A Novel DNA-Binding Motif Abuts the Zinc Finger Domain of Insect Nuclear Hormone Receptor FTZ-F1 and Mouse. Embryonal Long Terminal Repeat-Binding ...
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1992, p. 5667-5672
Vol. 12, No. 12
0270-7306/92V125667-06$02.00/0 Copyright © 1992, American Society for Microbiology
A Novel DNA-Binding Motif Abuts the Zinc Finger Domain of Insect Nuclear Hormone Receptor FTZ-F1 and Mouse Embryonal Long Terminal Repeat-Binding Protein HITOSHI UEDA,`* GUAN-CHENG SUN,2 TAKEHIDE MURATA,' AND SUSUMU HIROSE2 Genetic Stock Research Center' and DNA Research Center,2 National Institute of Genetics and Department of Genetics, The Graduate University forAdvanced Studies, Mishima, Shizuoka-ken 411, Japan Received 12 May 1992/Returned for modification 12 June 1992/Accepted 21 September 1992
Fruit fly FTZ-Fl, silkworm BmFTZ-Fl, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGG PyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-Fl box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-Fl box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-Fl to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-Fl box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-Fl recognizes and binds to DNA as a monomer. Occurrence of the FTZ-Fl box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer. The FTZ-F1 protein is
a
polymerase chain reactions (8) with FTZ-F1 cDNA as the template. Amplification with the N-terminal primer 5'-tata catATGGAGGAGC'l'l'GCCCCGTGTG (NdeI restriction site underlined; coding region represented in capital letters) and the C-terminal primers 5'-agccggatcCTAACCCATCGA GTTGCGCAGCG, 5'-agccggatcCTATCGAACAGCCTCTA GC(TCAT, and 5'-agccggatcC7ATCCGAATVfGTTGCG TCCACC (BamHI restriction sites underlined; coding regions [including stop codons] represented in capital letters) resulted in 369-, 246-, and 282-bp fragments, respectively. After digestion with NdeI and BamHI, the fragments were cloned between the NdeI and BamHI sites of a pET-3c vector (7). The resultant clones were designated pFTZ622, pFTZ581, and pFTZ593 according to the position of the last amino acid residue. Amino acid substitution mutants were made from pFTZ622 by polymerase chain reactions, using primers carrying base substitutions at desired amino acid positions as described by Higuchi (3). pFTZ622PK was the same as pFTZ622 except that the C-terminal primer contained the additional sequence AGCAACTGAAGCTCT TCT, coding for Arg-Arg-Ala-Ser-Val-Ala, a cyclic AMP (cAMP) protein kinase phosphorylation site (5), between the last glycine codon and the stop codon. Expression of peptides in E. coli. Peptides were expressed in E. coli by using a T7 phage expression system (9, 12). The peptides were recovered from inclusion bodies by denaturation with buffer D (6 M guanidium hydrochloride, 20 mM N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.9], 50 mM NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol [DTE], 12.5 mM MgCl2, 0.1 mM ZnSO4, 0.1% Nonidet P-40) and renaturation by dialysis against 20 mM HEPES (pH 7.9)-50 mM NaCl-0.1 mM EDTA-0.1 mM DTT-5 mM phenylmethylsulfonyl fluoride-20% glycerol. Insoluble materials were removed by centrifugation at 10,000 x g for 15 min. Protein labeling with [f32PJATP. Fifty picomoles of FTZ622PK, expressed on pFTZ622PK, was labeled with 1,000 U of cAMP protein kinase catalytic subunit in a 50-,ul reaction mixture containing 20 mM HEPES (pH 7.9), 100
positive regulator of the fushi
Drosophila melanogaster (22). The BmFTZ-F1 protein, a factor present in the silkworm, Bombyx mon, is biochemically similar to FTZ-F1 (20). The embryonal long terminal repeat-binding protein (ELP), a mouse factor that is present in undifferentiated murine embryonal carcinoma cells but not in differentiated cells, represses transcription from the promoter of the long terminal repeat of Moloney leukemia virus (17). Molecular cloning of cDNAs for these three proteins revealed that they are members of the nuclear hormone receptor superfamily with Cys2-Cys2-type zinc fingers as DNA-binding domains (4, 13, 18). FTZ-F1, BmFTZ-F1, and ELP recognize the 9-bp sequence 5'-PyCAAGGPyCPu-3', which apparently does not have a direct or inverted repeat (16-18, 20-22). In contrast, other members of the nuclear hormone receptor superfamily usually bind to repeated sequences (1, 6, 24). Nevertheless, the FTZ-F1, BmFTZ-F1, and ELP proteins have high affinities to the binding-site DNA (23). These results indicate that the mechanism of binding of these proteins to DNA is somewhat different from that of other members of the nuclear hormone receptor superfamily. Using various mutant peptides in the DNA-binding domain of FTZ-F1, we demonstrated that in addition to the tarazu gene in blastoderm-stage embryos of
zinc finger motif, the basic region abutting the C-terminal end of the zinc finger motif is involved in sequence-specific DNA binding and that the 9-bp binding site is recognized by a single polypeptide. MATERIALS AND METHODS Plasmid construction. To express peptides carrying various lengths of the DNA-binding region of FIZ-F1 in Escherichia coli, three DNA fragments coding for amino acids 507 to 622, 507 to 581, and 507 to 593 preceded by methionine as the translational start site were made by using *
Corresponding author. 5667
5668
MOL. CELL. BIOL.
UEDA ET AL.
A Sequence specificity-
Binding af finity-, FINGER2
FINGER1 ELP (87)
DELCPVCGDICVSGYHIYGLLTCESCIKGPTlRTVHYTESQSCXIDTQ
BmE'TZ-Fl
EELC PVCGDXVSGYHYGLLTC ESCKGrL- T^V
********************************* * ***
PFCRFQKCLTYGKLE.AVBADRMRBGG.RNliPMYlaDRAl^Q KKIAQIRANGFKETGPP
************* ******* ***************************** *
CVAERAC HIDK
C
PFCRKCLW4MEVBARGG~RQJMMYVR-RQ
*
MRHGSIVQL
************ ******* ********************************* **
(86)
********************************************
FTZ-F1
VAERSCHIDXTCPY CC VBA EELC_PVCGD_VSQYYGLLTCESC__
G
A
*
* * * ** * *
RRQ
ANG
0
(507)
B
520
540
560
FTZ622
FTZ581 FTZ593 ETZ565S
580
600
620
+1+ 0 0 ?
+++ 0
FTZ581Q FTZ584Q
FTZ585I FTZ586Q FTZ587A2 FTZ589Q
+ /\
FTZ591Q FTZ597Q5
++ 0 +++ 0
O140 0 ?
FTZ622PK ---------------------------------------------------------------------------------------------------------------------RRASVA
FIG. 1. Comparison of amino acid sequences of ELP, BmFTZ-Fl, and FTZ-F1 and design of peptides of the DNA-binding region of FTZ-F1. (A) Amino acid sequences of the predicted DNA-binding region of ELP, BmFTZ-F1, and FTZ-F1. Asterisks represent identical amino acids. Positions from the N terminus are shown in parentheses. Underlines represent basic amino acids. (B) Design of the peptides used for DNA-binding studies. Numbers on the top line represent amino acid positions of FTZ-F1. Bars indicate sequences identical to those of FTZ-F1. Positions of amino acid substitutions are indicated by letters representing substituted amino acids. Each peptide has a methionine at the N terminus (not shown). A summary of the binding studies is shown at the right. One plus sign represents an approximately 10-fold difference, and a small plus sign represents a roughly 2-fold difference, in binding affinity. 0, binding activity could not be measured because of very low affinity; 0 and A, sequence specificity the same as and similar to that of FTZ-F1, respectively; ?, sequence specificity could not be measured because of low affinity.
mM NaCl, 1 mM DTT, 12 mM MgCl2, and 400 ,uCi of [_y-32P]ATP at 37C for 30 min. The reaction mixture was applied to a 0.2-ml column of S-Sepharose equilibrated with buffer A (20 mM HEPES [pH 7.9], 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol) containing 0.1 M NaCl. The column was washed with buffer A containing 0.4 M NaCl, and the peptides were eluted with buffer A containing 0.6 M NaCl. Gel mobility shift assay. Probe and competitor DNAs were prepared as described by Ueda and Hirose (20, 21). Gel mobility shift assays were performed by incubating peptide(s) and DNA in binding buffer [37.5 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 100 p,g of poly(dI-dC) poly(dI-dC) per ml, 5% glycerol] at room temperature for 1 h and subjected to 0.8% agarose gel electrophoresis in 1 x Tris-borate-EDTA (21). For gel mobility shift competition assays with mutant peptides, 25 fmol of unlabeled FTZ-Fl binding-site I fragment (21) and 50 fmol of 32P-labeled FTZ622PK were used. Signals of the peptideDNA complexes and free DNA were measured with an Ultroscan XL laser densitometer (LKB). Whole cell extract from the posterior silk gland at the second day of the fifth-instar larvae (20) was used for determining the dissociation constant (Kd) of native BmFTZ-Fl. Methylation interference experiment. The lower strand of the site I fragment was labeled with [.y-32P]ATP (21) and partially methylated with dimethyl sulfate (9). Labeled and double-stranded site I fragment (2 pmol) were incubated with approximately 1 ng of wild-type and mutant peptides in binding buffer at room temperature for 1 h and subjected to
0.8% agarose NA (Pharmacia) gel electrophoresis in lx Tris-borate-EDTA (12). DNA fragments shifted by peptides were recovered by using an NA45 membrane (22). Recovered DNA was hydrolyzed with piperidine (9) and subjected to electrophoresis on a sequencing gel. Denaturation and renaturation of peptides. Peptides were denatured in buffer D by incubation at room temperature for 30 min and then renatured by dialysis against buffer A containing 0.1 M NaCl. RESULTS Conserved amino acid sequence abutting the C-terminal end of the zinc finger motif in FTZ-Fl, BmFTZ-Fl, and ELP. Comparison of amino acid sequences among FTZ-F1 (4), BmFTZ-F1 (13), and ELP (18) showed extensive homology in the zinc finger motif and relatively weak homology at the ligand-binding domain. In addition, a 23-amino-acid region abutting the C-terminal end of the zinc finger motif is identical among these three proteins. This region is quite basic, and the basic region further extends to a total of about 30 amino acids, constituting the FTl-F1 box (Fig. 1). This region is a unique feature of these proteins because no other members of the nuclear hormone receptor have such a long basic region just after the C terminus of the zinc finger motif. From these facts, we speculated that the zinc finger region and the FTZ-F1 box have a role in sequence-specific binding to DNA. The FTZ-F1 box and zinc finger motif are involved in DNA
VOL. 12, 1992
A NOVEL DNA-BINDING MOTIF ABUTS THE ZINC FINGER
5669
B
A
0.8.
0.6
IL
:ayc 0 0
000
m
0.4
0.2.
0
0.01 0
0.1
0.2
0.3
0.4
0.02 0.04 0.06
0.5
0.08 0.10 0.12 0.14
Bound (nM)
Bound (nM)
FIG. 2. High-affinity binding of FTZ622 to the site I fragment. Scatchard plots of the results are shown. (A) FTZ622 (Kd BmFI7Z-F1 (Kd = 0.12 nM).
binding. To test this possibility, we made a set of peptides harboring mutations around the zinc finger region of FT-Z-F1 by using a phage T7 expression system (Fig. 1B). Before comparing the binding affinities of these peptides, we measured the dissociation constants of the wild-type peptide, FTZ622, by using a gel mobility shift assay. Scatchard plots showed that FTZ622 and the oligonucleotide carrying the FTZ-F1 binding site (the site I fragment; see Fig. 3A) form a stable complex with a dissociation constant of 0.33 nM, which is slightly higher than that of the native BmFIZ-F1 (Fig. 2). Relative binding capacities of bacterially expressed peptides were measured by gel mobility shift competition analyses. The binding of 3 P-labeled wild-type peptide FTZ622PK to the site I fragment was competed for with unlabeled wild-type and mutant peptides. Competition of FTZ622PK binding with FTZ622 indicated that the binding affinity of FTZ622PK, which has six extra amino acids at the C-terminal end of FTZ622, was the same as that of FTZ622 (Fig. 3A, lanes 1 to 3). Peptide FTZ593, lacking the C-terminal half of the FTZ-F1 box, had a binding affinity five times lower than that of wild-type peptide FTZ622 (lanes 4 and 5 versus lanes 2 and 3). FTZ581, lacking most of the FTZ-F1 box, had a binding affinity more than 1,000 times lower than that of FTZ622 (lanes 6 and 7 versus lanes 2 and 3). These results suggest that the region between amino acid positions 582 and 593 and the region between positions 594 and 622 are involved in DNA binding. To examine the importance of the amino acid residues in these regions, we made mutant peptides carrying amino acid substitutions and analyzed them as described above (Fig. 3B). FTZ565S, carrying a single amino acid substitution at the last cysteine of the second zinc finger, had a binding affinity 100 times lower than that of FTZ622, suggesting that the zinc finger region is involved in the binding of FTlZ-Fl. FTZ597Q5, which has five glutamine residues instead of lysine or arginine residues, showed a binding affinity five times lower than that of FTZ622, almost the same level as that of FTZ593, which lacks amino acid residues from positions 594 to 622. Thus, the basic amino acids in this region appear to stabilize the FTZ-F1-DNA complex. FTZ591Q, harboring a lysineto-glutamine substitution at position 591, had no effect on the binding affinity. Conservation of the lysine residue at this position among FT`Z-F1, BmFTZ-Fl, and ELP may implicate a function other than DNA binding. FTZ1589Q or FTZ586Q (an arginine-to-glutamine substitution at position
=
0.33 nM); (B)
589 or 586) had a binding affinity 10 times lower than that of FTZ622. Similar substitution peptides, FTZ584Q and FIZ581Q, showed a binding affinity 100 times lower than that of FTZ622. These results suggest the importance of basic residues in this region, although we cannot exclude the
A CtD)
N
o
cm
0
o
0
-wIL
x
d
xL
X
e
X
FTZ622-site I fragment DNA complex
_ _
I
12 3 4 56 7 8 9
B
e
N
cN
r. 0
00
CO
U)
IU.
u
-
o
e
00o
0o 8
U)
U.
U
.
In
00
xx xx x
0
U) 0
L
co °
O
U.
0
xxxx xxx xx x xx xxx
4MR40~ .An~~ 1 2 3 4 5 6 7 8 9 1011 1213141516171819 FIG. 3. DNA-binding affinities of mutant peptides. (A) Gel mobility shift competition assay using deletion mutants in the DNAbinding domain of FTZ-Fl. Twenty-five femtomoles of the site I fragment DNA and 50 fmol of 32P-labeled FTIZ622PK were incubated without (lanes 1, 8, and 9) or with (lanes 2 to 7) various amounts of unlabeled mutant peptides. Control samples lacking the site I fragment DNA (lane 8) and lacking the site I fragment DNA and poly(dI-dC)- poly(dI-dC) (lane 9) are also shown. Numbers represent amounts of competitor peptides relative to the amounts of labeled FT622PK. (B) Gel mobility shift competition assay using amino acid substitution mutants. Experiments were performed as for panel A. Lane 1 contained no competitor.
5670
MOL. CELL. BIOL.
UEDA ET AL.
A 123456789
position:
site I:5 -GCAGCACCGTCTCAAGGTCGCCGAGTAGGAGAA-3 RftzlE: ftz2E
-D-
B--
ftz3E:
ftz4E:-B ----ftz5E:-H
ftz6E:-H-
ftz7E: ftz8A: ftz9E:
R-T -------------------Y
-
--------
best binding site:
YCAAGGYCR
Y=T+C, R=G+A, D=G+A+T, B=G+T+C, H=A+T+C
B ww
-
w w
'N
N
N
CLO
0
N
w w
Uw
N N
N
w
N
N
*en r- *: = = r ;r ;r = rLO X
x
N N N x xx
O0 0 Ce ClTx
x x X
X
BmFTZ-Fl FTZ622 FTZ5890
FTZ565S 1 2 3 4 5 6 7 8 9 1011 FIG. 4. Comparison of sequence specificities among wild-type and mutant peptides. (A) Design of the mutant oligonucleotides used for the gel mobility shift matrix competition assay. Site I indicates the sequence of the site I fragment DNA which is located between positions -299 and -267 (relative to the transcription initiation site) of the Drosophila ftz gene. Mutant oligonucleotides have base substitutions at one position within the 9-bp sequence recognized with FTZ-F1. (B) Gel mobility shift matrix competition for the formation of DNA-peptide complexes with various oligonucleotides. Different amounts of competitor DNA and fixed amounts of 32P-labeled site I fragment (numbers represent their ratios) were incubated with each peptide in binding buffer and subjected to a gel mobility shift assay as described in the legend to Fig. 3. Only shifted bands are shown.
possibility that these effects are due to the conformational change of peptides resulting from these substitutions. When the two glycine residues at positions 587 and 588 are changed to alanine residues (FTZ587A2), the binding affinity decreased more than 1,000-fold, suggesting the importance of the flexible structure in this region. These results clearly show that the FTZ-F1 box and the zinc finger region are responsible for the high-affinity binding of FIZ-F1 to DNA. The FTZ-Fl box and zinc finger motif recognize different parts of the 9-bp binding site. To examine the effects of amino acid substitutions on sequence-specific recognition, a gel mobility shift matrix competition assay was performed. The binding of a 32P-labeled site I fragment to native protein or to wild-type or mutant peptide was competed for with various mutant oligonucleotides carrying base substitutions within the 9-bp recognition sequence of FTZ-F1 (Fig. 4A). The results of these analyses are shown in Fig. 4B. Wild-type peptide FTZ622 showed the same competition pattern as did
native BmFTZ-F1, suggesting that FTZ622 contains enough information for sequence-specific DNA binding. FTZ593, FTZ597Q5, FTZ591Q, FTZ586Q (see above), and FTZ585I (a methionine-to-isoleucine substitution at position 585) showed the same competition patterns as did BmFTZ-F1 and FTZ622 (data not shown). Binding affinities of FTZ581, FTZ584Q, FIZ581Q, and FTZ587A2 to the site I fragment were so weak that we could not examine the binding specificity. When binding to the site I fragment was competed for with oligonucleotides ftz5E, ftz6E, and ftz8A, FTZ565S showed a more severe effect than did BmFTZ-F1 and FIZ622. On the other hand, FTZ565S gave the same competition pattern as did BmFTZ-F1 and FTZ622 upon competition with oligonucleotides ftz1E, ftz2E, and ftz3E. These results suggest that the zinc finger region governs the recognition of nucleotide positions 5 to 8 but is not involved in recognition of nucleotide positions 1 to 3. Binding of FTZ589Q to the site I fragment was more efficiently competed for with oligonucleotides ftzlE, ftz2E, and ftz3E than was binding of native BmFTZ-Fl or FTZ622, although FTZ589Q gave the same competition patterns as did BmFTZ-Fl and FTZ622 upon competition with other oligonucleotides. These results suggest that the amino acid sequence around the arginine at position 589 is involved in the sequence-specific recognition of nucleotide positions 1 to 3 without affecting the recognition of nucleotide positions 4 to 9. These results are summarized in Fig. 1. To confirm the contact points by an independent procedure, methylation interference experiments were performed with peptides FTZ622, FT7Z589Q, and FTZ565S. As shown in Fig. 5, clear methylation interference by FTZ622 was observed at nucleotide positions 0, 2, 8, and 10. Binding of FTZ622 to the site I fragment was interfered with by methylation of guanine nucleotides at positions 0, 2, 8, and 10 of the lower strand, suggesting that the FTZ-Fl DNAbinding region makes a contact at least through the major groove of DNA. The degree of methylation interference at nucleotide positions 0 and 2 by FTZ589Q was slightly weaker than that by FTZ622. Densitometric tracing of autoradiographs showed an approximately 1.5-fold difference between the band intensities of FTZ589Q and FTZ622. Although the difference was subtle, the results were quite reproducible. The two peptides showed the same interference at the other nucleotide positions. On the other hand, the degree of methylation interference at nucleotide position 8 by FIZ565S was weaker than that by FTZ622, without affecting the interference at position 2. These results suggest that the FTZ-F1 box is involved in sequence recognition at the sequence around nucleotide positions 0 and 2, whereas the zinc finger region interacts at the sequence around position 8. The DNA-binding domain of FIZZ-F1 binds as a monomer. Conventional nuclear hormone receptor recognizes repeated sequences and binds to DNA as a dimer (1, 6, 24). It is possible that the 9-bp sequence 5'-PyCAAGGPyCPu-3' is recognized as two direct repeats and that the DNA-binding domain of FTZ-F1 binds as a dimer because the first three bases (PyCA) are similar to the last three bases (PyCPu). We believe that this possibility is remote for the following reasons. First, the sequence preceding PyCA is not important for the binding of FTZ-Fl, whereas the sequence AGGPyCPu is essential (21). Second, the 586S mutation reduces sequence specificity at nucleotide position 8 but does not affect sequence-specific recognition at nucleotide position 2. To test the possibility rigorously, we tried to detect a heterodimer of FTZ622 and FTZ593, which recog-
A NOVEL DNA-BINDING MOTIF ABUTS THE ZINC FINGER
VOL. 12, 1992
5671
Ocb)
M 0o)LO
XII
NNN
N N
1-1-4-
OU.LLI
CO)
LL
CM
X
CM
cm
LUL LIL
A
O-MG0 A 1 -~---~ G2 M
T 3 T 4 C 5 C 6 A 7 G 8 C
9
G 10 G
1 2 34 FIG. 5. Effects of methylation of guanine residues on binding of wild-type and mutant peptides. Shown is an autoradiograph of a sequencing gel demonstrating chemical cleavages at sites of guanine methylation in the site I fragment bound to FTZ622 (lane 2), FTZ589Q (lane 3), and FTZ565S (lane 4) and unbound (lane 1). The nucleotide sequence of the lower strand of the site I fragment and nucleotide positions of site I are shown at the right.
nize the same 9-bp sequence, by gel mobility shift analysis (Fig. 6). We could not detect the heterodimer when these peptides were mixed and subjected to gel mobility shift analysis. Inclusion of a denaturation-renaturation step after mixing of these peptides did not alter the results. From these results, we conclude that the DNA-binding region of FTZ-F1 can recognize and bind to the 9-bp sequence as a monomer.
DISCUSSION In this study, we showed that the FTZ-F1 box and the zinc finger region are responsible for the high-affinity binding of FTZ-F1 to its target site. The DNA-binding region of FTZ-F1 can bind as a monomer to target DNA. These results suggest that single polypeptides of native FTZ-F1 and BmFTZ-F1 can recognize the 9-bp sequence through their DNA-binding domains, although we cannot rule out the possibility that native FTZ-F1 binds as a dimer. The FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also demonstrated that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. From these observations, we propose the following model for the recognition by FTZ-F1. The zinc finger region recognizes and binds to the sequence 5'AGGPyCPu-3' with moderate affinity. The FTZ-F1 box extends the recognition sequence by three bases to the 5' side and allows the high-affinity binding. In this model, the C
probe
1
2 3 4
FIG. 6. Heterodimerization analysis of the DNA-binding region of FTZ-F1. Lanes: 1, FTZ622; 2, FTZ593; 3 and 4, FTZ622 and FTZ593 mixed before (lane 4) and after (lane 3) the denaturationrenaturation step. After mixing, samples were subjected to a gel mobility shift assay.
terminus of the zinc finger is placed at the 5' side of the recognition sequence, as reported for the zinc finger region of the estrogen receptor, which recognizes a similar sequence, 5'-AGGTCA-3' (10, 11). Two consecutive glycine residues in the FTZ-F1 box at amino acid positions 587 and 588 might provide the flexible structure needed and hence facilitate the proper contacts of the zinc finger and the FTZ-F1 box onto DNA. The fact that two alanine-to-glycine substitutions in FTZ587A2 resulted in a dramatic reduction of the binding affinity supports this notion. Sequence comparison of the FI-Z-F1 box with other proteins revealed conserved sequences (Fig. 7). A 13-aminoacid sequence within the DNA-binding region from positions 597 to 609 shows 69% homology and 85% similarity with a part of the basic region of GCN4, one of the yeast basic leucine zipper proteins (15). In GCN4, this region forms an a-helical structure and is involved in DNA binding (14). A 23-amino-acid sequence from positions 579 to 601 of FTZ-F1 is identical with the corresponding region of mouse orphan nuclear receptor LRH-1 (19). Between LRH-1 and ELP, a 40-amino-acid region abutting the C terminus of the zinc finger shows 95% identity and 95% similarity, although the
FTZ-F1 BmFTZ-F1 ELP LRH-1 hERRl hERR2
GCN4
(579) (158) (159) (228) (245) (172) (222)
FTZ-F1 box of the FTZ-F1 box with other 7. comparison FIG. Sequence DNA-binding proteins. The positions of the first amino acid residues are noted in parentheses. Numbers on the top line represent amino acid positions of FTZ-Fl. Asterisks represent identical amino acids, and shaded letters represent similar amino acids.
5672
UEDA ET AL.
homology in the zinc finger motifs is not as high (87% identity and 90% similarity) and the surrounding sequences do not show significant homology. A 14-amino-acid sequence from positions 579 to 592 of FTZ-F1 showed 64% homology and 93% similarity with hERR1 and hERR2 (2), members of the orphan nuclear receptor family. Because this region recognizes the first three bases of the 9-bp binding site of FTZ-F1, hERR1 and hERR2 may also bind to a sequence longer than 6 bp which is recognized by a conventional Cys2-Cys2-type zinc finger. These conserved amino acid sequences within the FTZ-F1 box suggest the functional importance of the sequence. It is most likely that in the nuclear hormone receptor superfamily, FTZ-F1, BmFTZFl, ELP, LRH-1, hERR1, and hERR2 constitute a unique subfamily whose members recognize a sequence of more than 6 bp as a single polypeptide. Wilson et al. (25) reported that NGF1-B also requires a region after the C terminus of the zinc finger in addition to the zinc finger motif for specific binding to its target sequence, 5'-AAAGGTCA-3', probably as a monomer, although the relevant amino acid sequence and the recognition sequence of DNA are quite different from those of FTZ-F1. ACKNOWLEDGMENTS We thank K. Umesono for valuable discussion and C. Wu for helpful advice on preparation of the manuscript. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, by the Joint Studies Program of the Graduate University for Advanced Studies, and by a grant from Saito Foundation. REFERENCES 1. Berg, J. M. 1989. DNA binding specificity of steroid receptors. Cell 57:1065-1068. 2. Giguere, V., N. Yang, P. Segui, and R. M. Evans. 1988. Identification of a new class of steroid hormone receptors. Nature (London) 331:91-94. 3. Higuchi, R. 1989. Using PCR to engineer DNA, p. 61-70. In H. A. Erlich (ed.), PCR technology. Stockton Press, New York. 4. Lavorgna, G., H. Ueda, J. Clos, and C. Wu. 1991. FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu. Science 252:848-851. 5. Li, B.-L., J. A. Langer, B. Schwartz, and S. Pestka. 1989. Creation of phosphorylation sites in proteins: construction of a phosphorylation human interferon a. Proc. Natl. Acad. Sci. USA 86:558-562. 6. Naar, A. M., J.-M. Boutin, S. M. Lipkin, V. C. Yu, J. M. Holloway, C. K. Glass, and M. G. Rosenfeld. 1991. The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors. Cell 65: 1267-1279. 7. Rosenberg, A. H., B. N. Rade, D.-S. Chui, S.-W. Lin, J. J. Dunn, and F. W. Studier. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125-135.
MOL. CELL. BIOL.
8. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 9. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 10. Schwabe, J. W. R., D. Neuhaus, and D. Rhodes. 1990. Solution structure of the DNA-binding domain of the oestrogen receptor. Nature (London) 348:458-461. 11. Schwabe, J. W. R., and D. Rhodes. 1991. Beyond zinc fingers: steroid hormone receptors have a novel structural motif for DNA recognition. Trends Biochem. Sci. 16:291-296. 12. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130. 13. Sun, G.-C., S. Hirose, and H. Ueda. Submitted for publication. 14. Talanian, R. V., C. J. McKnight, and P. S. Kim. 1990. Sequence-specific DNA binding by a short peptide dimer. Science 249:769-771. 15. Thireos, G., M. D. Penn, and H. Greer. 1984. 5'-untranslated sequences are required for the translational control of a yeast regulatory gene. Proc. Natl. Acad. Sci. USA 81:5096-5100. 16. Tsukiyama, T., and 0. Niwa. 1992. Isolation of high affinity cellular targets of the embryonal LTR binding protein, an undifferentiated embryonal carcinoma cell-specific repressor of Moloney leukemia virus. Nucleic Acids Res. 20:1477-1482. 17. Tsukiyama, T., 0. Niwa, and K. Yokoro. 1989. Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells. Mol. Cell. Biol. 9:4670-4676. 18. Tsukiyama, T., H. Ueda, S. Hirose, and 0. Niwa. 1992. Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-F1, a member of the steroid receptor superfamily. Mol. Cell. Biol. 12:1286-1291. 19. Tugwood, J. D., I. Issemann, and S. Green. 1991. Liver receptor homologues protein. GenBank DNA data base accession number M813985. 20. Ueda, H., and S. Hirose. 1990. Identification and purification of a Bombyx mon homologue of FTZ-F1. Nucleic Acids Res. 18:7229-7234. 21. Ueda, H., and S. Hirose. 1991. Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mon, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis. Nucleic Acids Res. 19:3689-3693. 22. Ueda, H., S. Sonoda, J. L. Brown, M. P. Scott, and C. Wu. 1990. A sequence-specific DNA-binding protein that activates fushi tarazu segmentation gene expression. Genes Dev. 4:624-635. 23. Ueda, H., C. Wu, and S. Hirose. Unpublished data. 24. Umesono, K., K. K. Murakami, C. C. Tompson, and R. M. Evans. 1991. Direct repeat as selective response elements for the thyroid hormone, retinoic acid and vitamin D3 receptors. Cell 65:1255-1266. 25. Wilson, T. E., R. E. Paulsen, K. A. Padgett, and J. Milbrandt. 1992. Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors. Science 256:107-110.
