©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
GATA-3 Dominant Negative Mutant
FUNCTIONAL REDUNDANCY OF THE T CELL RECEPTOR alpha AND beta ENHANCERS (*)

(Received for publication, August 22, 1994; and in revised form, October 21, 1994 )

Virginia M. Smith Perry P. Lee Shannan Szychowski Astar Winoto (§)

From the Department of Molecular and Cell Biology, Division of Immunology and Cancer Research Laboratory, University of California, Berkeley, California 94720-3200

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The GATA family of transcription factors regulates a wide variety of genes, including those involved in differentiation of erythrocytes and T lymphocytes. We report here the creation of a dominant negative mutant of GATA-3, KRR, which effectively blocks wild-type GATA-1, GATA-2, and GATA-3 transactivation when co-expressed in transient assays. KRR was generated by site-directed mutagenesis while investigating a putative activation domain of GATA-3, located between its two zinc fingers. The GATA-3 KRR mutation does not affect expression, nuclear translocation, or the ability to bind to a consensus GATA sequence. KRR can suppress the activity of the minimal T cell receptor (TCR) alpha and beta enhancers by 12- and 3.4-fold, respectively. However, KRR did not have a significant effect on the activity of larger TCR-alpha and -beta enhancer fragments. Thus, functional redundancy in the TCR-alpha and -beta enhancers can compensate for the loss of GATA-3 activity.


INTRODUCTION

GATA-3 is the third member of a family of transcription factors that contain two zinc fingers of the sequence CX(2)CXCX(2)C and recognize the core consensus sequence (A/T)GATA(A/G) (for review, see Orkin(1992)). The best characterized member of the family is GATA-1, the primary regulator of erythoid-cell specific gene expression (Evans and Felsenfeld, 1989; Tsai et al., 1989; Whitelaw et al., 1990). It binds to and regulates several erythroid-specific genes, including the alpha- and beta-globin genes. The GATA-1 DNA binding site can be found not only in the promoters and enhancers but also in the locus control regions of the globin genes. GATA-2 functions in the erythrocyte lineage by promoting proliferation and maintaining an immature state of early erythrocytes; it may also play a role in the development of the nervous system (Yamamoto et al., 1990; Lee et al., 1991). GATA-4 is present in heart, intestinal epithelium and primitive endoderm, and gonads (Arceci et al., 1993).

GATA-3 is expressed in embryonic brain, kidney, early stage erythrocytes, and early in the development of T cells (Yamamoto et al., 1990; Ho et al., 1991; Joulin et al., 1991; Ko et al., 1991; Marine and Winoto, 1991; Orkin, 1992). GATA-3 can be found in the thymus at day 12.5 of murine fetal development, as early as T cells can be detected in the thymus and before the expression of the T cell receptor on the surface of T cells (Oosterwegel et al., 1992). The expression of a functional T cell receptor (TCR) (^1)is critical for differentiation and maturation of T cells with the appropriate expression controlled by their respective enhancers (Krimpenfort et al., 1988; McDougall et al., 1988; Ho et al., 1989; Winoto and Baltimore, 1989; Bories et al., 1990; Gottschalk and Leiden, 1990; Redondo et al., 1990; Kappes et al., 1991; Spencer et al., 1991). Binding sites for the GATA-3 protein have been identified in the TCR-alpha, -beta, and - enhancers (Gill et al., 1991; Ho et al., 1991; Joulin et al., 1991; Ko et al., 1991; Marine and Winoto, 1991; Leiden, 1993), as well as in the promoter and enhancer to the CD8alpha gene (Hambor et al., 1993; Landry et al., 1993), CD4 enhancer (Wurster et al., 1994), and in the interferon- promoter (Penix et al., 1993). The GATA site from the human TCR-beta enhancer, Tbeta2, can act as a T cell-specific enhancer when multimerized in T cells (Marine and Winoto, 1991). The importance of GATA-3 in regulating T cell receptor expression in the context of the entire gene, however, is not entirely clear.

To address the function of the GATA-3 protein in TCR gene regulation and ultimately in T cell development, we have identified a GATA-3 dominant negative mutant. While none of the N- or C-terminal GATA-3 deletions was able to suppress wild-type GATA-3 function, a site-directed mutant in a putative transactivation domain of GATA-3 between the two zinc fingers can act as a strong dominant negative mutant. In addition, the GATA-3 dominant negative mutant can also dramatically suppress activation by both GATA-1 and GATA-2. This mutant KRR, amino acids 305-307 changed from KRR to alanine, is transcriptionally inactive but is able to bind to the GATA consensus site and can translocate to the nucleus. KRR overexpression can suppress the activity of the minimal TCR-alpha and -beta enhancers, but has only a small effect on larger enhancer-containing fragments. Thus, redundancy in the TCR-alpha and -beta enhancers can compensate for the loss of GATA-3 function.


EXPERIMENTAL PROCEDURES

Plasmids for Transactivation

The reporter construct J214E4 contains four copies of the GATA-3 site from the human T cell receptor enhancer in the J21 vector (c-fos -71 to +109 promoter driving a CAT gene) and a transcription terminator placed in front of the GATA-3 sites. The plasmid was created in several steps. First, two copies of the oligonucleotides containing E4 of the T cell receptor enhancer (Marine and Winoto, 1991) were inserted into the SalI site of J21 (J21-E4-2A). The SV40 trancriptional terminator from p1642 plasmid (Kingsley and Winoto, 1992) was then placed into the AccI site of the J21-E4-2A. A clone, which contains the SalI site from the polylinker placed 3` of the terminator was selected (J21-E4T-B). Two more copies of the E4 oligonucleotides were inserted into the SalI site of J21-E4T-B to yield the final J214E4 construct. pG8 was created by inserting the 600 bp HaeIII fragment of the Valpha11.1 promoter (from G8, S. Hedrick) into the SmaI site of pCAT3`L. pG8 0.2alpha was created by inserting the 235-bp BglII/PvuII fragment containing the murine alpha promoter into the EcoRV site of pG8. pG8 0.5alpha was created by inserting the 0.5-kb PvuII fragment containing the murine alpha enhancer into the EcoRV site of pG8. pG8 1.6beta was created by inserting the 1.6-kb BglII fragment containing the murine beta enhancer into the EcoRV site of pG8. pG8 0.8beta was created by inserting the 0.8-kb PvuII/NcoI fragment containing the murine beta enhancer into the EcoRV site of pG8. pcGATA-3 was created by inserting the 2.6-kb EcoRI fragment from pSP72 R1A containing a full-length human GATA-3 cDNA (Marine and Winoto, 1991) into the EcoRI site of pcDNA-1 (Invitrogen). pcGATA-1 was created by inserting the 1.6-kb EcoRI fragment containing the human GATA-1 cDNA (a generous gift of Dr. G. Felsenfeld) into the EcoRI site of pcDNA-1. pCDGATA-2, which contains a 3.3-kb human GATA-2 cDNA in the vector pCDM8 that is the parent vector of pcDNA-1, was a generous gift of Dr. Mu-En Lee. pc-DeltaC2 was created by inserting the 1.6-kb EcoRI/MaeI fragment from pSP72 R1A into the EcoRV site of pcDNA-1. pE-DeltaH was created by inserting the 1.6-kb HincII/EcoRI fragment of pSP72 R1A into the SmaI site of pEVRF2. pE-DeltaN was created by inserting the 1.4-kb StyI/EcoRI fragment of pSP72 R1A into the SmaI site of pEVRF2. For transactivation of the GATA-3 site-directed mutants, mutations were introduced in mp18-GATA-3 (see below). The EcoRI fragment was then transferred into pcDNA-1. Mutations were confirmed by DNA sequencing.

Transfections and CAT Assays

Transfections were performed using the DEAE/chloroquine method. CAT assays were performed as described previously using equal amount of protein extracts (Marine and Winoto, 1991). All transfections were repeated at least two times with equal molar and equal weight of DNA. The ratios of plasmid DNA transfected represents a comparison of molar equivalents used. One molar equivalent represents the transfection in µg of the size of the plasmid in kilobases. In this way, an equal number of each plasmid is transfected. The DNA for each sample was adjusted to an equal amount using sheared calf thymus DNA. Error bars were computed using Microsoft's Excel program.

Gel Shift Analysis

Gel shift analysis using the E4 oligonucleotide (containing the GATA site from the human T cell receptor enhancer (Redondo et al., 1990)) or the Tbeta2 oligonucleotide (containing the GATA site from the human T cell receptor beta enhancer, Gottschalk and Leiden, 1990) was performed as described previously (Marine and Winoto, 1991). Equal amount of nuclear extracts from 293 cells transfected with GATA-3 or GATA-3 mutant expressing plasmids were used.

Antibody Production

New Zealand white rabbits were injected subcutaneously every other week for a total of five injections with glutathione-agarose beads coated with approximately 50 µg of glutathione S-transferase-GATA-3. In the first injection, the antigen was mixed 1:1 with Freund's complete adjuvant (Sigma) in a total volume of 1 ml. For all subsequent injections, the antigen was mixed 1:1 with Freund's incomplete adjuvant (Sigma) in a total volume of 1 ml. The rabbits were bled prior to the first (preimmune sera), third, fourth, and fifth injection. The sera were tested for GATA-3 reactivity on a gel mobility shift assay using Tbeta2 oligonucleotide and either Molt13 or BJA-B nuclear extract. Two weeks after the fifth injection, the rabbits were terminally bled, and the sera were collected. A similar injection schedule was followed for injection of CD-1 mice.

Sequencing

Sequencing was performed using Sequenase version 2.0 DNA sequencing kit as per the manufacturer's instructions. The reactions were subsequently run on a 5% acrylamide, 8 M urea gel, fixed for 10 min with 5% methanol, 5% acetic acid, dried down, and exposed to Kodak XAR film. For sequencing the site-directed mutants of GATA-3, the following primer was used: 5`-GGTCTGACAGTTCGCACA-3`.

Site-directed Mutagenesis

Site-directed mutagenesis was performed using the oligonucleotide-directed in vitro mutagenesis system version 2.1 (Amersham Corp.) as per the manufacturer's instructions. A 2.6-kb EcoRI fragment containing the human GATA-3 cDNA from pSP72 R1A was subcloned into the EcoRI site of M13mp18. Mutants in M13mp18-GATA-3 were confirmed by sequencing. The site-directed GATA-3 mutants were subcloned into the pcDNA-1 by excising the 2.6-kb EcoRI GATA-3 containing fragment from M13mp18-GATA-3 mutant into the EcoRI site of pcDNA-1. The site-directed mutations in pcDNA-1 were re-confirmed after subcloning by sequencing. The following oligonucleotides were used to generate the following mutants: KRR, 5`-GGCTGCAGACAGCGCTGCCGCGGGCTTAATGAG-3`; YHKM, 5`CGGTTCTGTCCGTTCGCTGCGGCAGCGAGCCCGCAGGCGTT-3`; NGQN, 5`-TTAATGAGGGGCCGGGCCGCTGCGGCCATTTTGTGATAGAG-3`; RPLI, 5`-CTTCGCTTGGGCTTAGCGGCGGCCGCGTTCTGTCCGTTCAT-3`.


RESULTS

Truncated GATA-3 Proteins Are Not Dominant Negative Mutants

To investigate the role of GATA-3 in TCR gene regulation, we first sought to identify a GATA-3 dominant negative mutant. We reasoned that a nonfunctional, truncated GATA-3 protein may bind to the cognate DNA sequence, and prevent the wild-type GATA-3 protein from binding. Several truncated proteins were made with deletions either N- or C-terminal to the zinc finger DNA binding domain (shown schematically in Fig. 1). These truncations do not affect protein expression or recognition of a consensus GATA site (data not shown). To examine the transactivation capabilities of these mutants, transient transfection experiments were performed in 293 cells. The reporter construct used, J214E4, contains the CAT reporter gene under the control of a minimal c-fos promoter (-71 to +109) and four GATA-3 DNA binding sites from the TCR- enhancer (Redondo et al., 1990). The GATA-3 mutants were expressed from the cytomegalovirus promoter in either the expression vector pcDNA-1 (Invitrogen) or in the pEVRF plasmid which contains additionally the translational initiation site from the hamster sarcoma virus thymidine kinase gene. As shown in Fig. 1, the smallest N-terminal deletion, DeltaN, had only background transactivation activity, whereas DeltaH, which has some of its N-terminal intact, had 14-fold less transactivation activity compared to its wild-type counterpart. The C-terminal deletion DeltaC2 had 10-fold less transactivation activity. To test for their ability to shut off the wild-type GATA-3 function, we performed triple transient transfection experiments using the J214E4 reporter, wild-type GATA-3, and the various GATA-3 deletions. Equal numbers of GATA-3 and each GATA-3 mutant plasmid were transfected. The results of such experiments are shown in Fig. 2. None of the deletions, when co-expressed, was able to shut the activity of the wild-type GATA-3. Hence, despite their proper expression and DNA binding, none of the GATA-3 truncated proteins acted as a dominant negative mutant.


Figure 1: GATA-3 deletions N- and C-terminal to the zinc fingers have a greatly decreased transactivation ability. Expression constructs containing wild-type GATA or various deletions were transfected into 293 cells and tested for transactivation using the J214E4 reporter construct, containing four copies of the functional GATA site from the TCR- enhancer upstream of the CAT gene and c-fos minimal promoter. One molar equivalent of each expression construct was used; 1 molar equivalent represents the transfection in micrograms of the size of the plasmid in kilobases. In this way, an equal number of each plasmid is transfected. Total DNA transfected was standardized with sheared salmon sperm DNA. Approximately 48 h after transfection, cell extracts were prepared and tested for CAT activity. Relative CAT activity as compared to GATA-3 (=100) is shown; these numbers represent the average of at least two independent experiments. The two solid boxes represent the two zinc fingers of GATA-3.




Figure 2: None of the GATA-3 truncated proteins functions as a dominant negative. Triple transfections were performed in 293 cells using 3 µg of J214E4 reporter, 1/2 molar equivalent of GATA-3, and 1/2 molar equivalent of vector control or GATA-3 mutant as described in the legend for Fig. 1. The plasmids transfected in each sample are listed on the left of the CAT assay. Shown is a representative CAT assay demonstrating the lack of dominant negative activity in the GATA-3 deletions.



In chicken GATA-1, the region between the two GATA-1 zinc fingers is also a transactivation domain (Yang and Evans, 1992). As the region between the GATA-3 fingers is highly conserved to that of GATA-1, it might also be functionally important. In further search for a dominant negative mutant, we performed mutagenesis in this region to see if mutation here will affect the GATA-3 function and hence can serve as a putative dominant negative mutation. The following amino acids in the region between the two zinc fingers were mutated individually to alanines: YHKM (amino acid 291-294), NGQN(295-298), RPLI(299-303), and KRR(305-307). The mutants were then introduced into pc-GATA-3 expression plasmids and tested first for their abilities to transactivate a reporter gene under the GATA-3 control (J214E4). All the mutants exhibited decreased transactivation ability (Fig. 3). In comparison to the wild-type GATA-3, which has a 45-fold transactivation activity (compare pc-GATA-3 to pcDNA-1 vector alone), mutations at YHKM, NGQN, or RPLI all resulted in a 6.7-10-fold decrease of transactivation. Mutation at KRR was the most dramatic of all, resulting in complete inactivation of the GATA-3 protein (Fig. 3).


Figure 3: Mutations between the zinc fingers affect GATA-3 transactivation. The site-directed mutant GATA-3 cDNAs, shown schematically, were subcloned into pcDNA-1 and were tested for transactivation in HeLa cells using the J214E4 reporter construct as described in the legend for Fig. 1. Relative CAT activity as compared to GATA-3 (=100) is shown; these numbers represent the average of at least two independent experiments.



DNA Binding and Nuclear Translocation Activities of the Site-directed GATA-3 Mutants

The decreased transactivation of the above site-directed GATA-3 mutants could be due to an inability to bind DNA or an inability to translocate to the nucleus. To explore these possibilities, we transfected 293 cells with the wild-type or mutant GATA-3 expression plasmids and tested the nuclear extracts for the presence of GATA-3 DNA binding activity by gel shift analysis. An oligonucleotide containing the GATA-3 DNA binding site from the TCR beta enhancer, Tbeta2, was used (Fig. 4). Since there are nonspecific bands co-migrating with the GATA-3bulletTbeta2 complex, mouse antisera against GATA-3 was also added to the samples. After antisera addition, the GATA-3bulletTbeta2 complex was supershifted from the extract transfected with wild-type GATA-3 (Fig. 4, lane 5, arrow), but not with vector pcDNA-1 alone (lane 3). This specific supershifted complex is seen for each mutant as well, albeit lower for the RPLI mutant (lanes 7, 9, 11, and 13). In addition, these mutants expressed as fusion proteins in Escherichia coli could bind to the Tbeta2 or E4 oligonucleotide probe as well as the wild-type GATA-3 protein (data not shown). Thus, a GATA-3 protein with mutations in between the zinc fingers can still translocate to the nucleus and bind to its cognate element.


Figure 4: Mutations between the zinc fingers do not affect recognition of a consensus GATA site. The human kidney cell line 293 cells were transfected with equal molar equivalents of either pc-GATA-3 or the pcKRR. Approximately 48 h after transfection, the cells were harvested and nuclear extracts were made. The extracts were tested in a gel shift assay using an oligonucleotide containing the GATA element. In addition, each nuclear extract tested had either 1 µl of preimmune or immune mouse sera against GATA-3. Upon addition of immune sera, a supershifted complex of GATA-3 bound to the DNA probe is seen in samples transfected with wild-type (lane 5) or mutant GATA-3 (lanes 7, 9, 11, and 13), but not when the expression vector pcDNA-1 was transfected (lane 3). An arrow marks the supershifted GATA-3/DNA complex.



KRR Is a Dominant Negative Mutant of GATA-3 Which Can Inhibit GATA Family Function

As mutation at KRR(305-307) resulted in complete inactivation of the GATA-3 protein but can still bind DNA and translocate to the nucleus, we tested its ability to act as a dominant negative mutant. Triple transient transfections as described above were performed in 293 cells with the KRR mutant (J214E4, pcGATA-3, and pcKRR). Different ratios of mutant to wild-type GATA-3 were used and the results of several independent transfections are shown in Fig. 5A. The addition of pcDNA-1 vector alone did have some affect on GATA-3 transactivation that was most likely due to competition between pcDNA-1 and pcGATA-3 for the same factors required for transcriptional expression. The co-transfection of KRR with the wild-type GATA-3 drastically affected the ability of GATA-3 to transactivate a reporter. At a 1:1 ratio (GATA-3 to KRR), transactivation by GATA-3 was decreased by 10-fold. Even at a 1:1/2 and 1:1/4 ratio, transactivation by GATA-3 was decreased by 4- and 2-fold, respectively. Thus, KRR can act very effectively as a dominant negative mutant. In addition, the ability of the KRR dominant negative to inhibit the activity of other GATA family was examined. As shown in Fig. 5, B and C, the KRR dominant negative mutant can effectively inhibit the activity of both GATA-1 and GATA-2, respectively. At a 1:1 ratio, the KRR dominant negative can inhibit GATA-1 transactivation by 4-fold and GATA-2 transactivation by 10-fold. Therefore, the KRR dominant negative can generally suppress activity of GATA family members.


Figure 5: KRR can function as a dominant negative to suppress GATA family function. A, KRR can inhibit the activity of wild-type GATA-3. 293 cells were transfected with the reporter J214E4, pcGATA-3, pcDNA-1, or pcKRR as described in Fig. 2. The plasmids transfected in each sample is shown in the table below the plot. One-half molar equivalent of pcGATA-3 was used (as described in Fig. 1). The numbers in the table represent the ratio of each plasmid transfected. Within each replicate, CAT activity was standardized to the transactivation of wild-type GATA-3 alone (=100). The results represent the average of four independent transfections. B, KRR can inhibit the activity of GATA-1. 293 cells were transfected with the reporter J214E4, pcGATA-1, pcDNA-1, or pcKRR as described in Fig. 2. The plasmids transfected in each sample are shown in the table below the plot. One-half molar equivalent of pcGATA-1 was used. The numbers in the table represent the ratio of each plasmid transfected. Within each replicate, CAT activity was standardized to the transactivation of GATA-1 alone (=100). The results represent the average of three independent transfections. C, KRR can inhibit the activity of GATA-2. 293 cells were transfected with the reporter J214E4, pCDGATA-2, pcDNA-1, or pcKRR as described in Fig. 2. The plasmids transfected in each sample is shown in the table below the plot. One-half molar equivalent of pCDGATA-2 was used. GATA-2 is expressed from the pCDM8 expression plasmid, which is the parent vector of pcDNA-1 and is equivalent in expression to pcDNA-1. The numbers in the table represent the ratio of each plasmid transfected. Within each replicate, CAT activity was standardized to the transactivation of GATA-2 alone (=100). The results represent the average of three independent transfections.



KRR Does Not Significantly Affect the Activity of the TCR-alpha and -beta Enhancers

Utilizing the KRR dominant negative, we were able to assess the contribution of GATA-3 to the function of the T cell receptor alpha and beta enhancers. Either pcDNA-1 or pcKRR were transiently co-transfected into TAg Jurkat cells (human alphabeta T cell line containing endogenous GATA-3 which has the SV40 large T antigen stably transfected, a generous gift of Dr. Gerald Crabtree) along with an enhancer construct. As a control, an enhancerless reporter construct with the Valpha11 promoter alone was used (pG8). The following enhancers were tested: the minimal 0.2-kb TCR-alpha enhancer (as originally defined in Winoto and Baltimore(1989)), minimal 0.8-kb TCR-beta enhancer (as originally defined in McDougall et al.(1988)), larger 0.5-kb TCR-alpha enhancer or larger 1.6-kb TCR-beta enhancer. Each enhancer construct was co-transfected with either one or two molar equivalents of either pcDNA-1 or pcKRR. No wild-type GATA-3 was transfected, since TAg Jurkat cells already endogenously express GATA-3. The relative amount of overexpressed KRR dominant negative to endogenous GATA-3 should be great, since the KRR dominant negative expression construct has an SV40 origin and the TAg Jurkat cell line used in these experiments has the SV40 large T antigen stably transfected. As shown in Fig. 6, the KRR mutant had no effect on the enhancerless vector pG8 alone (columns 1-5). The addition of KRR dramatically decreased the minimal 0.2-kb alpha enhancer activity (columns 6-10), by approximately 12-fold when 2 molar equivalents of pcKRR was co-transfected. However, pcKRR decreased activity of a larger 0.5-kb fragment containing the TCR-alpha enhancer (columns 11-15) by less than 2-fold. Similarly, KRR overexpression decreased activity from the minimal TCR-beta enhancer by 3.4-fold (column 16-20), but by less than 2-fold when a larger enhancer fragment was used (columns 21-25). Therefore, although loss of GATA-3 function dramatically decreases the activity of the minimal alpha and beta enhancers, it has no appreciable effect on the larger enhancer fragments, demonstrating redundancy in TCR-alpha and -beta enhancer function.


Figure 6: Effect of KRR overexpression on TCR-alpha and -beta enhancer function. TAg Jurkat human alphabeta T cells were transfected with either 3 µg of the enhancer reporter construct alone, or with either the vector control pcDNA-1 or the dominant negative expressing plasmid pcKRR as described in Fig. 1. The plasmids transfected in each sample is shown in a table below the plot. The numbers represent the amount in molar equivalents of either pcKRR or pcDNA-1 transfected. Within each replicate, CAT activity was standardized to the the expression from pG8 1.6beta alone (=100). The results represent the average of three independent experiments.




DISCUSSION

We report that GATA-3, similar to chicken GATA-1 (Yang and Evans, 1992), has regions important for its function within its zinc finger linker region. This is in addition to the transactivation domains located N-terminal and C-terminal to the zinc fingers (Yang et al., 1994; this paper). Four different mutations were created in the linker region between the two zinc fingers of GATA-3. All of the mutations were dramatically affected in their ability to transactivate a reporter construct, from 1 to 15% of wild-type without affecting the ability to bind to a consensus GATA sequence. This is consistent with the findings from chicken and mouse GATA-1 that only the C-terminal zinc finger is required for binding to a consensus GATA sequence (Martin and Orkin, 1990; Yang and Evans, 1992; Merika and Orkin, 1993; Whyatt et al., 1993). One of the mutations in this newly described transactivation domain located between the two zinc fingers, KRR (for a substitution of KRR for AAA), creates a dominant negative mutant of GATA-3. When co-transfected with wild-type GATA-3, it strongly inhibits not only the ability of GATA-3 but also the ability of GATA-1 and GATA-2 to transactivate a reporter construct. The mutation in KRR does not affect expression, its ability to recognize a consensus GATA sequence or translocate to the nucleus. Yang et al.(1994) created specific deletions of two basic regions of human GATA-3 in an attempt to study the nuclear localization signal. One of their mutations, construct 21, deleted amino acids 249-258 and 303-311, which include the KRR sequence from amino acids 305-307. Our results confirm that this basic stretch of amino acids is not responsible for nuclear translocation. Surprisingly, their construct 21 had 93% transactivation ability as compared to the wild-type GATA-3. It would appear that the deletion of these residues does not have as deleterious an effect as a substitution to alanine. Perhaps these functional differences between our KRR and their construct 21 could be due to conformation changes in GATA-3 which may affect the position of GATA-3, or proteins with associate with GATA-3, relative to RNA polymerase to affect transcription. The double deletions of construct 21 therefore may retain an appropriate conformation to maintain near wild-type levels of transactivation.

The KRR mutant could decrease transcription from the minimal 0.2-kb alpha enhancer approximately 12-fold. The activity of the larger alpha enhancer-containing fragment, however, is not significantly affected by KRR overexpression. Apparently, the larger alpha enhancer contains additional activity which can compensate for the loss of GATA-3 function. Similarly, KRR overexpression had a much greater effect on the minimal TCR-beta enhancer than on the larger TCR-beta-containing fragment, 3.4-fold and less than 2-fold, respectively. Therefore, lack of GATA-3 function alone most likely will not lead to a significant loss of TCR-alpha and -beta transcriptional function. The effect of the KRR mutant on TCR- function could not be assessed as the murine TCR- enhancer is extremely weak (Gill et al., 1991). We could not detect any transcriptional enhancement to the pG8 vector by the addition of the TCR- enhancer (data not shown). The effect on the minimal TCR-beta enhancer function is in agreement with the results from Henderson et al.(1994). Mutation of the TE4 GATA site in the minimal TCR-beta enhancer resulted in decreasing transcriptional activity decreased by 85%. Overexpression of KRR, eliminating any contribution to enhancer function from GATA-3, decreased activity from the same minimal enhancer fragment by 70%.

The ability of an enhancer to compensate for the lack of one transcription factor binding has been demonstrated in the human TCR-alpha enhancer and the mouse intronic heavy chain enhancer (Ho and Leiden, 1990; Libermann et al., 1990; Nelsen et al., 1990; Staudt and Lenardo, 1991). Within the minimal human alpha enhancer fragment, loss of either the CRE or LEF-1 sites dramatically decreases enhancer activity (Ho and Leiden, 1990). However, within the context of a larger enhancer fragment, enhancer activity remains after mutation of either the CRE or LEF-1 sites. The presence of additional elements are able to compensate for the loss of CRE or LEF-1 sites. Similar redundancy is also present within the immunoglobulin intronic heavy chain (IgH) enhancer. Mutation of either the µB (Ets) site or the OCTA site alone has a small effect on total enhancer function. However, when both sites are mutated, the effect on total enhancer function is dramatic (Libermann et al., 1990; Nelsen et al., 1990; Staudt and Lenardo, 1991). Therefore, a mutation at one site could be compensated for by the second; redundancy within enhancers appears to be a common phenomenon. GATA-3 redundancy is also seen in the CD4 and CD8alpha enhancers (Hambor et al., 1993; Wurster et al., 1994), where in the absence of GATA-3 binding sites the enhancer retain at least 50% (CD8alpha) or full (CD4) activity.

The dominant negative GATA-3 identified here should be very useful for examining the role of GATA-3 in T cell-specific gene expression and function. Our attempts to generate stable T cell lines (Jurkat, Molt13, and CEM) with the KRR dominant negative has been unsuccessful, although control vectors without the KRR dominant negative were stably transfected successfully. Either the GATA-3 dominant negative protein is toxic, or the wild-type GATA-3 activity is critical to T cells. Contribution of GATA-3 to gene expression and T cell function and development awaits the generation of dominant negative KRR transgenic mice. Preliminary data from J. D. Engel's laboratory indicated that the GATA-3 homologous recombination knockout is embryonic lethal (Yang et al., 1994). The dominant negative GATA-3 could be used instead to determine the effects of the GATA family on individual tissues at later stages of development by utilizing various promoters and enhancers that are active at these later stages.


FOOTNOTES

(^1)
The abbreviations used are: TCR, T cell receptor; CAT, chloramphenicol acetyltransferase; bp, base pair(s); kb, kilobase pair(s).

*
This work was supported by National Institutes of Health Grant RO1 AI31558 (to A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Searle Scholar and Cancer Research Institute Investigator. To whom correspondence should be addressed: Tel.: 510-642-0217; Fax: 510-642-0468.


ACKNOWLEDGEMENTS

We thank Dr. Michael Shapiro, John Woronicz, and Dr. Myung Shin for critical reading of the manuscript. We also thank Dr. Gerald Crabtree for the gift of TAg Jurkat cells, Dr. Mu-En Lee for the gift of the human GATA-2 expression plasmid, and Dr. G. Felsenfeld for the gift of the human GATA-1 cDNA.


REFERENCES

  1. Arceci, R. J., King, A. A., Simon, M. C., Orkin, S. H., and Wilson, D. B. (1993) Mol. Cell. Biol. 13, 2235-2246 [Abstract]
  2. Bories, J. C., Loiseau, P., d'Auriol, L., Gontier, C., Bensussan, A., Degos, L., and Sigaux, F. (1990) J. Exp. Med. 171, 75-83 [Abstract]
  3. Evans, T., and Felsenfeld, G. (1989) Cell 58, 877-885 [Medline] [Order article via Infotrieve]
  4. Gill, L. L., Zaninetta, D., and Karjalainen, K. (1991) Eur. J. Immunol. 21, 807-810 [Medline] [Order article via Infotrieve]
  5. Gottschalk, L. R., and Leiden, J. M. (1990) Mol. Cell. Biol. 10, 5486-5495 [Medline] [Order article via Infotrieve]
  6. Hambor, J. E., Mennone, J., Coon, M. E., Hanke, J. H., and Kavathas, P. (1993) Mol. Cell. Biol. 13, 7056-7070 [Abstract]
  7. Henderson, A. J., McDougall, S., Leiden, J., and Calame, K. L. (1994) Mol. Cell. Biol. 14, 4286-4294 [Abstract]
  8. Ho, I. C., and Leiden, J. M. (1990) Mol. Cell. Biol. 10, 4720-4727 [Medline] [Order article via Infotrieve]
  9. Ho, I. C., Yang, L. H., Morle, G., and Leiden, J. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6714-6718 [Abstract]
  10. Ho, I. C., Vorhees, P., Marin, N., Oakley, B. K., Tsai, S. F., Orkin, S. H., and Leiden, J. M. (1991) EMBO J. 10, 1187-1192 [Abstract]
  11. Joulin, V., Bories, D., Eleouet, J. F., Labastie, M. C., Chretien, S., Mattei, M. G., and Romeo, P. H. (1991) EMBO J. 10, 1809-1816 [Abstract]
  12. Kappes, D. J., Browne, C. P., and Tonegawa, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2204-2208 [Abstract]
  13. Kingsley, C., and Winoto, A. (1992) Mol. Cell. Biol. 12, 4251-4261 [Abstract]
  14. Ko, L. J., Yamamoto, M., Leonard, M. W., George, K. M., Ting, P., and Engel, J. D. (1991) Mol. Cell. Biol. 11, 2778-2784 [Medline] [Order article via Infotrieve]
  15. Krimpenfort, P., de Jong, R., Uematsu, Y., Dembic, Z., Ryser, S., Von Boehmer, H., Steinmetz, M., and Berns, A. (1988) EMBO J. 7, 745-750 [Abstract]
  16. Landry, D. B., Engel, J. D., and Sen, R. (1993) J. Exp. Med. 178, 941-949 [Abstract]
  17. Lee, M. E., Temizer, D. H., Clifford, J. A., and Quertermous, T. (1991) J. Biol. Chem. 266, 16188-16192 [Abstract/Free Full Text]
  18. Leiden, J. M. (1993) Annu. Rev. Immunol. 11, 539-570 [CrossRef][Medline] [Order article via Infotrieve]
  19. Libermann, T. A., Lenardo, M., and Baltimore, D. (1990) Mol. Cell. Biol. 10, 3155-3162 [Medline] [Order article via Infotrieve]
  20. Marine, J., and Winoto, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7284-7288 [Abstract]
  21. Martin, D. I., and Orkin, S. H. (1990) Genes & Dev. 4, 1886-1898
  22. McDougall, S., Peterson, C. L., and Calame, K. (1988) Science 241, 205-208 [Medline] [Order article via Infotrieve]
  23. Merika, M., and Orkin, S. H. (1993) Mol. Cell. Biol. 13, 3999-4010 [Abstract]
  24. Nelsen, B., Kadesch, T., and Sen, R. (1990) Mol. Cell. Biol. 10, 3145-3154 [Medline] [Order article via Infotrieve]
  25. Oosterwegel, M., Timmerman, J., Leiden, J., and Clevers, H. (1992) Dev. Immunol. 3, 1-11 [Medline] [Order article via Infotrieve]
  26. Orkin, S. H. (1992) Blood 80, 575-581 [Medline] [Order article via Infotrieve]
  27. Penix, L., Weaver, W. M., Pang, Y., Young, H. A., and Wilson, C. B. (1993) J. Exp. Med. 178, 1483-1496 [Abstract]
  28. Redondo, J. M., Hata, S., Brocklehurst, C., and Krangel, M. S. (1990) Science 247, 1225-1229 [Medline] [Order article via Infotrieve]
  29. Spencer, D. M., Hsiang, Y., Goldman, J. P., and Raulet, D. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 800-804 [Abstract]
  30. Staudt, L. M., and Lenardo, M. J. (1991) Annu. Rev. Immunol. 9, 373-398 [CrossRef][Medline] [Order article via Infotrieve]
  31. Tsai, S. F., Martin, D. I., Zon, L. I., D'Andrea, A. D., Wong, G. G., and Orkin, S. H. (1989) Nature 339, 446-451 [CrossRef][Medline] [Order article via Infotrieve]
  32. Whitelaw, E., Tsai, S. F., Hogben, P., and Orkin, S. H. (1990) Mol. Cell. Biol. 10, 6596-606 [Medline] [Order article via Infotrieve]
  33. Whyatt, D. J., deBoer, E., and Grosveld, F. (1993) EMBO J. 12, 4993-5005 [Abstract]
  34. Winoto, A., and Baltimore, D. (1989) EMBO J. 8, 729-733 [Abstract]
  35. Wurster, A. L., Siu, G., Leiden, J. M., and Hedrick, S. M. (1994) Mol. Cell. Biol. 14, 6452-6463 [Abstract]
  36. Yamamoto, M., Ko, L. J., Leonard, M. W., Beug, H., Orkin, S. H., and Engel, J. D. (1990) Genes & Dev. 4, 1650-1662
  37. Yang, H. Y., and Evans, T. (1992) Mol. Cell. Biol. 12, 4562-4570 [Abstract]
  38. Yang, Z., Gu, L., Romeo, P. H., Bories, D., Motohashi, H., Yamamoto, M., and Engel, J. D. (1994) Mol. Cell. Biol. 14, 2201-2212 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.