(Received for publication, August 22, 1994; and in revised form, October 21, 1994 )
From the
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) and
enhancers by 12- and 3.4-fold,
respectively. However, KRR did not have a significant effect on the
activity of larger TCR-
and -
enhancer fragments. Thus,
functional redundancy in the TCR-
and -
enhancers can
compensate for the loss of GATA-3 activity.
GATA-3 is the third member of a family of transcription factors
that contain two zinc fingers of the sequence
CXCX
CX
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
- and
-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) ()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-
, -
,
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 CD8
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-
enhancer, T
2, 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- and -
enhancers,
but has only a small effect on larger enhancer-containing fragments.
Thus, redundancy in the TCR-
and -
enhancers can compensate
for the loss of GATA-3 function.
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 J21
4E4 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 J21
4E4 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.
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.
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 J21
4E4, 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 J21
4E4, 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 J21
4E4, 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.
Figure 6:
Effect of KRR overexpression on TCR-
and -
enhancer function. TAg Jurkat human
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
1.6
alone (=100). The results represent the average of
three independent experiments.
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
enhancer approximately 12-fold. The activity of the larger
enhancer-containing fragment, however, is not significantly affected by
KRR overexpression. Apparently, the larger
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-
enhancer than on the larger TCR-
-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-
and -
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-
enhancer function is in agreement with
the results from Henderson et al.(1994). Mutation of the TE4
GATA site in the minimal TCR-
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- 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
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 CD8
enhancers (Hambor et al., 1993;
Wurster et al., 1994), where in the absence of GATA-3 binding
sites the enhancer retain at least 50% (CD8
) 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.