Interaction between the ZF proteins GATA-1 and FOG-1

7.3.1 Background

Interaction of the mammalian FOG-1 protein with the GATA-1 protein is a biologically important example of a protein-protein interaction mediated by zinc fingers. GATA-1 belongs to a small family of transcription factors that were originally found in erythroid cell lines (Evans and Felsenfeld 1989; Tsai et al., 1989; Trainor et al., 1990; Pevny et al., 1991).

FOG-1 is co-expressed with GATA-1 during embryonic and hematopoietic development and functions as an in vivo cofactor for GATA-1. It has been shown that the GATA-1/FOG-1 interaction is necessary for proper hematopoiesis and thrombopoiesis (reviewed in Cantor et al., 2002). The GATA-1/FOG-1 interaction is mediated by two different zinc finger motifs:

GATA-1 contains two zinc fingers of the Cys4 type (where zinc ligands are represented by four cysteines) that fold into a structure termed the treble clef motif which is composed of two irregular β-hairpins and an α-helix (e.g. Kowalski et al., 1999; Grishin, 2001; Krishna et al., 2002; Figure 7.1). While the C-terminal finger (GATA-1 CF) binds to DNA, the N-terminal finger (GATA-1 NF) mediates interaction with FOG-1 (Kowalski et al., 2002).

FOG-1 contains nine putative ZFs (F1-F9) that are of two different types: four belong to the classical C2H2 finger-type (F2, F3, F4 and F8) whereas the other five are of the C2HC type (F1, F5, F6, F7 and F9) (Figure 7.1). The sequence of the C2HC ZFs conforms well to the C2H2 ZF consensus with the exception of a cysteine replacing the final histidine (Tsang et al., 1997). Structural studies of C2HC fingers indicate that their overall fold is similar to the ββα fold of C2H2 fingers but with a more extended structure at the C-terminal end (Liew et al., 2000). Different reports suggest that C2HC fingers do not bind to DNA but instead mediate interactions with other proteins (Matthews et al., 2000).

It has previously been shown that the GATA-1 NF can interact with the C2HC fingers F1, F6, or F9 of FOG-1(Fox et al. 1999). By contrast, GATA-1 CF fails to bind to the FOG-1 zinc fingers. Interestingly, FOG-1 fingers that are involved in protein contact are exclusively of the C2HC type, whereas the four C2H2 FOG-1 fingers are unable to mediate protein-protein interactions with GATA-1. Using the Y2H system and alanine scanning mutagenesis, several residues important for this interaction were defined for both GATA-1 and FOG-1 (Fox et al., 1999; Fox et al., 1998). Although these amino acid residues were identified, additional study

and definition of the protein interface is still required to define the specific amino acid-amino acid interactions that occur at the interaction interface. Thus, we wished to determine whether this interaction can also be detected in the B2H system to further analyze and characterize the interaction surface.

7.3.2 Validation of the B2H system for studying GATA/FOG interactions

7.3.2.1 GATA-1 and FOG-1 interactions can be detected in the B2H system

To test whether GATA/FOG interactions can be detected in the B2H system a plasmid encoding a GATA-1/DBD hybrid protein consisting of the N-terminal zinc finger of GATA-1 (residues 200-254) was constructed. The DBD protein in this hybrid protein is the λcI protein.

Additionally, three plasmids encoding hybrid proteins consisting of either FOG-1 zinc finger 1 (residues 241-295), 6 (residues 677-760), or 9 (residues 945-995) fused to the α-subunit of the E. coliRNA-polymerase were created. All of these zinc fingers have been shown to be involved in protein-protein contact between GATA-1 and FOG-1 in yeast (see section 7.3.1).

Combinations of plasmids expressing these hybrid proteins were then introduced into a reporter strain bearing the λcI binding site positioned upstream of the lacZ gene and β-galactosidase activity was measured. None of the FOG-1 fingers was able to mediate transcriptional activation of lacZ when co-expressed with the GATA-1 fusion protein (data not shown). To vary the expression level of the fusion proteins, different concentrations of IPTG were used to induce the expression of the constructs in the reporter cells. It was found that IPTG concentrations above 30 μM inhibited the growth of the bacteria and the optical densities of these cells were extremely low even after 6 hr of growth. This suggested that high levels of the fusion proteins might be toxic to the bacterial strains. Experience with the B2H system has demonstrated that certain λcI-hybrid proteins can be toxic to bacterial strains (K. Joung, personal communication).

Therefore we decided to construct an additional plasmid encoding GATA-1 fused to the DNA binding domain of the Zif268 protein (see Chapter 3, section 3.2). Combinations of the α-fusion proteins together with this new plasmid were then introduced into a reporter strain containing the binding site for the Zif268 protein and β-galactosidase activity was assessed.

As shown in Figure 7.2A two of the FOG-1 fingers (F1 and F6) mediated transcriptional activation of lacZ when co-expressed with the GATA-1 NF fusion protein. The strongest

activation (4-fold) was observed with GATA-1 NF and FOG-1 F1 (Figure 7.2A). Control experiments expressing either the Zif-hybrid or the α-hybrid protein alone did not show increased β-galactosidase expression (data not shown).

To further test if activation of lacZ is depended on the expression of the two fusion proteins, we performed an IPTG titration experiment where the expression of the fusion proteins was induced using increasing concentrations of IPTG. Increased lacZ expression only occurred when the amount of IPTG used for induction reached a certain level (>25 μM) indicating that the activation depended on expression of both fusion proteins (Figure 7.2B).

Since activation of lacZ depends on the distance and orientation of the DBD-binding site relative to the promoter, the interaction between GATA-1 NF and FOG-1 F1 was further analyzed using various Zif268 binding site reporter strains. As shown in Figure 7.2C, the strongest activation was observed with reporter strains bearing the Zif268 binding site at position -61 and -65 relative to the promoter of the reporter gene. Further decreasing or increasing of the distance relative to the promoter did abolish the ability of GATA-1 and FOG1 to activate lacZ (Figure 7.2C and data not shown). Interestingly, the binding site at -61 was positioned such that the C-terminal part of the Zif268 fusion protein is most proximal to the promoter while the orientation of the -65 binding site brought the N-terminal part of the Zif268 fusion protein close to the promoter. Although different from each other, both orientations seemed to provide the essential geometry for the GATA-1-Zif268 fusion protein to mediate interaction with FOG-1 (Figure 7.2C and data not shown).

7.3.2.2 Mutations in GATA-1 and FOG-1 disrupted the interactions

To obtain additional evidence that the transcriptional activation detected in the B2H system reflects the physiological GATA-1/FOG-1 interaction, various GATA-1 NF and FOG-1 F1 mutations known to disrupt their interaction (Fox et al., 1998; 1999) were introduced into our hybrid proteins using site-directed PCR mutagenesis (as described in section 2.1.2.2). We tested whether these mutations would affect the ability of GATA-1 NF and FOG-1 F1 to mediate transcriptional activation of the lacZ reporter gene. Indeed, substitution of FOG residue I262 with alanine resulted in a strong inhibition of the interaction with wild type GATA-1 whereas replacement of R265A had a weaker impact on binding (Figure 7.2D). The same differential effects were found when these mutants were tested in the Y2H system (Fox et al., 1999). Similarly, substitution of the GATA-1 residues E203 with valine and V205 with threonine also disrupted the interaction just as they did in the yeast-based system, with the

mutation at residue position E203 displaying a stronger impact on interaction then the mutation at residue position 205 (Figure 7.2D, Fox et al., 1998).

Taken together, these results suggest that interactions between GATA-1 NF and fingers from FOG-1 interaction can mediate transcriptional activation in the B2H system. Importantly, these experiments further establish the feasibility of using the B2H system for studying protein-protein interactions mediated by different types of ZF proteins.

0 fold-activation of β- galactosidase expression

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fold-activation of β- galactosidase expression

-61 -63 -65 -67 -69 -71 fold-activation of β- galactosidase expression

0 fold-activation of β- galactosidase expression

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fold-activation of β- galactosidase expression

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fold-activation of β- galactosidase expression

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Figure 7.2 Analysis of the interaction between GATA-1 and FOG-1 in the B2H system. B2H reporter strains expressing pairwise combinations of plasmids encoding domains from GATA-1 and FOG-1 were assayed for β-galactosidase activity. In these assays GATA-1 NF was fused to the DBD of Zif268 while ZFs 1, 6 and 9 from FOG-1 were connected to the α-subunit of the RNAP. If not mentioned otherwise an IPTG concentration of 50 μM was used. (A) The reporter strain harboring the Zif268 DBS at position -61 in orientation 123 relative to the promoter was transformed with combinations of plasmids and β-galactosidase assays were performed. A control expressing Zif268 and the α-subunit alone is shown to the right. (B) β-galactosidase activities of -61 reporter cultures grown at various IPTG concentrations were measured to analyze the effect of different hybrid-protein expression levels. Fold activation was normalized to cells expressing only Zif268 and the α-subunit. (C) The interaction between GATA-1 NF and FOG-1 F1 was further analyzed using reporter strains that differ in their location and orientation of the Zif268 DBS relative to the promoter. Position of the binding site is indicated on the X-axis. Purple bars represent reporter strains with the Zif268 binding site in orientation 123 and blue bars indicate orientation 321. (D) Specific mutations in FOG-F1 (m1=I262A, m2=R265A) and in GATA-1 (m1= E203V, m2= V205T) were analyzed for their ability to abolish the interaction between GATA-1 NF and FOG-1 F1. In this assay, the reporter strain harboring the Zif268 DBS at position -65 in orientation 321 relative to the promoter was used.