(Received for publication, September 1, 1995; and in revised form, October 23, 1995 )
From the
EVI1 is a zinc finger oncoprotein that binds via fingers 1-7 to the sequence GACAAGATAA. The target genes on which EVI1 acts are unknown. This binding motif overlaps with that for the GATA transcription factors, (T/A)GATA(A/G), and GATA-1 can bind to and activate transcription via a GACAAGATAA motif. The possibility has been raised that, when overexpressed in leukemogenesis, EVI1 may function by interfering with the differentiation-promoting action of GATA factors. To explore this, we have assessed the affinity of EVI1 for the GATA binding sites derived from erythroid-specific GATA-1 target genes, and found only low affinity interactions. We examined the contacts between EVI1 and DNA by methylation interference studies, which revealed extensive contacts between EVI1 and its binding site. The importance of the contacts for high affinity binding was shown by in vitro quantitative gel shift studies and in vivo cotransfection studies. To examine what types of sequences from mouse genomic DNA bind to EVI1, we isolated and sequenced five EVI1-binding fragments, and each showed the GACAAGATA site. The data presented contribute to our knowledge of the binding specificity of EVI1, and yield a clearer picture of what sequences can, and cannot, act as targets for EVI1 action.
The current molecular understanding of myleoid leukemogenesis has come largely from the identification and characterization of genes that contribute to this multistep process. Proviral tagging in retrovirally induced tumors has been a powerful way of identifying myeloid-specific oncogenes. One such oncogene, Evi1, was identified as a common site of retroviral insertion in murine myeloid leukemias(1, 2) . Rearrangements in Evi1 have also been documented in human myelodysplasias and leukemias, indicating its involvement in human disease(3, 4, 5, 6, 7, 8, 9) . Both the retroviral insertions and the chromosomal alterations at Evi1 result in transcriptional activation of the gene, suggesting that the gene acts as a dominant oncogene in leukemia. Interestingly, most acute myeloid leukemias bearing EVI1-activating alterations are of the M0, M1, or M2 class, and are usually CD34-positive, suggesting an immature phenotype. Cell lines with such alterations are dependent on hematopoietic growth factors, indicating that Evi1 does not abrogate growth factor requirements(4, 10) .
cDNA cloning and analysis showed that the murine Evi1 encodes a 1042-residue, 145-kDa protein with 10 zinc finger motifs that are separated into two domains by 481 amino acids(10) . There is also an acidic domain at the C-terminal end, which may function in transcriptional activation, although, as presented in this paper, Evi1 does not appear to function as a transcriptional activator. A shorter isoform of Evi1, which migrates as 88 kDa, is produced via alternative splicing in both human (11) and mouse (12) . This form lacks zinc fingers 6 and 7, as well as 269 of the adjacent C-terminal amino acids. Since these fingers are important for DNA binding(13) , it is likely that this isoform has different DNA binding characteristics than the 145-kDa isoform, although this has not been carefully examined.
The finding of 10 zinc fingers in the protein argues that Evi1 encodes a sequence-specific DNA-binding protein that plays a role in nucleic acid regulation, most likely RNA transcription. The consensus binding sites for both the first and second sets of zinc fingers have been identified; fingers 1-7 bind to TGACAAGATAA (14) or GACAAGATAAGATAA(13) , and the second set of fingers, numbers 8-10, bind to GAAGATGAG(15) . The first EVI1 binding site, TGACAAGATAA, shows overlap with the binding site for the GATA family of transcription factors, which bind to the consensus motif (A/T)GATA(A/G)(16, 17) . This site was first identified as a common motif present in cis-acting elements of erythroid-specific genes, and through mutagenesis studies was found to be functionally important for erythroid gene transcription(18, 19, 20, 21) . The GATA family of transcription factors now includes GATA-1(22, 23) , GATA-2(24, 25, 26) , GATA-3(27, 28, 29) , GATA-4(30, 31) , GATA-5(32) , and GATA-6(32) , each of which has a distinct pattern of expression. While GATA-2 expression can be detected in early myeloid precursors(33) , it is expressed in other cells as well. To date, no myeloid-specific GATA family member has been reported.
The overlap in binding sites for EVI1 and the GATA factors suggested the possibility that EVI1 may bind to GATA sites located in cis to GATA target genes and influence their transcription. Support for this theory comes from the observation that ectopic expression of Evi1 in primary bone marrow cells inhibits differentiation in response to erythropoietin(34) . Since differentiation along the erythroid lineage is dependent on GATA1 activity(35) , and since there is overlap between the GATA-1 and EVI1 binding sites, it is possible that loss of erythropoietin responsiveness results from EVI1-induced repression of GATA-1-responsive gene(s) harboring an EVI1 binding site. Also in support of this, as presented below, Evi1 can repress GATA1-mediated activation of responsive reporter constructs in cotransfection studies in NIH 3T3 cells. Bona fide target genes for EVI1 have not been reported, and the importance of Evi1-induced repression of GATA factor function within cells has not been carefully addressed. In this paper, we report the further characterization of the interaction between zinc fingers 1-7 of EVI1 and its binding site, both in vitro and in vivo. These studies indicate a high degree of specificity of EVI1's interaction with DNA and show that EVI1 has negligible affinity for known GATA sites. Our data suggest that, theoretically, only a small subset of GATA sites, those conforming to the full EVI1 binding site (TGACAAGATAA or GACAAGATAAGATAA), can be bound in vivo by both proteins. However, none of the known GATA target sites conforms to this motif.
The missing base contact probing was performed as described (42) and was essentially the same as that described above, except that, instead of modification with dimethylsulfate, the DNA was treated with 0.1 M formic acid for depurination, or hydrazine for depyrimidation. These modified DNAs were then bound to MBP-EVI1(1-254), fractionated, cleaved with piperidine, analyzed on a 6% sequencing gel, and autoradiographed as described(42) .
Figure 1: Cotransfection studies in NIH 3T3 cells showing that GATA-1 activates reporters containing the GACAAGATAA motif and that Evi1 can repress this activation. 10-cm plates (in duplicate) were transfected with a tk-CAT reporter (5 µg/plate), with or without the EVI1 binding site as indicated, along with GATA-1 (2.5 µg/plate) and Evi1 (0.5 µg/plate) expression plasmids as indicated. CAT activity in cells is expressed as a percentage of the activity in cells transfected with tk-CAT (no binding site) alone.
The binding of MBP-EVI1(1-254) for the radiolabeled wild type
oligonucleotide was competed with varying amounts of different cold
competitor oligonucleotides, each corresponding to a candidate target
site, bearing the GATA core together with the surrounding sequences
from the indicated genes. Surprisingly, none of these sequences bound
to EVI1 with appreciable affinity (Table 1). The chicken
globin GATA site, having sequence GATAAGATAA, deviates from the EVI1
core only at position 3. Despite this degree of similarity to the EVI1
consensus, the chicken
globin sequence bound with poor avidity to
EVI1 (Table 1). However, when a C was substituted for that T in
the chicken
globin sequence, it bound with wild type affinity (Table 1), indicating the importance of C at position 3.
These experiments indicate that none of the reported GATA target sites adjacent to either erythroid-specific genes, or other genes that we tested, are likely to be high affinity targets of EVI1 binding. To assure accurate determination of EVI1-DNA affinities, each value for relative affinity was derived from the data of binding reactions done in triplicate with three competitor concentrations. In addition, both protein and DNA titrations were done prior to competitive gel shift to assure that reactions were performed in DNA excess, and most of the affinity measurements were performed on separate occasions with essentially the same results.
Figure 2: Methylation interference and missing contact probing studies reveal multiple contact points between EVI1 and its binding site. Depicted are denaturing PAGE analysis of the piperidine cleavage products following chemical modification and EVI1 binding. To the right of each panel is the EVI1 binding motif. Panel A shows results obtained with the top strand radiolabeled. The type of modification is as indicated, along with the specific base(s) modified. B and F denote the fractions that bound to EVI1, or remained free, respectively. For the formate and hydrazine reactions, there are two bound lanes presented. Panel B shows results obtained with the bottom strand radiolabeled.
Figure 3: Summary of the methylation interference and missing contact probing analysis. The arrows indicate the points of contact between EVI1 and the 11-bp binding site. The size of the arrow depicts the relative importance of the contact, as assessed by visual assessment of band intensities shown in Fig. 2.
Figure 4:
A, quantitative gel shift studies reveal
the affinity of MBP-EVI1(1-254) for various oligonucleotides with
single base changes to the GACAAGATAA motif, relative to the wild type (WT) 11-bp motif. To the left is indicated the
mutation in the competing oligonucleotide. The bars indicate
the affinity expressed as a percentage of that for the 11-bp wild type
oligonucleotide. Also shown (WT(15 BP), second bar from top) is the relative affinity for the 15-bp motif
identified by Delwel et al.: (GACAAGATAAGATAA). B, cotransfection studies to show the in vivo activity of the wild type (WT (11 BP)) or mutant binding
sites (as indicated). Plates (10 cm) of NIH 3T3 cells (in triplicate)
were cotransfected with tk-CAT reporters (5 µg/plate) containing
the binding site indicated (inserted at bp -109 relative to the
start of transcription), along with either an Evi1, Evi1-VP16, or GATA-1 expression vector, as indicated. The CAT
activity in cell lysates is expressed as the percent acetylation of the
[
C]chloramphenicol substrate, plus and minus the
standard error.
These data support and extend the missing contact probe and methylation interference experiments, and argue that changes at positions 1, 2, 3, 5, 6, or 8 are not compatible with high affinity binding to EVI1. Together with in vitro binding selection studies(13, 14) , the data presented here indicate a minimum core EVI1 binding site of GACAAGATA, which extends by 4 bases on the 5` side the GATA consensus sequence, (A/T)GATA(A/G).
Since Evi1 proved ineffectual on these
promoters, we constructed a dominantly acting chimeric Evi1 activator, comprised of the VP16 activation domain of herpes
simplex virus linked downstream of the first zinc finger region of Evi1 (fingers 1-7, amino acids 1-250). This
construct encoded a protein with potent transactivating capacity that
was specific for reporters bearing a wild type EVI1 binding motif (Fig. 4B). The reporters having mutant binding sites
(G
T, C
A, or G
T) were not responsive to the Evi1-VP16 chimera,
indicating that EVI1 had little if any affinity for these sites in
vivo. As expected, GATA-1 was able to activate reporters
containing the wild type motif, as well as the G
T
and C
A mutations, but not the G
T mutation. Since G
is part of the GATA binding motif, it
is not surprising that reporters bearing this mutant fail to be
activated by GATA-1. These data show that the mutant reporters are
functional and respond appropriately to GATA-1.
Figure 5: Sequence analysis of the EVI1 binding sites isolated from mouse genomic DNA. Depicted are five EVI1 binding sites, each from the clone indicated, identified within the Sau3A1 subclones of plasmids selected for EVI1 binding. The core region of identity between the sequences is indicated in bold.
The molecular role of EVI1 in leukemogenesis is not clear. In
certain studies, it appears to interfere with differentiation program
induced by certain cytokines in cultured and primary hematopoietic
cells(34, 44) . EVI1 is a sequence-specific
DNA-binding protein possessing 10 zinc fingers in two separate domains.
In our previous studies, we found that EVI1 binds with high affinity to
the sequence TGACAAGATAA(14) . Given the DNA binding properties
of EVI1, a possible mechanism for its role in leukemogenesis is to
dominantly interfere with the expression of genes required for normal
hematopoiesis. To arrive at a mechanistic understanding of the role
that EVI1 plays in leukemia, we are conducting studies to determine its
genetic targets within the cell, and its action on those targets. One
possible set of genetic targets in this mechanism is the GATA target
genes, whose proper regulation is essential for the erythroid lineages.
The GATA motif (T/A)GATA(A/G) is a cis-acting element that
plays an important role in the regulation of erythroid-specific genes.
It is present adjacent to a variety of globin (16) and
non-globin(18, 45) erythroid-specific genes, and is
present within a minimal 3`--globin gene enhancer(17) .
Functional importance of the GATA motif in the regulation of these
genes has emerged from numerous
studies(20, 21, 46, 47, 48) .
The presence of a (T/A)GATA(A/G) motif within the EVI1 binding site
suggested to us the possibility that EVI1 may play a role in the
regulation of genes that are transcriptionally controlled via this
motif. In addition, by searching the DNA data bases, we identified near
matches of the EVI1 binding site adjacent to several genes, including
proliferating cell nuclear antigen.
In this paper we report our
studies on the ability of the first set of zinc fingers of EVI1
(numbers 1-7) to bind to various GATA binding sites, and found
that EVI1 binds poorly if at all to known GATA sites, despite only
single base pair differences between the EVI1 motif (GACAAGATA) and
certain GATA target sites, such as that for the chicken globin
gene (GATAAGATA) (Table 1). From our quantitative studies
presented here, it is clear that EVI1 cannot bind with any appreciable
affinity to a AGATAA monomer. This makes it very likely that, without
accessory factors that could increase affinity, EVI1 does not play any
significant role in the regulation of genes that contain AGATAA but
lack the additional bases that would yield a high affinity site for
EVI1. This includes most of the (T/A)GATA(A/G) sequences that have been
found to be important for erythroid-specific gene transcription. It is
likely that GATA proteins can bind to endogenous EVI1 sites, since they
are likely to contain the (T/A)GATA(A/G) motif.
The high degree of
specificity that EVI1 shows for its binding motif was probed further by
performing methylation interference and missing base contact probing
experiments. These revealed that EVI1 makes contacts at multiple bases
in its recognition sequence (Fig. 3). These findings were
confirmed and extended by studying the binding both in vitro and in vivo of EVI1 to a series of oligonucleotides
bearing single base changes to the GACAAGATA motif (Fig. 4, A and B). Interestingly, we observed the same
affinity between EVI1 and the short motif that we described previously
(GACAAGATAA) and the longer one described by Delwel et al.(13) (GACAAGATAAGATAA). Since both of these motifs were
identified by in vitro binding to random oligonucleotides, and
may not be representative of EVI1 binding sites in the nucleus, we
examined what sites EVI1 binds to within genomic mouse DNA, by
performing binding selection experiments with genomic fragments
(average size 3.5 kilobases). This led to the identification of EVI1
binding sites with the shorter rather than the longer motif. Indeed, in
a larger scale selection performed with full-length EVI1 protein
produced in insect cells, we have isolated 16 more EVI1 binding sites
in genomic mouse DNA, and have never found the longer motif. ()These findings suggesting that the selection of the longer
15-bp motif from the pool of random oligonucleotides was a function of
the method used, and may not represent a physiologically relevant
motif.
One consistent finding in our studies was that Evi1 fails to activate transcription of any reporter, despite the
presence of an acidic region, which commonly acts as an activation
domain in transcription factors. Reporters that contained multimers of
the GACAAGATAA motif exhibited only basal levels of transcription in
most cell lines tested, including NIH3T3, Ltk cells,
choriocarcinoma cells (JEG), hepatoma cells (HepG2), and HeLa cells (Fig. 4, data not shown). In these cells, cotransfection of Evi1 had little effect (Fig. 4, data not shown). When
these reporters are activated by GATA-1, then addition of Evi1-expressing plasmids results in transcriptional
repression. Similarly, in WEHI cells, a myelomonocytic leukemic cell
line that does not express Evi1, the GACAAGATAA-containing
reporter was expressed at significantly higher levels; in these cells,
cotransfection of Evi1 resulted in transcriptional repression
(data not shown). Thus, while Evi1 did not appear to act as a
repressor of basal transcription, it did appear to repress higher level
transcription, suggesting that EVI1 does not interact with the basal
transcription machinery, but rather with other transcription factors or
coactivator proteins. This repressive effect of Evi1 appeared
to depend on the presence of a GACAAGATAA sequence; no effect was seen
on viral promoters such as the SV40 early region promoter or the
metallothionein promoter (data not shown). Tanaka et al.(49) have reported that Evi1 overexpression in
NIH 3T3 cells leads to increased AP-1 activity, and that this activity
is dependent on the presence of the second set of zinc fingers on the
protein. This suggests that EVI1 may be acting as a transcriptional
activator; the effect may be indirect, however, since, although Evi1 appears to increase c-fos transcription, there
is no evidence of EVI1 binding to the c-fos promoter.
Structure-function studies on EVI1 have shown that high affinity binding to the GACAAGATA sequence is mediated by fingers 4-7(13) . The structural studies on the zinc finger have showed that each finger interacts with three nucleotides(50) , which argues that the key fingers (4, 5, 6, 7) would interact with 12 bp. The work we present here, specifically the methylation interference and missing base contact probing, indicates an essential core of 9 base pairs: GACAAGATA, which might interact with only three of these fingers. Based on a comparison of the zinc finger amino acid sequences with their cognate DNA binding sites, as well as the information from structural studies, a set of rules to define these interactions has been proposed(50, 51, 52, 53) . It has been difficult to use these rules to assign specific zinc fingers in the first domain of EVI1 with base pairs in the binding site. As suggested previously, the RN residues in fingers 4 and 6 may interact with the GA residues of the EVI1 binding site(13) . In this regard, it is informative to consider the crystal structure of the Tramtrack (TTK) protein, the finger 1 of which has a recognition helix sequence very similar to zinc fingers 4 and 6 of EVI1 (given N-terminal to C-terminal): HISNFCR for TTK, QFSNLCR for finger 6, and DPSNLQR for finger 4. The TTK finger binds to the sequence GAT, with the R contacting the G, the N contacting the A, and the S interacting with T. Interestingly, the GAT is followed by A in the TTK binding site, and the A-T-A triplet is readily deformable (54) , allowing the short side-chained S to make contact with T. Even with this added insight, however, it is difficult to phase the interactions of specific fingers with specific triplets to satisfy the methylation interference studies and the predictions made by the recognition code. What this suggests is that further analysis of EVI1-DNA interactions will provide information on novel types of zinc finger-DNA interactions and allow a better understanding of this important class of DNA binding motif. Necessary additional information may come from ongoing mutagenesis studies.
Our data clearly indicate that in a variety of settings
EVI1 can act as a transcriptional repressor. Other leukemogenic nuclear
proteins that appear to function as transcriptional repressors in their
oncogenic state are ErbA, a truncated and mutated form of the thyroid
hormone receptor, and PML-RAR, the protein encoded by the t(15;17)
translocation of acute promyelocytic leukemia. The target genes for
none of these three leukemogenic oncoproteins is known with certainty,
but it appears that ErbA functions in oncogenesis by blocking the
effects of retinoic acid(55) . Likewise, PML-RAR
may
interfere with the action of RAR
on its normal
targets(56) . Alternatively, PML-RAR
may act in
oncogenesis by interfering with intranuclear localization of
PML(57, 58) . It is intriguing that these three
leukemogenic zinc finger proteins all potentially act as
transcriptional repressors, and may act to block normal cellular
differentiation(44, 59, 60, 61, 62) .
While the genetic targets of these factors that are relevant to
leukemogenesis are not known, it could be that similar pathways of
differentiation are blocked by the different proteins, but via a
different target with each factor. Future studies aimed at the
identification of target genes for these proteins should yield insight
into this possibility.