FOG noninteracting GATA-1. Three such FOG variants were identified. When coexpressed with V205G GATA-1, S706R FOG (S706R) rescued erythroid cell maturation, thereby establishing the critical role of physical interaction of GATA-1 and FOG for in vivo function. This allele, as well as the two others we isolated, very likely does not compensate directly at the site of the mutation in the GATA-1 N finger, but rather creates new contacts. The two other FOG suppressor alleles contained more complex alterations of finger 6 (data not shown). Since E203V GATA-1 was the only mutant unable to interact with S706R FOG, it seems likely that the glutamic acid that normally resides at this position

forms a salt bridge with the newly introduced arginine. In the E203V mutant, this compensatory interaction is not possible, and thus, this noninteracting phenotype cannot be reversed. Our success in using altered specificity mutants selected in yeast to dissect the contribution of FOG to GATA-1 function in erythroid gene expression and differentiation should encourage their wider use for evaluating the requirement of other pairwise associations of regulatory factors in hematopoietic (and other cellular) pathways.

The GATA:FOG Complex Superficially, the association of cell-type specific factors, such as GATA-1 and FOG, is reminiscent of other partnerships in hematopoietic development. In B-lymphoid cells, the coactivator OCA-B (Bob-1, OBF-1) interacts with POU-containing transcription factors Oct-1/Oct-2 (Gstaiger et al., 1995; Luo and Roeder, 1995; Strubin et al., 1995) at a subset of octamer sites (Cepek et al., 1996; Gstaiger et al., 1996). While both Oct-2 and its coactivator are essential for proper B cell function, the contribution of the interaction alone remains unclear (Corcoran et al., 1993; Feldhaus et al., 1993; Kim et al., 1996; Nielsen et al., 1996). In erythroid cells, a pentameric complex containing GATA-1, LMO2, SCL/tal-1, E2A, and Ldb1 can assemble on a composite GATA E box DNA element (Wadman et al., 1997). While the leukemia oncoproteins SCL/tal-1 and LMO2 are individually essential for development of the entire hematopoietic system, the contribution of physical association of LMO2 to overall SCL/tal-1 function is uncertain. In these instances, complexes assemble at a subset of sites recognized by a DNA-binding factor, such as Oct-1/2, GATA-1, or SCL/tal-1. The GATA-1:FOG complex is distinctive in two respects. First, FOG does not appear to contribute a typical activation domain (Tsang et al., 1997). It seems that the principal role of FOG may be to recruit additional, as yet unknown, nuclear proteins. Besides the two fingers of FOG (fingers 1 and 6) that individually are sufficient to mediate interaction with GATA-1, an additional seven fingers are available to facilitate other protein– protein interactions. Remarkably, the specificity of fingers 1 and 6 for GATA-1 is indistinguishable; mutants of GATA-1, such as V205G, selected for noninteraction with FOG finger 6 also do not associate with finger 1 (data not shown). Second, FOG does not appear to modulate the DNAbinding specificity of GATA-1. CASTing experiments using erythroleukemia cell extracts fail to suggest that a FOG:GATA-1 complex recognizes a subset of GATA consensus sequences (A. Tsang and S. H. O., unpublished data). If FOG’s principal function is to couple GATA-1 to the transcriptional machinery, why might two cell-restricted proteins be necessary when a single DNA-binding protein would, in principle, suffice? While appearing unnecessarily complex, this strategy may afford an additional means of regulation. Transcription could be controlled at the level of the GATA-1:FOG protein–protein interaction rather than by the binding of GATA-1 to DNA. In maturing erythroid cells, GATA-1

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might be poised at specific elements. Transcription would ensue only upon FOG interaction and recruitment of other components. This scenario is consistent with the observation that locus control regions of globin clusters exhibit DNase I hypersensitivity prior to activation of globin gene transcription (Jimenez et al., 1992). Implications of FOG-Independent Targets of GATA-1 Previously the extent to which GATA-1-dependent transcription in erythroid cells relies on the GATA-1:FOG complex was unknown. Use of altered specificity mutants discriminates between two classes of targets. The majority of downstream targets of GATA-1, represented by globins, band 3, DC11, and HD2, are not activated in the absence of FOG association. Repressed genes, such as GATA-2 or c-myc, are also not regulated normally. Nonetheless, a subset of targets, including EKLF, HRI, and FOG itself, are relatively FOG-independent. At these targets, GATA-1 acts either alone or in concert with a different cofactor(s). In the vast majority of cis elements of erythroid-expressed genes, GATA motifs are not found in a consistent relationship to other sequence motifs. We propose that the GATA-1:FOG complex is primarily employed at such sites. Rarely, GATA motifs are present in a specific orientation and distance from an E box motif, consistent with the assembly of a pentameric complex (Wadman et al., 1997). Recently it has been shown that an upstream enhancer of the EKLF gene relies on a composite GATA E box element (Anderson et al., 1998). In this setting, the E box–binding protein SCL/tal-1 and associated proteins may provide cofactor activity to GATA-1, obviating the need for FOG interaction. We speculate that GATA-1 functions in alternative complexes, one in which FOG is the cofactor, and another in which SCL/tal-1 (and associated components) fulfills this role. The precise architecture of regulatory elements of a given target gene is likely to dictate which complex is assembled in vivo. Implications for Other GATA:FOG Combinations The residues critical for FOG binding are conserved in all two-finger GATA factors. It seems likely that other vertebrate GATA factors will be found to require FOG or other FOG-like cofactors for their in vivo function. In this regard, it is of interest that FOG is also expressed in endodermal derivatives in the mouse (Tsang et al., 1997), sites of GATA-4, -5, and -6 expression. Moreover, a second FOG-like factor (Tevosian et al., 1999) is expressed in a variety of nonhematopoietic sites, where GATA factors are also coexpressed. Mutation of the N finger of GATA factors at residues critical for FOG association will provide a genetic means of testing the in vivo roles of pairwise GATA:FOG complexes in a variety of developmental contexts. Experimental Procedures Yeast Two-Hybrid Screens The split two-hybrid screen has been described (Shih et al., 1996). The N finger of murine GATA-1 (aa [amino acids] 200–248) was cloned by polymerase chain reaction (PCR) in-frame to both the activation domain of VP16 as well as b-galactosidase in pVP16 to create the wild-type GATA-1 bait. Mutagenic PCR was performed using DisplayTaq (Display Systems Biotech) including an imbalance

in nucleotide concentration (Vidal et al., 1996; Vidal, 1997). Fragments were subcloned into the parent pVP16 vector. To generate the FOG bait, residues 559–760, which includes zinc fingers 5 and 6, were fused in-frame to the DNA-binding domain of lexA in pBTM116. Plasmids were introduced into yeast strain YI671 (a gift from Anthony DiMaggio) following standard protocols (Vidal, 1997). Colonies were selected on Trp2, Leu2, and His2 synthetic drop-out media containing 10 mM 3-AT. The conventional two-hybrid system was performed with yeast strain Y190 (Clontech). GATA-1 mutant baits recovered from the split two-hybrid screen were used in conjunction with a FOG bait, in which fingers 5 and 6 were fused to the DNA-binding domain of Gal4 (plasmid pGBT9, Clontech). Transformants were taken from Leu2 Trp2 plates and streaked onto Leu2 Trp2 His2 plates with 10 mM 3-AT. Compensatory mutant screens were performed in yeast strain Y190. Mutant GATA-1 cDNA in pVP16 was cotransformed with pGBT9-FOG (described above). Residues in FOG between 677–760 were mutagenized by PCR and introduced into yeast by gap repair (Vidal, 1997). Transformants were selected on Leu2 Trp2 His2 synthetic media containing 20 mM 3-AT. Electrophoretic Mobility Shift Assays pXM GATA-1 was mutagenized using the Gene Editor kit (Promega). Mutant plasmids (10 mg) were transfected into COS cells by the DEAE-dextran method (Sambrook et al., 1989). Nuclear extracts were prepared from cells after 48 hr (Andrews and Faller, 1991) and assayed by Western blotting to confirm that extracts contained equivalent amounts of wild-type and mutant proteins. Oligonucleotides were radiolabeled and annealed to form a double-stranded, palindromic probe (Trainor et al., 1996). The mobility shift in Figure 3C was performed using published conditions (Martin and Orkin, 1990). For the dissociation assays in Figure 2, standard conditions were used, but a 100-fold excess of unlabeled DNA was added after the initial 20 min incubation. Aliquots were loaded onto an 8% polyacrylamide gel (19:1) run with 13 Tris-glycine buffer at increasing time points after the addition of competitor. To monitor the dissociation in the linear range, different sets of time points were used for different versions of GATA-1. Gel profiles were quantitated by Phosphorimager analysis. The percentage of probe bound by GATA-1 at different times after competitor addition was plotted on a log scale versus time after addition of competitor. Dissociation rate constants were determined from the slopes of the curves using the least-squares method. Northern Blot Analysis Total RNA was isolated from cells using Trizol reagent (GIBCO BRL), followed by poly A1 selection (Oligotex, Qiagen). Poly A1 RNA (2 mg/lane) was fractionated on 1% agarose-formaldehyde gels, transferred to nitrocellulose, and hybridized with various radiolabeled cDNA probes. Immunoprecipitations and Western Blot Analysis Flag-tagged FOG was made by inserting FOG residues 559–760 inframe with the Flag epitope in pEFrFLAGPGKpuropAV18 (a gift from D. Huang). Full-length GATA-1, or mutants (in pXM GATA-1), were cotransfected into COS cells with Flag-tagged FOG plasmid by the DEAE-dextran method (Sambrook et al., 1989). Nuclear extracts were prepared as described (Andrews and Faller, 1991). Immunoprecipitation was performed in binding buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 0.1% NP-40, 1 mM EDTA, 1 mM DTT, and 0.2 mM pefablock) for 2 hr at 48C with 1 ml anti-Flag antibody (Kodak). Complexes were precipitated with protein G–sepharose (Pharmacia), washed three times in binding buffer, and eluted by boiling in SDS gel loading buffer (Sambrook et al., 1989). Proteins were resolved by electrophoresis in 10% SDS-PAGE and blotted to nitrocellulose. GATA-1 was detected with a rat monoclonal GATA-1 antibody (N6; Santa Cruz Biotechnology) and HRP-conjugated secondary antibodies and developed by ECL (Amersham). Retroviral Infection of G1E Cells G1E cells were cultivated as described (Weiss et al., 1997). For transient assays, mutations in GATA-1 were introduced into the

Analysis of GATA-1:FOG Interaction 227

retroviral vector pMFG-GATA-1. A transient system (Pear et al., 1993) was used to generate viral supernatants for infection of G1E cells. Infected cells were cytospun and stained by benzidine reagent 48 hr after cocultivation (Orkin et al., 1975). Stable cell lines were generated using wild-type or V205G cDNA in the pGD-G1ER-puro construct (Daley et al., 1990; Tsang et al., 1997), in which GATA-1 cDNA is fused in-frame to the ligand-binding domain of the estrogen receptor. Two independent clones of the V205G mutant DNA were used to generate mutant cell lines. Cells were selected in liquid culture with puromycin (1 mg/ml) for seven days, and independent clones were isolated by limiting dilution. Stable cell lines were cultured in the presence of 1027 M b-estradiol and stained for hemoglobin after 48 hr. Expression of GATA-1 was monitored by Western blots. The dicistronic vector consisting of an LTR-driven FOG gene followed by an IRES-zeocin cassette in MFG (Tsang et al., 1997; Weiss et al., 1997) was mutagenized to create FOG mutant-5 by replacement of a wild-type Sac II fragment with that of pGBT9-FOG mutant-5 (obtained in the compensatory mutagenesis screen). The V205G/ER stable cells were infected with retrovirus harboring either wild-type FOG, FOG mutant-5, or the MFG IRES zeocin plasmid lacking the FOG cDNA. Infected cells were selected in media containing zeocin (90 mg/ml) for 7 days. FOG expression was confirmed by Western blot analysis. Zeocin-resistant pools were induced by addition of 1027 M b-estradiol, split after 24 hr into identical media, and assayed for hemoglobin accumulation after 72 hr. Acknowledgments We thank the laboratory for discussions, Paul Mead for manuscript review, Alice Tsang for continued input, and Richard Goodman for the split two-hybrid system. Dennis Hom contributed to the subcloning of the split two-hybrid vectors. J. D. C. is supported by a fellowship from the Jane Coffin Childs Fund for Medical Research. S. H. O. is an Investigator of the Howard Hughes Medical Institute. Partial support was provided by a grant from the NIH. Received October 21, 1998; revised November 23, 1998. References Anderson, K.P., Crable, S.C. and Lingrel, J.B. (1998). Multiple proteins binding to a GATA-E-Box-GATA motif regulate the erythroid Kruppel-like Factor (EKLF) gene. J. Biol. Chem. 273, 14347–14354. Andrews, N.C., and Faller, D.V. (1991). A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19, 2499. Bieker, J.J., and Southwood, C.M. (1995). The erythroid Kruppellike factor transactivation domain is a critical component for cellspecific inducibility of a b-globin promoter. Mol. Cell. Biol. 15, 852–860. Blobel, G.A., Nakajima, T., Eckner, R., Montminy, M., and Orkin, S.H. (1998). CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. Proc. Natl. Acad. Sci. USA 95, 2061–2066. Cepek, K.L., Chasman, D.I., and Sharp, P.A. (1996). Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B. Genes Dev. 10, 2079–2088. Corcoran, L.M., Karvelas, M., Nossal, G.J., Ye, Z.S., Jacks, T., and Baltimore, D. (1993). Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival. Genes Dev. 7, 570–582. Crosby, J.S., Lee, K., London, I.M., and Chen, J.-J. (1994). Erythroid expression of the heme-regulated eIF-2a kinase. Mol. Cell. Biol. 14, 3906–3914. Daley, G., Van Etten, R., and Baltimore, D. (1990). Induction of chronic myelogenous leukemia in mice by the p210 bcr/abl gene of the Philadelphia chromosome. Science 247, 824–830. Evans, T., and Felsenfeld, G. (1989). The erythroid-specific transcription factor eryf1: a new finger protein. Cell 58, 877–885. Feldhaus, A.L., Klug, C.A., Arvin, K.L., and Singh, H. (1993). Targeted

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