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© 7992 Oxford University Press Nucleic Acids Research, Vol. 20, No. 10 2485-2492 Mapping functional regions of the segment-specific transcription factor Krox-20 Christine Vesque and Patrick Charnay* Laboratoire de Genetique Moleculaire, CNRS D 1302, Ecole Normale Superieure, 46 rue d'Ulm, F-75230 Paris Cedex 05, France Received February 7, 1992; Revised and Accepted April 10, 1992 ABSTRACT Krox-20, a zinc finger transcription factor with similarity to Sp1, is likely to play an important role in the development of the vertebrate central nervous system. A knowledge of its molecular properties will help to understand its physiological functions. We have therefore performed a structure-function analysis of the protein to identify the regions involved in DNA-binding and transcriptional activation. Our data suggest that only the zinc fingers are required for high affinity, specific DNA-binding. Transcriptional activation was not affected by deletion of the C-terminal tail of the protein. In contrast, deletion of the N-terminal half, upstream of the zinc fingers, completely abolished transactivation without affecting DNA-binding or nuclear localization. Two transcriptional activation domains were identified in this region. They cooperate to establish full activity. They are rich in negativelycharged amino acids and are therefore may constitute acidic activation domains. Comparative analysis of the amino acid sequences of several zinc finger proteins belonging to the Krox-20 subfamily indicates that they contain acidic regions at similar locations within their N-terminal region, suggesting that the functional organization of these proteins has been conserved during evolution. INTRODUCTION The mouse gene Krox-20 was originally identified as a member of a subset of genes, termed immediate-early genes, whose expression is induced following serum stimulation of NIH3T3 cells (1). These genes are activated very rapidly and transiently at the transcriptional level by mechanisms that do not require de novo protein synthesis and they are thought to mediate long term cellular responses such as proliferation or differentiation. Krox-20 encodes a protein with three C2H2-type zinc fingers (1—5), which are similar to those of the transcription factor Spl (6). As anticipated from the presence of the zinc fingers, the Krox-20 protein was shown to bind to a specific DNA sequence and to act as a transcription factor (7). It belongs to a small * To whom correspondence should be addressed subfamily of proteins that have very similar zinc fingers and which recognize identical or very closely related GC-rich sequences. So far, three other members have been identified: Krox-24 (also known as Egr-1, Zif268, NGFI-A and TIS8 (8-12)), EGR-3 (13) and NGFI-C (14). Although these proteins are very closely related within their putative DNA-binding domains, they appear to be much less conserved elsewhere. The Wilm's tumor gene product WT1 (15) recognizes a similar DNA target, but it contains an additional zinc finger. The zinc fingers of Spl are more distant from those of Krox-20 and indeed this protein binds to a slightly different GC-rich sequence (16). Recently, site-directed mutagenesis of the zinc fingers of Krox-20 allowed the identification of the amino acids responsible for the difference in DNA binding specificity of Krox-20 and SP1 (17); the establishment of the structure of the three zinc fingers of Zif268/Krox-24 complexed with their target DNA sequence has provided a framework for understanding how zinc fingers recognize DNA (18). Further interest for Krox-20 arose from the analysis of the pattern of expression of the gene during development. During mouse embryogenesis, Krox-20 is specifically expressed in two non-adjacent rhombomeres of the developing hindbrain (19). Rhombomeres have been shown to constitute anterior-posterior metameric units within this region of the neural tube (20—22). This pattern of expression, which appears before morphological segmentation and partially overlaps with that of several homeobox-containing genes (23), raises the possibility that Krox-20 might play a role in the regulation of hindbrain segmentation (19) and participate in the control of the expression of some of the homeobox-containing genes (24). The role of Krox-20 is likely to have been conserved throughout evolution, since frog and zebra fish homologs of the gene have been cloned and show segmented patterns of expression during hindbrain development (25; D. Wilkinson, personal communication). Consequently, it has become increasingly important to perform a detailed analysis of Krox-20 molecular properties. We present here the results of a structure-function analysis designed to identify the role of the different regions of the protein. A series of external and internal deletions in the Krox-20 cDNA were built to analyze the involvement of the corresponding parts 2486 Nucleic Acids Research, Vol. 20, No. 10 of the protein in DNA-binding, nuclear localization and transcription control. Our data suggested that only the zinc fingers were required for high affinity, specific DNA recognition. Two transcriptional activation domains were identified within the Nterminal half of Krox-20. Rich in negatively-charged amino acids, they may be of the acidic type. Acidic regions are also observed at similar positions in other members of the Krox-20 subfamily. Such a conservation suggests that these acidic regions are involved in transcriptional activation like those of Krox-20. MATERIALS AND METHODS DNA constructions The reporter plasmid p6E-tkCAT was constructed as described previously (7). All the bacterial expression plasmids were derived from pET-Krox-20 (7). The deletions were created by polymerase chain reaction (PCR) amplification of a part of the cDNA and subsequent substitution to a wild-type cDNA fragment in pETKrox-20 (17). In the case of the N-terminal deletions, the 5' PCR primer included a Ndel restriction site for cloning into the vector and an in frame initiator ATG codon. In the case of the C-terminal deletions, the 3 ' PCR primer included an in frame stop codon and a BamHI restriction site. The internal deletion A186—331 was obtained by creation of a BamHI restriction site at the position corresponding to amino acid 331 by PCR amplification and proline-rich proline-rich DH subsequent deletion of the portion of the cDNA in between this site and a natural BamHI site corresponding to position 186. The PCR products were cloned and completely sequenced using a Pharmacia sequencing kit. The Drosophila cells expression constructs were derived from the corresponding pET constructs by purification of a Ndel-BamHI fragment containing the Krox-20 coding sequence and subsequent cloning into the expression vector pPacU + Ndel (generous gift of A.Courey and R.Tjian). This places the Krox-20 cDNA under the control of the Drosophila actin 5C promoter. In this case, the expected proteins contain two additional amino acids (methionine and histidine) at their N-termini as compared to the corresponding proteins produced in bacteria. Bacterial protein extracts and Western blotting The expression system and protocols of Studier and collaborators were used to produce the different proteins in E. coli (26, 27). Protein extracts were prepared according to Kadonaga and collaborators (6). For immunoblotting, 8 to 80 ng of bacterial protein extract were separated by SDS-PAGE. Blotting and immunodetection were carried out according to standard procedures (28). 539A is a rabbit antiserum raised against a Krox-20 fusion protein (M.Zerial, P.Chavrier, P.Charnay and R.Bravo, unpublished result). The 539A antiserum and the peroxydase conjugated anti-rabbit antibody (Jackson proline-rich ' Nimm basic acidic Meti ammo acid position _1_ ' 51 106 238 143 184 Krox-20 C 298 _U£/ SSS/SA 1 470 331 ZincFhingers / \ DNA-binding 418 424 nuclear localization transaclivation ++ mrnnniiiiiiiiiniiiiiitiiiitiii Krox-20/2ERC N51 ^ •'»"»' frMIIIM N10S Illlllll \D1LJ N184 + ND miinnniiimiiiiiiH + Tim—innim + " N143 Illlllllllllllllllllll Miiiniiimmiiiiliniimmii ND N238 N298 ND N331 ND C424 C C418 C A 186-331 i INIIIKI mmiiiniiiHiiitiiiimiimlir k\\\\1T- Figure 1. Schematic representation of the Krox-20 protein and of its deletion derivatives, and estimation of their DNA-binding, nuclear localization and transactivation properties. Several features of the Krox-20 protein are represented: zinc fingers, acidic, basic and proline-rich regions. The positions of the extremities of the different deletions are indicated along the wild-type sequence. The 'M' within Krox-20/2ER zinc fingers symbolize the double amino acid modification responsible for a change in DNA-binding specificity (17). DNA-binding was estimated by gel retardation experiments (see Figure 2) after synthesis of the protein in E. coli. + + , proportion of complex similar to that formed by the wild-type Krox-20 protein within a 3-fold range; - , proportion at least 50-fold lower than the control; ND, not determined. For deletions N238 and N298, the parentheses indicate that the relative concentration of the protein in the extract could not be precisely estimated. Cellular localization was determined by indirect immunofluorescence with an antiserum directed against the N-terminal part of Krox-20 (see Figure 3): + , the protein is mostly nuclear. Transcriptional activation was determined by transient cotransfection with a reporter CAT gene in SL2 cells (see Figures 4 and 5). + + , transactivates the reporter gene like the wild-type protein within a 2-fold range; + , transactivates 4- to 6-fold less efficiently than the wild-type Krox-20; - , no transactivation detected. Nucleic Acids Research, Vol. 20, No. 10 2487 Immunoresearch Laboratories) were diluted 2000- and 2500-fold respectively. Cell lines, DNA transfection and immunofiuorescence Schneider 2 cells (29) were grown and transfected as described (30, 31). Each plate received the following plasmids: 5 ng of reporter DNA, 100 or 250 ng of expression plasmid, 5 /*g of a control plasmid, pAdh-/3gal, which is capable of expressing the E. coli lac Z gene under the control of the Drosophila Adh promoter, and pUC19 DNA up to a total amount of plasmid DNA of 20 fig. For immunofluorescence analysis, the cells were grown on cover slides. Two days after transfection, they were washed twice with PBS and fixed by incubation in PBS (Phosphate Buffered Saline) containing 3.7% formaldehyde and 0.1 % NP40 for 15 min. After three washes in PBS containing 0.1% NP40, the cells were preincubated for 30 min in PBS containing 1 % Bovine Serum Albumin (BSA), before exposure for 1 hr to a Pel3A Krox-20 N106 N143 C418 C424 4186-331 B Pet3a Krox-20 N106 N143 C418 C424 A186-331 Figure 2. DNA-binding activity of the different Krox-20 derivatives synthesized in E. coli. A, estimation of the relative amount of Krox-20 present in the extracts by Western blotting using the 539A antiserum. For each derivative, three amounts (100 /ig, 30 tig, 10 ^g) of extract were tested. Pet3A, control bacterial extract containing no Krox-20 protein. The molecular weights of size markers are indicated. B, determination of the DNA-binding activity by gel retardation with an oligonucleotide including a consensus Krox-20 binding site. The amounts of extract used for each derivative were normalized according to the results of the Western blotting analysis. Two dilutions ( l x and 0.5x) were tested. F, free oligonucleotide; C, complexed oligonucleotide. 200-fold dilution of the 539A antiserum in PBS containing 0.5% BSA and 0.1 % NP40. The cells were washed three times and incubated for 30 min with a 100-fold dilution of the FITC antirabbit IgG antibodies (Biosys) in the same buffer. DNA was stained by exposing the cells to a 0.5 /xg/ml solution of DAPI (4',6-diamidimo2phenylindole 2HC1, Serva) in PBS. Cellular extracts, transactivation and gel retardation assays 44 hours after transfection, half of the cells were resuspended in 0.25 M Tris-HCl pH 7.8, to prepare the extracts for chloramphenicol acetyl transferase (CAT) and /3-galactosidase assays (32, 33). The amount of extracts used for the CAT assays were normalized relative to the levels of /3-galactosidase. The other half of the cells were used for the preparation of extracts for gel retardation analysis. They were pelleted by centrifugation and frozen in liquid nitrogen. The frozen cells were resuspended in five volumes of extraction buffer (10 mM Hepes pH 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol) and 0.5 mM PMSF were added. The gel retardation assays (34-36) were performed as described (37) with the complementary oligonucleotides 5'-CTCTGTACGCGGGGGCGGTTA-3' and 5'-CTCTAACCGCCCCCGCGTACA-3' containing a Krox-20 consensus binding site. RESULTS Specific DNA-binding We have performed a detailed study of the regions of the Krox-20 protein responsible for transcriptional activation. It was based on the analysis of a series of Krox-20 deletion derivatives. Such a study required discrimination between regions involved directly in transcriptional control and those necessary for specific DNAbinding or nuclear localization. Concerning DNA-binding, the zinc fingers were shown previously to be involved in specific DNA recognition (17). The question here was whether any region of the protein was required in addition to the zinc fingers for high affinity binding to the cognate sequence. The DNA-binding activity and specificity of the Krox-20 protein were previously studied in bacterial extracts, using the expression system of Studier and collaborators (7,17, 26, 27). This approach allows an easy determination of the relative affinities of different derivatives since the amount of Krox-20 protein present in the extract can be quantitated by Western Figure 3. Nuclear localization of the N143 Krox-20 derivative. The expression construct was transiently transfected into SL2 cells and the cellular localization of the protein was studied by indirect immunofluorescence with the antiserum 539A directed against the N-terminal part of the protein. Three photographs were taken from the same field: A, phase contrast; B, immunofluorescence with the 589A antibody; C, DAPI coloration of the DNA, indicating the positions of the nuclei. 2488 Nucleic Acids Research, Vol. 20, No. 10 blotting. In order to evaluate the contributions of the different regions of the Krox-20 molecule to DNA-binding, a series of external and internal deletions were introduced into the Krox-20 cDNA. Figure 1 presents the schematic structure of the expected proteins. It also illustrates some of the features of the Krox-20 protein that can be identified simply by examining the amino acid sequence: i) the zinc fingers are located between positions 339 and 418; ii) they are immediately flanked, upstream and downstream by two short basic sequences. The upstream basic sequence is highly conserved between Krox-20, EGR-3 and Krox-24 (8, 13), while the downstream one is not, although basic amino acids are also observed at this position in the other members of the subfamily; iii) two relatively acidic regions can be identified between positions 23 and 67 (net charge - 8 ) , and 160 and 184 (net charge - 4 ) respectively; iv) finally, the protein is very rich in proline residues, except in the zinc finger domain. It contains a stretch of proline residues (positions 167-173) and three regions where the content in proline is around or above 30% (positions 7 0 - 8 5 , 204-264 and 306-340). Several deletions were selected to evaluate the contributions of these different regions to DNA binding. In particular, deletions N331, A186 —331 and C418, which eliminate the upstream or downstream basic flanking regions respectively (Figure 1), were designed to identify a possible role of these particular regions. The selected Krox-20 derivatives were expressed in E. coli and the amount of Krox-20 protein in the extracts was estimated by Western blotting with a rabbit polyclonal antiserum, 539A, directed against the N-terminal half of the wild-type protein (Figure 2A and data not shown). The polyclonal nature of the antiserum may have led to a slight underestimation of the amount of some of the N-terminal deletion mutants. However, this did not compromise our conclusions. The proteins N298 and N331 were not recognized at all by the antiserum, preventing estimation of their relative concentrations. The extracts were then used to evaluate the affinity of the different proteins for a consensus Krox-20 nucleotide target sequence by a gel retardation assay (34-36). In these conditions, formation of the complex between the Krox-20 derivatives and the oligonucleotide gave rise to major retarded bands (Figure 2B). In addition, in some cases, lower shifted bands were also observed. They were likely to correspond B A CD O ptkCAT C*} ^f tj 0 Z Z Z CO T CO CO 01 . - C 0 C N J CO CO cr> Z Z - C *-<t D T J CO •<* - J Q cO Z P6E-CAT SV40 p o l y - A E E E E E E, -ft s ~ vj © C O „ w e " - Figure 4. Transactivation by the different Krox-20 derivatives. SL2 cells were cotransfected with Krox-20 derivatives expression plasmids, CAT reporter plasmids and a normalization plasmid, pAdh-/3gal. A, structure of the reporter plasmids. ptkCAT contains the CAT gene (open box) driven by the HSV tk promoter (closed box) and followed by the SV40 polyadenylation signal (striped box). p6E-tkCAT was obtained by insertion of six Krox-20 binding sites of the E-type (7) into the BamHI site. B, estimation by gel retardation assay of the Krox-20 DNA-binding activity present in the cell extracts. pPacU-Ndel (Pac) is the expression vector. C and F refer to the positions of complexed and free oligonucleotide respectively . The arrow indicates a retarded band corresponding to an endogenous binding factor. C, transactivation assay. CAT assays were performed after normalization of the amounts of extracts according to /3-galactosidase activity. 'C' corresponds to the unreacted substrate [ I4 C] chloramphenicol and '3-AC to the major acetylated form (in position 3). Nucleic Acids Research, Vol. 20, No. 10 2489 to complexes involving partially degraded Krox-20 protein. The proportion of complexed versus free oligonucleotide obtained with each of the Krox-20 derivatives was determined and taken as an estimate of the relative affinity of the protein for the target sequence. The outcome of these experiments is summarized in Figure 1: none of the deletions tested significantly affected the binding activity. In contrast, a double amino acid change in finger 2 (construction Krox-20/2ER) has been shown previously to change the recognition specificity and to abolish binding to the oligonucleotide (Figure 1 and reference 17), which illustrates the involvement of the zinc fingers in DNA recognition. The Cterminal endpoint of deletions A186-331 abutted the zinc fingers, eliminating the basic region located upstream. The deletion C-418 eliminated the entire C-terminal part of the protein, including the basic region located immediately downstream to the zinc fingers. The fact that these deleted proteins bound efficiently to the cognate sequence (Figure 1 and 2) indicated that the basic regions were not required simultaneously for DNA-binding. In fact, our data suggest that the DNA-binding domain consists of only the zinc fingers and they are consistent with the results of Pavletich and Pabo indicating that a similar region of Krox-24/Zif268 binds efficiently to the target sequence (18). Nuclear localization We have previously studied transactivation by Krox-20 by transient transfection into Drosophila Schneider line 2 (SL2) cells (7). These cells were selected because they are devoid of Spl and Krox-20 endogenous activities that might have interfered in the assay (31) and since they allow a high level of transactivation by Krox-20 (7). We have used the same system to evaluate the transcriptional properties of the different Krox-20 derivatives. An essential point in the analysis was to determine whether the mutations affected nuclear localization. Therefore, the cellular localization of the Krox-20 derivatives that were recognized by the 539A antibody was determined by immunofluorescence after transient expression in SL2 cells. The coding sequences of the different mutants were placed under the control of the Drosophila actin 5C promoter as described previously (7). Representative results for the deletion mutant N143 are shown in Figure 3. Examination of the same field under phase contrast, immunofluorescence and DAPI staining, which labels the DNA, indicated that the protein was found localized preferentially within the nucleus. The background fluorescence observed in the cytoplasm of all cells was also obtained with preimmune serum or untransfected cells (data not shown). The same pattern was observed for all the other deletion mutants tested (data not shown and Figure 1). Although the N-terminal external deletions N-298 and N-331 could not be tested in this assay, because they were not recognized by the antiserum, the combination of the data obtained with the other external deletions and with the internal deletion A186—331 indicated that any region outside of the zinc fingers, including the two flanking basic regions, could be omitted without affecting nuclear localization. plasmids are represented in Figure 4A: ptkCAT includes the herpes simplex virus thymidine kinase (tk) gene promoter driving the chloramphenicol acetyl transferase (CAT) gene; it contains no Krox-20 binding site; p6E-tkCAT was derived from ptkCAT by insertion of six Krox-20 binding sites immediately upstream of the tk promoter. Extracts were prepared from transfected cells and used to measure /3-galactosidase and CAT activities as well as to estimate the amount of Krox-20 protein by measuring its DNA-binding activity by gel retardation analysis with the oligonucleotide carrying the consensus Krox-20 binding site. CAT activity, after normalization relative to /3-galactosidase activity, was taken as a measure of the transcription of the CAT gene, which allowed an estimation to be made of the capacities of the different Krox-20 derivatives to promote transcription from the reporter gene promoter. As shown in Figure 4B, there were important variations in the amounts of oligonucleotide-binding activity present in cells transfected with the different derivatives. Nevertheless, these levels were always equal or higher than the level observed with the wild-type protein, which was sub-saturating for transcriptional activation. The results obtained with proteins produced in bacteria suggested that the deletions did not affect the DNA-binding activity of the proteins (Figure 2). Therefore the differences in DNA-binding activity observed in SL2 cells were likely to be due to changes in the amount of Krox-20 derivatives. Increases in the amount of some of the deleted proteins may reflect higher stability compared to the wild-type protein, which has been shown to have a very short half-life in eukaryotic cells, in the order of 20 min (Chavrier, Zerial, Bravo and Charnay, unpublished result). In any case, our data suggested that the amount of each derivative present in transfected cells was at least equal to the amount of wild-type Krox-20. Measure of the expression of the reporter gene indicated that wild-type Krox-20 transactivated the construct containing its binding site by a factor of 20- to 30-fold, while it had no effect on ptkCAT (Figure 4C). Analysis of the relative levels of transactivation by the different derivatives (Figure 5) revealed 1.2 1 1.0 • 0.8 - I 0.6 H is 0,4 - 0.2 - o Transcriptional activation For the determination of the transcriptional activities of the different Krox-20 derivatives, we used a transient cotransfection assay developed in SL2 cells by Courey and Tjian (31). The Krox-20 expression constructs were cotransfected with reporter plasmids and the plasmid pPAdh-|3gal, which contained the E. coli lacZgene under the control of the Drosophila Adh promoter and was used to normalize the experiments (7). The reporter I.I. o z to n CO CO z Z o z 00 o CM z Z z O o Figure 5. Quantification of transactivation. The ratio of CAT activities from the p6E-tkCAT reporter construct obtained with each Krox-20 derivative and with the wild-type Krox-20 was taken as a measure of the transcriptional activity of the derivative. The bars correspond to the means of the values obtained in at least three independent experiments. The standard deviations are indicated. 2490 Nucleic Acids Research, Vol. 20, No. 10 the presence of two important regions located in the N-terminal half of the protein. Elimination of the first 50 N-terminal amino acids led to a 4- to 5-fold reduction in transactivation (Figures 4C and 5). This deleted protein was particularly interesting since it corresponded precisely to the shorter Krox-20 protein that is expected to be synthesized from a splice variant mRNA observed in NIH3T3 cells (2). Further deletion of N-terminal sequences up to position 143 did not produce additional significant changes (Figure 4C). In contrast, elimination of 41 additional amino acids, up to position 184, completely abolished transactivation, although the amount of protein present in the cells was dramatically increased, as judged from the DNA-binding activity (Figures 4B and C). Proteins with larger N-terminal deletions behaved similarly. In contrast to the N-terminal external deletions, the internal deletion A186—331, which eliminated almost half of the sequence upstream of the zinc fingers including the conserved basic region (Figure 1), affected only marginally transactivation (85% of the wild-type level, Figure 5). However, DNA-binding was also increased (Figure 4B). Analysis of dose-response curves indicated that the transcriptional activity of the A186-331 mutant is at least 40% of the wild-type level (data not shown). Finally, we tested the importance of the C-terminal tail of the protein by specifically deleting it. Complete elimination, including the downstream basic region, had no effect on DNA-binding activity or on transcriptional activation (Figures 4 and 5). In conclusion, our data pointed to the existence of two regions required for transactivation that are located, respectively, between positions 1 and 51 and 143 and 184. These regions did not appear to be involved in DNA-binding (Figure 1). They did not affect nuclear localization, since the proteins N184 and N238, although inactive in the transactivation assay (Figures 4 and 5), were located in the nucleus (Figure 1). Therefore, our results suggested that these two regions were involved directly in transcriptional activation. Evolutionary conservation of the functional organization As indicated above, Krox-20 belongs to a small subfamily of zinc finger proteins with closely related DNA-binding domains. Comparative analysis of the amino acid sequences indicated that the degree of similarity is much lower outside of the zinc finger domain (data not shown). The family members closest to Krox-20 in these regions are EGR-3 (13) and Krox-24 (8). In these cases, some similarity is observed throughout the region upstream of the zinc fingers (Figure 6), while no significant similarity is detected downstream (data not shown). Since the two Krox-20 transactivation domains are located in the N-terminal region and since they overlap with the acidic regions, we compared the distribution of charges within the sequences of the three proteins (Figure 6). Although the overall sequence conservation is low (24% identity between Krox-20 and Krox-24, from position 1 to position 318, with numerous insertions and deletions), there is a striking conservation of the charge distribution. In particular, the two acidic regions that correlate with the transcriptional activation domains in Krox-20 are also found in the two other proteins, although in the case of Krox-24 the N-terminal acidic region is interrupted by the insertion of a sequence rich in serine and glycine. We believe that this conservation is indicative of a functional significance of the acidic regions and suggests that, like in the case of Krox-20, the two acidic regions of EGR-3 and of Krox-24 may constitute transactivation domains. A limited deletional analysis of Krox-24 has been performed and the results are consistent with these evolutionary considerations: while the wild-type Krox-24 protein was able to activate transcription from the p6E-tkCAT plasmid in SL2 cells by a factor of approximately 20-fold, deletion of the entire region upstream of the zinc fingers, including the two acidic regions, completely abolished transactivation without affecting DNA-binding (Nardelli and Charnay, unpublished results). In addition, deletion of the region located downstream to the zinc fingers also slightly reduced transcriptional activation (2—3-fold effect). These data indicate that, like in the case of Krox-20, the N-terminal part of Krox-24 contains a transactivation domain. They are consistent with an analysis of hybrid proteins that suggested that the N-terminal part of Krox-24 was involved in transcriptional activation (38). DISCUSSION Our analysis of the regions of the Krox-20 protein responsible for transcriptional activation has first involved the identification of the requirements for DNA binding. The data eliminated the PVTLSGFMHQLPSSLYP—VggLA-ASSVTIFPNGg KROX20 EGR3 KROX24 PVTMSSLLNQLfftlLYP—gl—IPSALNLFSGS LGGPFBR-MNGVAGg 57 ^SWH-YNQMATg 53 * ,PYPSSFAPISAP3SQTFTYMGHFSIgPQ-YPGASCYPgGIINIVSAGlLQGVTPPASTTASSSVTSASPNPlATCPL0VCT«SQ 156 KROX20 EGR3 KROX24 KROX20 EGR3 KROX24 •SYSGSFOP—APG^TVTYFGKFAFB SPSNWCXENUSLMSAGIL-GVPPAS GALSTQTSTASHVQP 133 SYPSQ TTBLPPITYTGHFS^PAPNSGNTLHPBPLFSLVSGLVSMTNPPTSSSSAPSPAASSSSSASQSPPLS-CAV— 192 LYSPPPPPPPYSi Y PALPPYSNC-i PSITOSSPIYSAAPT PPT; KROX20 EGB3 HV-PPPLTPLST: KROX24 HTQQPSLTPLST: SAFLSPPSTTSTSSLAYQPPPSYPSpRpAjffiPGLIPMIPraXPGFFPSPCCgJ'HGAAG SFH3PQGNPG LAY-SPO3VQSMi>AlSsNLFPMIPMfN-LY HHPNOXGSI QS—QAFPGSAGTALQY-PPPAYPATyMFQ IPGAGVTGPGASGGGSSPBI.PGSGSAAVTATPYNPHHLPIS'I RVNPPPTTPIST: •A B3QIHPGFGSLP QPPLTLQPI TQ.SGSCSlBALNTTYQ.SQ LlgP VPHIpfi*-- LFPQ—QQGEJLSLGT 338 277 337 Figure 6. Alignment of the amino acid sequences of the N-terminal regions of three members of the Krox-20 subfamily of zinc finger proteins: Krox-20 (2), EGR-3 (13) and Krox-24 (8). The sequences were aligned using the program CLUSTAL (57). Gaps are marked by dashes. Amino acids identical in the three sequences are indicated by stars. Acidic amino acids are shown in reversed characters and basic amino acids are boxed. The two acidic regions of Krox-20 are overlined. Nucleic Acids Research, Vol. 20, No. JO 2491 possibility that both basic regions were required simultaneously for high affinity binding. Indeed, they suggested that only the zinc fingers were necessary for DNA recognition. Nevertheless, our data did not exclude an influence of regions outside the zinc fingers on aspects of DNA-binding like association rate or salt stability of DNA-protein complexes. The next question concerned the regions required for nuclear localization. Proteins are targeted to the nucleus by specific signals, encoded within their primary sequence (39). The nuclear localization signals (NLS) have been delineated in a large number of cases (40). No single, strict consensus NLS has been derived, but these sequences are generally short (8 — 10 amino acids) and contain a high proportion of positively charged amino acids. The two basic sequences flanking the Krox-20 zinc fingers fulfill these conditions and they are the only highly basic sequences in the protein, apart from the zinc fingers. They could therefore constitute an NLS. Our deletion analysis does not allow an identification of the Krox-20 NLS. Nevertheless, it indicates that elimination of all sequences upstream or downstream of the zinc fingers does not affect nuclear localization, at least as judged by our immunofluorescence assay. These data are consistent with the following possibilities: each of the flanking basic regions constitutes a NLS and both signals are not required simultaneously; the zinc fingers themselves contain an NLS. Confirmation of one of these hypotheses will require the analysis of the behavior of mutant proteins consisting of only the zinc fingers or specifically mutated within both basic regions. Different types of transactivation domains have been identified through the functional analysis of several transcription factors: acidic regions, which may be the most common (41 -43), and proline-, glutamine- or serine/threonine-rich regions (31, 44, 45). Other types certainly await to be discovered. These regions are thought to be involved in protein-protein interactions with other elements of the transcription complex (46,47) and distinct classes may function by different mechanisms (48,49). Their properties are not well defined and their primary structures are poorly conserved through evolution. Krox-20 does not contain any glutamine-rich region, but several acidic or proline-rich regions can be identified (Figure 1). Analysis of the transcriptional activation of a reporter gene carrying multiple Krox-20 binding sites, revealed the existence of at least two transactivation domains located in the N-terminal half of the protein and overlapping with the regions 1 - 5 0 and 143—183. Examination of Krox-20 amino acid sequence indicated that the region 1-50 overlapped with the first acidic region while the region 143-183 contained the second acidic region and overlapped with a portion of a prolinerich region (Figure 1). Comparison of the mouse and Xenopus laevis (D.Wilkinson, personal communication) Krox-20 amino acid sequences over these regions indicates that, although there is an about 50% divergence between these sequences, all the negative charges except one are conserved (data not shown). In contrast, the 7 proline residue stretch present in region 143-183 in the mouse sequence is reduced to 3 residues in the frog sequence. These observations are consistent with the two major transcriptional activation domains of Krox-20 being of the acidic type. In addition, the limited effect (less than 2-fold decrease) of the internal deletion A186-331, which eliminates the two major proline-rich regions of the protein (Figure 1), suggests that these latter regions are not important for transcriptional activation, at least in the present assay system. The comparison of the amino acid sequence of Krox-20 with those of the closest members of the subfamily, EGR-3 and Krox-24, also supports the involvement of the two acid regions of the N-terminal half in transcriptional activation. These regions are maintained in these proteins, despite a low degree of sequence conservation. Furthermore, this suggests that the acidic regions may also constitute transcription activation domains in EGR-3 and Krox-24. The demonstration that the major activation region of Krox-24 is located upstream of the zinc fingers is consistent with this idea. The definitive demonstration of the involvement of the acidic regions in transcriptional activation will require modification of the charge distribution by site-directed mutagenesis. In contrast to the N-terminal half, no function was found for the C-terminal part of Krox-20 downstream of the DNA-binding domain. This is again consistent with the outcome of the comparative analysis of mouse and frog amino acid sequences (D.Wilkinson, personal communication), which indicates no significant similarity between the C-terminal parts of the two proteins. This rapid divergence may reflect a less important role of this region of the Krox-20 molecule. The sequence of the Cterminal region is also not conserved between Krox-20, EGR-3 and Krox-24. However, unlike Krox-20, elimination of this region in Krox-24 leads to low but significant reduction in transcriptional activation. This difference may indicate that the highly divergent C-terminal regions of the members of the Krox-20 subfamily have acquired distinct functions in the course of evolution. In this respect, it is interesting to note that the Cterminal region of Krox-24 contains a series of short tandemly repeated sequences rich in proline, serine, threonine and tyrosine and which are similar to the repeats present in the C-terminal region of the largest subunit of eukaryotic RNA polymerase II (50, 51). These repeats could play a role in transcriptional activation. An heterologous system has been used in this study for the evaluation of transcriptional activity. Originally, Drosophila cells were chosen because, unlike mammalian cells, they are devoid of Spl activity (31). Although Spl recognizes a DNA sequence slightly different from the Krox-20 target sequence, there is some cross-reactivity that might have interfered with our assay. In addition, using the p6E-tkCAT reporter construct, we have observed much lower levels of transactivation by Krox-20 in the mammalian cell lines tested so far than in SL2 cells (only 3—4-fold in Cos7 cells, data not shown). We do not understand presently the basis of this difference, which may be due to the presence of a specific repressor or the absence of a necessary activator or coactivator in the latter cells. In any case, this low level of transactivation has prevented a quantitative analysis of the effect of the different mutations in mammalian cells. Nevertheless, the conclusions that have been reached in term of nuclear localization and identification of transcription activation domains are likely to stand in an homologous system: posttranslational processing (except N-glycosylation) and cellular localization of recombinant proteins are very similar in insect and mammalian cells (52, 53); the general mechanisms of transcription control have appeared to be conserved and a number of mammalian transcription factors have been shown to function correctly in insect cells and vice versa (31, 54-56). The present work suggests additional investigations and provides some tools for further analysis of the physiological functions of Krox-20. It is interesting that a protein encoded by an alternatively spliced version of Krox-20 mRNA (2) has a reduced transcriptional activity. This effect raises the possibility that alternative splicing is physiologically involved in the 2492 Nucleic Acids Research, Vol. 20, No. 10 regulation of Krox-20 activity. The distribution of the two forms of the mRNA during development will therefore deserve further investigation. Deletions which increase DNA-binding and abolish transactivation might act as dominant negative mutations. Expression of such mutant genes in specific tissues or at specific stages of development in transgenic mice might create phenotypes informative on the role of Krox-20 or of Krox-20-related molecules. 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