Transcriptional Activities of the Zinc Finger Protein Zac Are

MOLECULAR AND CELLULAR BIOLOGY, Feb. 2003, p. 988–1003 Vol. 23, No. 30270-7306/03/$08.00�0 DOI: 10.1128/MCB.23.3.988–1003.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Transcriptional Activities of the Zinc Finger Protein Zac AreDifferentially Controlled by DNA Binding

Anke Hoffmann,1 Elisabetta Ciani,1 Joel Boeckardt,2 Florian Holsboer,1Laurent Journot,2 and Dietmar Spengler1*

Molecular Neuroendocrinology, Max Planck Institute of Psychiatry, D-80804 Munich, Germany,1 andUPR 9023 Centre National de la Recherche Scientifique,

F-34094 Montpellier Cedex 5, France2

Received 6 May 2002/Returned for modification 18 June 2002/Accepted 30 October 2002

Zac encodes a zinc finger protein that promotes apoptosis and cell cycle arrest and is maternally imprinted.Here, we show that Zac contains transactivation and repressor activities and that these transcriptionalactivities are differentially controlled by DNA binding. Zac transactivation mapped to two distinct domains.One of these contained multiple repeats of the peptide PLE, which behaved as an autonomous activation unit.More importantly, we identified two related high-affinity DNA-binding sites which were differentially bound byseven Zac C2H2 zinc fingers. Zac bound as a monomer through zinc fingers 6 and 7 to the palindromic DNAelement to confer transactivation. In contrast, binding as a monomer to one half-site of the repeat elementturned Zac into a repressor. Conversely, Zac dimerization at properly spaced direct and reverse repeatelements enabled transactivation, which strictly correlated with DNA-dependent and -independent contacts ofkey residues within the recognition helix of zinc finger 7. The later ones support specific functional connectionsbetween Zac DNA binding and transcriptional-regulatory surfaces. Both classes of DNA elements were iden-tified in a new Zac target gene and confirmed that the zinc fingers communicate with the transactivationfunction. Together, our data demonstrate a role for Zac as a transcription factor in addition to its role ascoactivator for nuclear receptors and p53.

Zac is a seven-zinc-finger protein which potently promotesapoptosis and cell cycle arrest upon expression in mesenchymaland epithelial cell lines. Conversely, ablation of Zac gene ex-pression increased cell proliferation, further supporting Zac’santiproliferative role (19, 24). Concordant with these findings,the rat ortholog Lot1 was isolated from ovarian surface epi-thelial cells in a screen for genes showing reduced expressionupon spontaneous transformation in vitro, hence the designa-tion “lost on transformation.” Moreover, treatment with epi-dermal growth factor receptor ligands rapidly downregulatedLot1 expression in these cells. In this view, Lot1 may controlmitogenic signal transduction pathways by counteracting cel-lular transformation (1, 2).

The human ortholog ZAC/LOT maps on chromosome 6q24,a region known to contain a tumor suppressor gene for severaltypes of neoplasms, including breast cancer (28). We recentlyshowed that ZAC is expressed in normal mammary and ante-rior pituitary epithelial cells and that its expression is down-regulated in primary breast and pituitary tumors (4, 20). Treat-ment of some mammary cell lines with 2-deoxyazacytidinerestored ZAC expression and suggested that ZAC may beinactivated by methylation of regulatory promoter sequences.The recent finding that ZAC/Zac is imprinted (see below) (3,21) supports our observations and suggests an additionalmechanism for inactivation of ZAC in tumors with loss of

heterozygosity at 6q24-25, a condition observed in up to 80%of breast tumors.

Recent studies (7, 13) evidenced an additional role for ZACin the etiology of transient neonatal diabetes mellitus, a rareform of childhood diabetes which usually resolves in the first 6months of life and strongly predisposes to type 2 diabetes ofadult onset (OMIM *601410; Online Mendelian Inheritance inMan; NCBI). Transient neonatal diabetes mellitus is associ-ated with intrauterine growth retardation, dehydration, andlack of insulin due to overexpression of a maternally imprintedgene localized on 6q24 that interferes with pancreatic devel-opment and/or glucose homeostasis in general.

Patients with transient neonatal diabetes mellitus show pa-ternal uniparental disomy of chromosome 6, paternal duplica-tions of the critical region, or a defect in a methylation imprinton chromosome 6 (3, 7). This methylation imprint mapped toa CpG island within the promoter region of ZAC, and itsmethylated state silenced promoter activity (27). In fibroblastsfrom patients with transient neonatal diabetes mellitus, themonoallelic expression of ZAC is relaxed, providing strongsupportive evidence that two unmethylated alleles of this locusare indeed associated with transient neonatal diabetes mellitus(16).

Taken together, accumulating evidence demonstrates a rolefor Zac/ZAC in the regulation of proliferation and differenti-ation, and altered expression most likely contributes to cancerand diabetes. These findings agree with the notion that im-printed genes are intricately involved in fetal development andunderlie numerous human diseases (6).

Interestingly, Zac has recently again been isolated in ascreen for proteins that bind the nuclear receptor coactivator

* Corresponding author. Mailing address: Molecular Neuroendocri-nology, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10,D-80804 Munich, Germany. Phone: 49 89 30622 559. Fax: 49 89 30622605. E-mail: [email protected].

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GRIP1 (12). More importantly, Zac potently coactivated orcorepressed the hormone-dependent activity of nuclear recep-tors, including androgen, estrogen, glucocorticoid, and thyroidhormone receptors, which are key regulators of cell growth anddifferentiation, homeostasis, and development in a cell-specificmanner. In further support of Zac’s role in coactivation, theauthors additionally observed that Zac enhanced transactiva-tion of p53 on synthetic and endogenous promoters andstrongly reversed E6 inhibition of p53 (11). Similarly, Zaccoactivated p53 transactivation of the Apaf-1 promoter (23)but in both studies was unable to confer transactivation byitself.

At odds with these findings, we previously suggested thatZac/ZAC DNA binding is indicative of a role as a transcriptionfactor (5, 28). Therefore, it appeared mandatory for us tofurther elucidate Zac’s status in transcriptional regulation. Inparticular, we reasoned that Zac’s status as transcription factorwill be instrumental to placing Zac/ZAC within the network ofgenes regulating cell proliferation, pancreatic development,and/or glucose metabolism.

MATERIALS AND METHODS

Cell culture and transfection. Saos-2 cells were kept as described previously(24). For immunoblots, 100 ng of each Gal4-Zac fusion construct was trans-fected; results represent three to four independent experiments. Gal4-Zac (10ng), adjusted amounts of deletion constructs, and Gal4-(PXX)n plasmids (100ng) were cotransfected with the reporter p(GAL4)5E1BLUC (0.5 �g) into 2 �106 cells. To prepare nuclear extracts (5), 50 ng of wild-type or mutated ZaccDNAs were transfected. Half-maximal transactivation of the palindromic andrepeat Zac DNA-binding site, their derivatives, and the CK14 promoter motifsby wild-type and mutated Zac cDNAs was determined from dose-responsecurves obtained from three independent cotransfections.

Plasmids, GST pulldown, and in vitro translation assays. Zac, synthetic re-porter, and CK14 promoter constructs were generated and sequenced by stan-dard methods; details are available on request. Oligonucleotides encoding ZacDNA-binding sites were inserted into the vector pGL2TATA at the SacI andKpnI sites. Restriction fragments used in electrophoretic mobility shift assayswere released by digestion with XbaI (present as an internal site) and XhoI.Glutathione S-transferase (GST)-Zac fusion proteins were expressed frompGEX2TK and purified as described previously (28). Equivalent amounts ofprotein, as judged by Coomassie blue staining, were used. In vitro translationswere done with the TNT kit (Promega). Results represent at least three inde-pendent experiments.

Electrophoretic mobility shift assays. Conditions for electrophoretic mobilityshift assays were as described previously (5). Additionally, Zac proteins in nu-clear extracts were measured by immunoblotting, and aliquots were adjusted andincubated with 20,000 cpm of labeled probe. Results represent three to fourindependent experiments quantified in a scintillation counter.

Immunoblots and immunohistochemistry. Western blots were done with anti-Zac (24) and anti-HA (Roche) antibodies. Immunostaining with anti-CK14 an-tibodies (BioGenex) was carried out as described previously (19).

Tetracycline-regulated Zac. Stable cell clones for tetracycline-regulated ex-pression of Zac�ZF were generated as described previously (24). Total RNAswere purified and hybridized with a cDNA expression macroarray according tothe manufacturer’s instructions (Clontech 7742). Reverse transcription-PCR ex-periments were done as reported before (4).

RESULTS

Zac transactivation and autonomous minidomains. The ZacN-terminal zinc finger domain contains seven canonical zincfingers of the C2H2 type (amino acids 1 to 208). This region isflanked by 65 amino acids (amino acids 209 to 274) withoutknown protein motifs. The central part of Zac (amino acids275 to 382) harbors 34 repeats of the motifs PLE, PMQ, PML,and PLQ. The C terminus (amino acids 383 to 667) is rich in

single and clustered P and Q residues, which commonly occurin the transactivation domains of transcription factors. Theextreme C terminus includes several PE repeats and E clusters(amino acids 583 to 654), which cause high acidity (29%). Werefer here to these regions as the zinc finger, linker, prolinerepeat, and C-terminal domains (Fig. 1A).

To investigate Zac transactivation potency, we fused differ-ent segments to the heterologous DNA-binding domain ofGal4 (Fig. 1B), which we additionally tagged at its C terminuswith the HA epitope to compare expression of the correspond-ing proteins (Fig. 1C). Absence of the zinc finger domain(G-Zac�ZF) reduced protein levels fivefold compared to Zac(G-Zac), whereas the remaining constructs were similarly ex-pressed. Note the aberrant migration of the proline repeatdomain at a higher molecular weight than predicted. Doses ofplasmids adjusted to obtain equal levels of protein expressionwere cotransfected with a luciferase reporter gene, which con-tained five GAL4 DNA-binding sites in front of the TATAelement derived from the E1B promoter. Zac (G-Zac) mod-erately (21-fold) activated transactivation, whereas absence ofthe zinc finger domain (G-Zac�ZF) strongly improved trans-activation (908-fold) (Fig. 1B). The entire C terminus (G-Zac-C) was inactive, which was also the case when shortersegments (G-Zac-1572/2004 and G-Zac-1141/1572) were sep-arately expressed (data not shown). The isolated linker (G-Zac-L) and proline repeat region (G-Zac-PR) each weaklyactivated (7- and 11-fold, respectively), while joint expression(G-Zac-LPR) multiplied (384-fold) transactivation. EqualDNA-binding activities were measured for the entire set ofconstructs in an electrophoretic mobility shift assay with anoligonucleotide encoding one GAL4 DNA-binding site as theprobe (data not shown). Therefore, transactivation by the jointlinker and proline repeat region demonstrated strong cooper-ativity between two independent and unrelated transactivationdomains.

To confirm the relevance of these putative transactivationdomains for Zac function, we deleted them individually withinthe full-length Zac fusion protein. Absence of either the linker(G-Zac�L) or the proline repeat (G-Zac�PR) region dimin-ished transactivation by 5-fold and 10-fold, respectively. Insupport of their role, the absence of both domains (G-Zac�LPR) completely abolished transactivation (Fig. 1B). Tofurther validate these results, we additionally studied nativeZac bound to a reporter plasmid containing two copies of acognate Zac DNA-binding site (see below). In agreement withthe data obtained from the Gal4 fusion system, transactivationwas clearly diminished or abolished in the absence of either orboth of the identified transactivation domains (Fig. 1D).Amounts of Zac, Zac�L, Zac�PR, and Zac�LPR were ad-justed to similar levels of protein expression in this experiment(inset).

The structural basis for the ability of activators to stimulatetranscription, the importance of particular residues, and theirposition within the transactivation domain are only partly un-derstood (9). The Zac proline repeat domain has a character-istic modular structure which consists of 13 PLE, 15 PMQ, 5PML, and 1 PLQ amino acid triplet (Fig. 1E). In view of thisunusual organization, we asked whether individual motifs au-tonomously confer transactivation. In order to test this, weserially fused oligonucleotides encoding four copies of each

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PXX triplet to the heterologous DNA-binding domain of Gal4.Four PLE triplets transactivated 4-fold, whereas insertion of asecond and third oligonucleotide showed 69-fold and 596-foldtransactivation, respectively (Fig. 1F).

To test the positional restraints of the proline residuewithin this motif, we designed oligonucleotides in which theyfaced each other (LEPPLELEPPLE). Fusion proteins in-cluding two, four, and six copies of this motif transactivated3-fold, 92-fold, and 539-fold, respectively, thus behavingindistinguishably from the parent PLE polypeptide (Fig.1F). PLE and LEPPLE fusion proteins containing multiplecopies of each motif were less expressed, emphasizing theiractual activities (Fig. 1G). These results led us to testwhether negatively charged residues are critical to function.When lysine replaced glutamic acid (PLK), transactivationwas abolished despite efficient expression of this fusion pro-tein. Likewise, the motif PMQ, which contains the polarresidue glutamine, conferred no transactivation. Also, the

construct PLQ, in which leucine substituted for methionine,activated only weakly, although both the PMQ and PLQfusion proteins were well expressed.

Finally, we compared transactivation of the PLE minido-main to the one reported for homopolymeric prolines (8). Inagreement with this study, we noted a threefold transactivationby PPP polypeptides, which was, however, at a fraction of theactivity obtained for the PLE and LEPPLE constructs and didnot significantly change with the number of proline residuespresent or the amount of DNA cotransfected. We concludethat the proline repeat domain of Zac contains a genuinetransactivation function based on the strong transcriptionalsynergism of discrete PLE minidomains which strictly de-pended on the acidic charge of the glutamic acid residue. Ourfindings further suggest that a recently reported Zac variant(AF324471), which lacked amino acids 343 to 362 representingfive PLQ and two PML motifs, is unlikely to differ in transac-tivation potency.

FIG. 1. Zac transactivation and autonomous minidomain. (A) Scheme of the Zac protein. Numbers indicate amino acid residues. ZF, zincfingers (amino acids 1 to 208); L, linker (amino acids 209 to 274); PR, proline repeat (amino acids 275 to 382); C, C terminus (amino acids 383to 667). (B) Zac confers transactivation in the Gal4 fusion system. Scheme of the fusion proteins; transactivation (TA) for concentrations ofplasmids adjusted to the expression of G-Zac was set to 100%. (C) Representative immunoblot of fusion proteins for doses of 100 ng each. (D) Zactransactivation of cognate DNA-binding site depends on the linker and proline repeat domains. The reporter plasmid contains two tandempalindromic DNA elements. Activation by Zac (50 ng) was set to 100% and compared to adjusted doses of Zac�L, Zac�PR, and Zac�LPR (inset).(E) Amino acid sequence of the proline repeat region. Amino acid triplets are 13 PLE (red), 15 PMQ (green), 4 PML (blue), and 1 PLQ (black).Residues 343 to 362 are absent in a variant of Zac (AF324471). (F) Amino acid triplets selectively transactivate. The indicated peptide motifs wereserially fused in steps of four (PLE, PLK, PMQ, PLQ, and PPP) or two (LEPPLE) copies to Gal4 and tested for transactivation (TA).(G) Representative immunoblot of Gal4 alone (Gal) and the indicated Gal4 fusion proteins. The number of amino acid triplets is noted. Note theaberrant migration of the PLK polypeptides.

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Zac DNA-binding and transcriptional activities. With a ran-dom oligonucleotide selection assay we previously (28) recov-ered two related consensus sequences: (i) a G-rich motif (5�-GGGGGGnnnnnnGGGGGG-3�) following three sequentialcycles of gel shift purification, and (ii) a GC-rich motif (5�-nacGGGGGGCCCCtttan-3�) which prevailed after additionalrounds of selection. To assess DNA binding in vitro and trans-activation in vivo in parallel, oligonucleotides derived fromthese consensus sequences were inserted in front of the TATAelement of a luciferase reporter plasmid. For DNA binding invitro, restriction fragments containing individual motifs wereexcised from the respective reporter plasmids, fractionated onpolyacrylamide gels, and purified. Labeled fragments weretested in electrophoretic mobility shift assays with nuclear ex-tracts of Zac-transfected Saos-2 cells, which were adjusted tothe results from the respective Zac immunoblots. Results arerepresentative of at least three experiments varying by lessthan 10%. Half-maximal transactivation of these reporter plas-mids by cotransfected Zac (varying by less than 5%) was de-termined from three dose-response curves and referred to theactivity of the parent motif, which was set to 100%.

Zac binding to a GC-rich DNA element confers transacti-vation. In order to define the core for Zac DNA binding andtransactivation, we equilaterally reduced the number of G andC nucleotides within the GC-rich consensus. The motif G4C3

showed about half of the DNA-binding and transactivationactivity measured for the motif G4C4, whereas the motif G3C3

was inactive (Fig. 2A). The sequence G4C2 retained someDNA-binding and transactivation activity, which was not thecase for the motif G4C1.

We also tested whether Zac transactivation was orientationdependent. Indeed, transactivation conferred by one copy ofthe reverse motif C4G4 was only half of that measured for themotif G4C4 (Fig. 2B). This difference grew larger in the case offour tandem copies of each motif and was also observed forcotransfection of human ZAC or the related zinc finger pro-teins KIAA and PLAG (28) (data not shown).

We also investigated the authenticity of the G4C4 bindingsite by determining its specificity and affinity. Addition of a100-fold excess of unlabeled self-oligonucleotide but not of themutated oligonucleotide G4AC4 (see below) completely inhib-ited the Zac-DNA complex (Fig. 2C). Moreover, preincuba-tion with a polyclonal Zac antiserum efficiently prevented theformation of the complex observed in the presence of preim-mune serum or an unrelated p53 antibody. Lastly, the Zac-specific DNA-protein complex was not observed for nuclearextracts from mock-transfected cells in any of these motifs(data not shown).

We determined Zac affinity for the motif G4C4 by Scatchardanalysis of saturation binding isotherms and measured an equi-librium binding dissociation constant (KD) of �2.7 nM (Fig.2D). Taken together, these results demonstrated specific andhigh-affinity binding of Zac to the palindrome G4C4, whichconferred transactivation in an orientation-dependent manner.

The specificity of DNA-binding sites involves primary nucle-otide sequences as well as spacing of half-sites. To assess bothpossibilities at the same time, we inserted two complementarybase pairs at the center of the palindrome G4C4 to obtain themotif G4CGC4. This inversion strongly impaired Zac transac-tivation and DNA binding (Fig. 2E). To analyze spacing re-

straints alone, we tested the motif G4AC4, in which the com-plementary half-sites were separated by one unrelatednucleotide. This insertion strongly weakened Zac DNA-bind-ing and transactivation activity, which declined further andprogressively in the case of two or three intervening nucleo-tides (Fig. 2E). These results demonstrated that the centralpositions within the palindrome G4C4 were conserved and thatchanges either in primary sequence or in spacing interferedwith Zac function.

Although similarities in DNA recognition by related zincfingers have been pointed out, these do not provide rules whichcould explain DNA-binding specificity in general. The proto-typical C2H2 zinc finger binds a single zinc ion that is sand-wiched between the two-stranded antiparallel �-sheet and the�-helix (Fig. 2H, dark and light grey residues, respectively),and at least two zinc fingers are necessary to establish high-affinity and stable contacts with DNA (30). In this view, Zaccould recognize G4C4 (i) by more than two zinc fingers, (ii) bytwo zinc fingers with key residues within each �-helix contact-ing single or multiple bases, and (iii) by at least one zinc fingerin case Zac binds as a dimer or as an oligomer.

To distinguish between these possibilities, we studied pro-gressive N-terminal deletions of the zinc finger domain. Step-wise truncation of zinc fingers 1 to 5 (�ZF1 to �ZF1-5) re-duced neither Zac DNA binding nor transactivation. In thelatter case, we observed a twofold enhancement in the pres-ence of unchanged protein levels (Fig. 2F, bottom). In con-trast, Zac DNA binding and transactivation abruptly disap-peared upon deletion of zinc fingers 6 and 7. These proteinswere undetectable in nuclear extracts (Fig. 2F, bottom) butwere even more highly expressed in whole-cell extracts (datanot shown), demonstrating a defect in nuclear localization thatprecluded further analysis of the role of zinc fingers 6 and 7 atthis step.

Therefore, we generated a second set of constructs withsubtler defects in DNA binding by replacing the first cysteineresidue with alanine, which destroys the zinc-dependent tetra-hedral coordination of the zinc finger structure (broken zincfinger, Fig. 2H). Analysis of these mutated Zac cDNAs con-firmed that zinc fingers 1 to 5 were dispensable to DNA-binding and transactivation of the palindrome G4C4 (Fig. 2G).Importantly, if either zinc finger 6 or 7 was broken, this com-pletely abolished binding of the G4C4 element in the presenceof protein levels undistinguishable from those of wild-type Zac.In conclusion, Zac zinc fingers 6 and 7 were necessary torecognize and transactivate the palindrome G4C4.

Amino acid residues at positions 1, 2, 3, and 6 (numberingwith respect to the start site of the �-helix, Fig. 2H) typicallymake key base contacts in the major groove that define se-quence specificity. Possible pairings between the 20 aminoacids and the four DNA bases and stereochemical rules whichdescribe the base and amino acid positions in contact havebeen compiled into a “recognition code” that describes thepreferred side chain base interactions of key residues withinthe recognition helix (30). The scheme of amino acids at po-sition 1, 2, 3, and 6 within the �-helices of zinc fingers 6 and7 compares anticipated nucleotides to the palindrome G4C4

(Fig. 2K). Provided that position 2 of zinc finger 7 and posi-tions 2 and 6 of zinc finger 6 contact bases in the secondarystrand, a code/consensus correlation of 100% is achieved. The


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FIG. 2. Zac transactivates the palindromic DNA element dependent on zinc fingers 6 and 7. (A) Determination of the core sequence withinthe GC-rich consensus motif. Zac DNA-binding activity (DB%) was measured by electrophoretic mobility shift assays with the motifs indicatedat the top. Half-maximal transactivation (TA%) was measured with the corresponding reporter plasmids. Activities are referred to the consensusmotif, which was set to 100%. (B) Zac transactivates in an orientation-dependent manner. Single or four tandem copies of G4C4 or of the reversemotif C4G4 were tested for transactivation (TA) with increasing doses of Zac. (C) Zac binds specifically to G4C4. Zac-DNA complexes wereinhibited by unlabeled self-oligonucleotide (100� cold) or by anti-Zac serum (�-Zac). The mutated motif G4AC4 (100� cold mt) and preimmuneand anti-p53 serum (�-p53) were inactive. (D) The palindrome G4C4 is a high-affinity DNA-binding site. A constant amount of Zac protein wasincubated with increasing concentrations of G4C4 until equilibrium occurred. The dissociation constant (KD, �2.7 nM) was estimated as thenegative reciprocal of the regressed slope by Scatchard analysis (inset). (E) The central nucleotide positions of the palindrome G4C4 are conserved.Substitutions in the primary sequence or the spacing at central positions of G4C4 prevented Zac DNA binding and transactivation. Motifs are notedat the top. (F) Zac N-terminal zinc fingers are dispensable for DNA binding and transactivation. Successive truncations C-terminal to each zincfinger as noted at the top were tested for DNA binding (DB%) and transactivation (TA%). Protein expression levels of these cDNAs are shownin the bottom panel. (G) Zac DNA binding and transactivation depend on zinc fingers 6 and 7. Zac proteins containing single broken zinc fingersas noted at the top were tested for DNA binding (DB%), half-maximal transactivation (TA%), and protein expression levels. (H) Scheme of theC2H2 zinc finger. Residues forming the �-sheet and the �-helix are shown in dark and light gray, respectively. Key positions within the �-helix arenumbered (1, 2, 3, and 6). Conserved residues are cysteine (Cys) and histidine (His) coordinated by one zinc ion (Zn), phenylalanine (F) ortyrosine (Y), and any hydrophobic amino acid (). (I) Key residues within the recognition helix of zinc fingers 6 and 7 participate in DNA binding.Mutated key residues within the �-helices (positions 1, 2, 3, and 6) of zinc fingers (ZF) 6 and 7 were tested for DNA binding (DB%),half-maximal transactivation (TA%), and protein expression. (J) Zinc fingers 6 and 7 are sufficient for G4C4 DNA binding and transactivation.Native Zac, Zac containing solely zinc fingers 6 and 7 (Zac�ZF1-5), or additionally single broken zinc fingers (Zac�ZF1-5/6mt and Zac�ZF1-5/7mt) or �-helical mutations (Zac�ZF1-5/R173N and Zac�ZF1-5/R201N) were tested for DNA binding, transactivation, and protein expression.(K) Scheme comparing predicted amino acid (AA) base (nt) contacts of key residues (1, 2, 3, and 6) within the �-helices of zinc fingers (ZF)6 and 7 to the palindrome G4C4.


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conserved center of the palindrome G4C4 indicated a limitedflexibility of zinc fingers 6 and 7, compatible with position 6 ofzinc finger 6 and position 2 of zinc finger 7 reciprocally con-tacting bases on the primary strand of the respective up- anddownstream subsites.

To investigate this code/consensus correlation, we individu-ally replaced each key residue within the �-helices with aspar-agine, a structurally related amino acid containing a side chainwith a very low affinity for guanosine or cytidine residues.Replacement at position 1 within zinc finger 6 (T167N)weakly reduced G4C4 binding and transactivation, suggestingan auxiliary role of this residue in base recognition (Fig. 2I). Incontrast, mutation of position 2, 3, or 6 of zinc finger 6 (K169N,D170N, and R173N) or of position 1, 2, 3, or 6 of zinc finger7 (R195N, D197N, H198N, and R201N) each strongly reducedDNA binding. All key residues within the �-helices of zincfingers 6 and 7 were important to transactivation except posi-tion 3 of zinc finger 6, which preserved some activity. Probably,conditions for DNA binding in vitro were more stringent thanthose for transactivation in vivo, or additional protein interac-tions occurring in vivo mitigated the phenotype. Taken to-gether, our data conclusively showed that Zac DNA bindingand transactivation of the palindrome G4C4 involved all keyresidues within the �-helices of zinc fingers 6 and 7.

Lastly, to investigate whether zinc fingers 6 and 7 weresufficient for G4C4 binding, we studied broken zinc finger and�-helix mutations within the context of a Zac protein havingonly these two zinc fingers. As shown in Fig. 2J, a single brokenzinc finger (6 or 7) abolished G4C4 DNA binding and transac-tivation. Noticeably, however, nuclear protein levels of theseconstructs (Zac�ZF1-5/6mt and Zac�ZF1-5/7mt) were weaklyor strongly reduced compared to the corresponding mutationscontained within the entire zinc finger domain (Fig. 2G). Thisfinding points to a second nuclear translocation signal in theN-terminal half of the zinc finger domain that could compen-sate for the impaired nuclear localization in case of broken zincfingers 6 and 7, respectively. In contrast, single �-helical mu-tations in either zinc finger 6 or 7 (Zac�ZF1-5/R173N andZac�ZF1-5/R201N) led to a complete loss of G4C4 DNA bind-ing without changes in nuclear protein expression levels anddemonstrated that each was necessary and sufficient for DNAbinding and transactivation.

Zac binding to a G-rich DNA element differentially regu-lates transcriptional activities. With respect to the G-rich con-sensus motif, we equilaterally decreased the number ofguanosine residues to determine bases essential to Zac DNAbinding. Formation of Zac-DNA complexes in the case of themotif G4N6G4 were undistinguishable from the consensus mo-tif but disappeared in the case of the motifs G3N6G3 andG2N6G2 (Fig. 3A, left). The asymmetric motifs G4N6G3,G4N6G2, G4N6G1, and G4N6G0 showed similar behaviors, de-fining G4N6G4 as the recognition core (Fig. 3A, right). Unex-pectedly, none of these motifs conferred transactivation, whichled us to test the G4 clusters adjacent to each other or sepa-rated by an increasing number of unrelated nucleotides. Incomparison to the parent motif G4N6G4, Zac-DNA complexeswere reduced twofold in the case of G4N3G4. In contrast, theywere enhanced twofold in the case of G4N12G4 and unalteredin the case of the motifs G4N0G4 and G4N24G4 (Fig. 3B).

Again, none of the corresponding reporter plasmids conferredtransactivation by Zac.

To validate the core motif G4N6G4 as an authentic DNA-binding site, we determined its specificity and affinity. A 100-fold excess of unlabeled self-oligonucleotide completely abol-ished the Zac-DNA complex, whereas the motif G4N6G3 wasinactive (Fig. 3C). Furthermore, preincubation with Zac anti-serum but not with preimmune serum or an unrelated p53antibody completely abolished the shift, demonstrating thespecificity of the Zac-DNA complex. Finally, Zac-specificDNA-protein complexes were not observed for any of thesemotifs when nuclear extracts from mock-transfected cells wereused (data not shown). By Scatchard analysis, we measured forthe motif G4N6G4 an equilibrium binding dissociation constant(KD) of �4.2 nM (Fig. 3D), which is similar to the one ob-tained for the palindromic Zac element (KD, �2.7 nM).

During the early phase of this study, we observed that Zacstrongly repressed a simian virus 40 early promoter-driven�-galactosidase reporter plasmid which we initially used tostandardize transfection efficiencies. The simian virus 40 pro-moter contains within each of its two 72-bp repeat enhancerregions the sequence 5�-CCCCAGGCTCCCC-3�, which per-fectly matched a complementary Zac DNA-binding site (Fig.3E). In fact, Zac efficiently bound the 72-bp fragment in elec-trophoretic mobility shift assay experiments (KD �4.1 nM),whereas a single nucleotide substitution in the upstream half-site (5�-CCACAGGCTCCCC-3�) completely abolished ZacDNA binding (A. Hoffmann and D. Spengler, unpublishedobservations). Importantly, the presence of this mutation ineach of the 72-bp enhancer regions largely reduced Zac-me-diated repression of the simian virus 40 promoter in vivo (Fig.3F). Moreover, we also observed Zac-dependent repressionwhen a number of G-rich and/or C-rich cellular promoterswere additionally tested (A. Hoffmann and D. Spengler, un-published observations). Taken together, these findings dem-onstrated that Zac possessed, in addition to transcriptionalactivation, a repressor function, which became disclosed uponbinding to a single G-rich DNA element.

In view of these observations, we speculated that Zac dimer-ization on two neighboring DNA elements might be necessaryto unlock transactivation. In order to test this idea, we studiedZac behavior on two adjacent G4N6G4 motifs separated by sixunrelated nucleotides. In fact, the presence of a second copy ofthis motif enabled vigorous transactivation (Fig. 3G). In thefollowing experiments, we refer to this direct and related re-peat motifs as (G4N6G4)2 and (GnN6Gn)2, respectively. Next,we reanalyzed the motif G4N6G4 and its derivatives in thecontext of two copies which were each placed tail to tail andspaced by six unrelated nucleotides. Zac strongly bound to thereverse repeats (G3N6G4)2 and (G2N6G4)2 with similar or evenhigher affinity than observed for the direct repeat (G4N6G4)2,but weakly to the direct repeat (G3N6G3)2 (Fig. 3H). In sharpcontrast, Zac transactivation of the reverse repeats (G3N6G4)2

and (G2N6G4)2 was 2-fold and 10-fold, respectively, dimin-ished compared to the direct repeat (G4N6G4)2.

Because loss of transactivation occurred despite unchangedDNA binding, we hypothesized that Zac transactivation is site-specifically controlled. Moreover, dimerization, althoughpartly necessary for DNA binding, was apparently not sufficientfor transactivation. In this view, different mechanisms of DNA


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binding and/or mechanisms distinct from DNA binding regu-late Zac transactivation. To test these assumptions for thisclass of repeat elements, we studied, first, DNA binding byeach of the Zac zinc fingers and, second, amino acid basecontacts by key residues within each DNA-binding recognitionhelix. Deletion of zinc finger 1 reduced transactivation twofoldcompared to wild-type Zac, although DNA binding of(G4N6G4)2 was unaltered (Fig. 3I). Deletions downstream ofzinc finger 1 collectively abolished DNA binding and transac-tivation.

Therefore, we next tested Zac cDNA constructs encodingsingle broken zinc fingers. In these experiments, zinc fingers 2to 4, 6, and 7 proved necessary to (G4N6G4)2 DNA binding andtransactivation, whereas zinc fingers 1 and 5 were dispensable(Fig. 3J). Importantly, we obtained identical results for a singleG4N6G4 element (data not shown). Based on these data, weconcluded that the absence or presence of transactivation inthe case of a single or a repeat element did not result fromnumerical differences in zinc finger contacts but, alternatively,

from mechanistic differences in the amino acid base contactsestablished by each DNA-bound zinc finger.

Because Zac binding to the repeat elements depended onzinc fingers 2 to 4 in addition to zinc fingers 6 and 7, weconsidered the former prime candidates for site-specific regu-lation of transactivation. The scheme of amino acids at posi-tions 1, 2, 3, and 6 within the �-helices of zinc fingers 2, 3 and4 compares anticipated nucleotides to the repeat element(G4N6G4)2 under the assumption that zinc fingers 2 to 4 bindto one half-site and zinc fingers 6 and 7 bind to the second one(Fig. 4A). We noticed a good code/consensus correlation,which in addition predicted that one base might be contactedby multiple amino acids.

To evaluate this correlation, we individually replaced eachkey residue within the �-helices of zinc fingers 2 to 4 withasparagine. Briefly, position 3 of zinc finger 2 (K48N) showedno base contacts, whereas substitution of position 6 (R51N)strongly reduced DNA binding and moderately reduced trans-activation (Fig. 4B). Similar results were obtained for position

FIG. 3. Zac DNA binding to a single or a repeat G-rich DNA element confers repression and transactivation, respectively. (A) Determinationof the core sequence within the G-rich consensus as described above. DNA-binding activity (DB%) is indicated. None of these motifs conferredtransactivation. (B) Distinct spacing of G clusters maintains DNA binding but enables no transactivation. G clusters (G4) were separated by 0, 3,6, 12, or 24 unrelated nucleotides and tested for DNA-binding activity (DB%). None of these motifs conferred transactivation. (C) Zac bindsspecifically to a single G4N6G4 motif. Zac-DNA complexes were abolished by unlabeled self-oligonucleotide G4N6G4 (100� cold) or by anti-Zacserum (�-Zac). The mutated motif G4N6G3 (100� cold mt) and preimmune and anti-p53 (�-p53) serum were inactive. (D) The G4N6G4 motif isa high-affinity DNA-binding site. The dissociation constant (KD, �4.2 nM) was measured as described above. (E) Schematic drawing of the simianvirus 40 (SV40) early promoter showing the two 72-bp enhancer regions, each containing a complementary Zac DNA-binding site and the three21-bp repeats encoding six canonical Sp1 sites. The sequence of the complementary Zac DNA-binding site is shown. (F) Zac dose-dependentlyrepresses simian virus 40 early promoter activity (SV40 wt). Repression was largely reduced for a simian virus 40 promoter containing a singlesubstitution (5�-CCACAGGCTCCCC-3�) in each of the cDNA binding sites (SV40 mt). (G) The direct repeat element (G4N6G4)2 conferstransactivation. Two copies of the motif G4N6G4 separated by six unrelated nucleotides conferred robust transactivation for increasing doses ofZac. (H) Reverse direct elements discriminate between Zac DNA binding (DB%) and transactivation (TA%). Zac binds to two copies of G3N6G4and of G2N6G4 with similar or even higher affinity than to G4N6G4 but not to the direct repeat G3N6G3, whereas transactivation progressivelydeclines. (I) Multiple zinc finger contacts are necessary to bind the repeat element. Successive truncations C-terminal to each zinc finger weretested for DNA binding (DB%) and transactivation (TA%). (J) Zac N- and C-terminal zinc fingers participate in binding of the direct repeatelement. Single broken zinc fingers were tested with (G4N6G4)2 for DNA binding (DB%) and transactivation (TA%), which depends on zincfingers 2 to 4, 6, and 7.


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1 (R73N) within zinc finger 3, whereas replacement of posi-tions 2 (D75N) and 3 (H76N) caused moderate defects inDNA binding and small impairments in transactivation. Mu-tation of position 6 within zinc finger 4 again showed an inter-mediate phenotype for DNA binding and transactivation. Im-munoblots demonstrated that this set of constructs wereexpressed at similar protein levels (Fig. 4B, bottom).

In general, changes in DNA binding and transactivation ofthese �-helical mutations of zinc fingers 2 to 4 were less thanthose obtained from the corresponding mutations of zinc fin-gers 6 and 7 when measured on the palindromic DNA element.These data suggest that multiple amino acids within the �-he-lices of zinc fingers 2 to 4 contact a single nucleotide base.More importantly, we again obtained identical results whenthis set of constructs were tested with a single G4N6G4 element(data not shown), which led us to conclude that base contactsestablished by zinc fingers 2 to 4 did not participate in site-specific control of transactivation.

In light of these findings, we went on to evaluate the effect ofindividually substituted key residues within the recognition he-lix of zinc fingers 6 and 7 for DNA binding to the single or therepeat G4N6G4 element. Replacement of positions 1 and 2by asparagine within zinc finger 6 (T167N and K169N) did notalter binding to a single G4N6G4 element, whereas a twofoldreduction occurred for mutation of positions 3 and 6 (D170Nand R173N) (Fig. 4C). In contrast, substitution of positions1, 2, and 6 within zinc finger 7 (R195N, D197N, and R201N)each strongly reduced DNA binding, whereas the effect ofposition 3 (H198N) was about twofold.

A strikingly different pattern of amino acid base contactsemerged when we tested this class of mutations of zinc fingers6 and 7 with the repeat element (G4N6G4)2. Of note, none ofthe mutations within the recognition helix of zinc finger 6interfered with efficient DNA binding (Fig. 4D). With respectto zinc finger 7, the function of positions 1, 2, and 3 withinthe recognition helix were conserved, with positions 1 and 2(R195N and D197N, respectively) being necessary and posi-tion 3 (H198N) being less required for DNA binding. Surpris-ingly, position 6 (R201N) within the �-helix of zinc finger 7 wascompletely dispensable for DNA binding to the repeat element

FIG. 4. Differential control of Zac transactivation. (A) Schemecomparing predicted amino acid base contacts of key residues (posi-tion 1, 2, 3, and 6) within the �-helices of zinc fingers 2, 3, and 4 toone half-site of G4N6G4. (B) Zac binding to the repeat element in-volves multiple amino acid base contacts of zinc fingers 2 to 4. Mutated

key residues within the �-helices of zinc fingers 2, 3, and 4 as noted atthe top were tested with (G4N6G4)2 for DNA binding (DB%) andtransactivation (TA%). Immunoblot shows the corresponding proteinexpression levels. (C to F) Site-specific control of transactivation oc-curs through zinc fingers 6 and 7. Mutated key residues within zincfingers 6 and 7 as noted at the top were tested for DNA binding(DB%) and transactivation (TA%) with the single element G4N6G4(C) or the repeat elements (G4N6G4)2 (D), (G3N6G4)2 (E), and(G2N6G4)2 (F). Positive control phenotypes (PC) are shaded and showa value of �1 for DNA binding divided by transactivation. (G) Zachybrids are selectively impaired in transactivation of the repeat ele-ment. The ectopic transactivation domain of VP16 replaces that of Zacin the hybrid Zac�LPR/VP and confers higher transactivation on(G4C4)2 compared to wild-type Zac, whereas transactivation on(G4N6G4)2 is strongly reduced. (H) Scheme summarizing the behaviorof mutated key residues within the �-helices of zinc fingers 6 and 7 ona single and the different repeat elements for DNA binding (DB) andtransactivation (TA). DNA motifs, zinc fingers (ZF), numbered andabbreviated amino acids, and their respective position within the �-he-lix are indicated. Activity scores are defined in the inset. Positivecontrol phenotypes are shaded.


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(G4N6G4)2, in sharp contrast to the behavior on a single ele-ment. Interestingly again, this zinc finger mutation conferredonly weak transactivation (10%) compared to wild-type Zac. Asimilar, albeit blunted phenotype was noted for mutation ofpositions 2 and 6 (K169N and R173N, respectively) of zincfinger 6, which reduced transactivation by 40% and 70%, re-spectively.

Based on these results, we concluded that this class of mu-tations distinguished contacts essential for transactivation fromthose involved in DNA binding. This phenotype, known as apositive control mutant (10), was clearly illustrated by dividingvalues for DNA binding by those for transactivation. Mutationof positions 2 and 6 within the �-helix of zinc finger 6 andposition 6 within the �-helix of zinc finger 7 gave results of 2.2,3.3, and 12, respectively. In contrast, all other �-helical muta-tions within the Zac zinc finger domain showed values of 1 orless, probably because analysis of transactivation in vivo wasless stringent than analysis of DNA binding in vitro. Takentogether, these results evidenced a clear mechanistic differencein Zac binding to a single versus a repeat element. Thus, DNAsequences specifically controlled transactivation, first at thelevel of amino acid base contacts necessary for DNA bindingand second at the level of contacts distinct from DNA bindingbut necessary for transactivation. Based on these findings, wehypothesized that the different degrees of transactivation con-ferred by Zac on the individual repeat elements result from thedifferential use of DNA-dependent and -independent contactsin response to the specific nucleotide sequences.

To further test this prediction, we studied this class of mu-tations on the reverse repeat element (G3N6G4)2, which con-ferred half of the Zac transactivation noted for the directrepeat element (G4N6G4)2. Changes in DNA binding andtransactivation preferentially mapped to the positive controlphenotypes (Fig. 4E). Specifically, position 2 of zinc finger 6was dispensable for transactivation of the reverse repeat motif(G3N6G4)2. In contrast, position 6 of zinc finger 6 and zincfinger 7 was partly or strongly required for DNA binding.These results marked a transition in the function of position 6within the �-helix of zinc finger 7 with an exclusive role intransactivation on the direct repeat element (G4N6G4)2 and anadditional role in DNA binding on the reverse repeat element(G3N6G4)2. We reasoned that this transition should advancefurther if Zac is DNA bound but deficient in transactivation. Infact, Zac binding to the reverse repeat element (G2N6G4)2

strongly depended on position 6 within the �-helix of zincfinger 7 (Fig. 4F), whereas minor changes occurred for posi-tions 1, 2, and 3. These results on site-specific control of Zactransactivation are schematically summarized in Fig. 4H.Taken together, they imply that the DNA-binding domain ofZac specifically communicates with its transactivation functionand that this communication is tightly regulated by the under-lying amino acid base contacts within the �-helices of zincfinger 6 and, more importantly, zinc finger 7.

These findings led us to ask whether an unrelated transac-tivation domain could respond to this mode of regulation. Toaddress this issue, we replaced the Zac transactivation domainwith herpesvirus VP16 to create Zac�LPR/VP and comparedZac to this hybrid protein on both classes of reporter plasmids.Remarkably, Zac�LPR/VP strongly induced two copies of thepalindromic DNA element (G4C4)2, exceeding wild-type Zac

activity, but was about 10-fold less active than wild-type Zac onthe direct repeat element (G4N6G4)2, although it bound sim-ilarly to both sites (Fig. 4G and data not shown). Likewise, thehybrid proteins Zac�LPR/p53 and Zac�LPR/E1A, containingthe activation domains of human p53 (amino acids 1 to 64) andof adenovirus E1A (amino acids 121 to 223), respectively, wereselectively about 15-fold less active than wild-type Zac on therepeat element but not on the palindromic one (data notshown).

We suggest that DNA binding to the palindromic elementimposed no or few restraints on these Zac hybrids, whereasthey behaved as dysfunctional activators on the repeat ele-ment. These results further support the idea that Zac DNAbinding and transactivation of the repeat elements are care-fully coordinated and suggest that conformational restraints orregulatory protein interactions of the zinc finger domain inter-fered with the function of the unrelated transactivation do-mains.

Cooperative DNA binding and dimerization. Our resultsstrongly indicated that Zac molecules interact with each otheron adjacent DNA binding sites and raised the possibility thatsuch interactions also increase the affinity of DNA binding.Concordant with this prediction, we observed that Zac boundabout eightfold more to the repeat motif than to a single site,corresponding to a fourfold cooperativity in DNA binding(Fig. 5A). Preincubation with Zac antiserum completely abol-ished these shifts and proved their specificity. We furthertested whether an enlarged spacing between the two half-sitesof the repeat element could disrupt dimerization. Indeed, Zaccooperative DNA binding was strongly reduced when the twohalf-sites were separated by 12 instead of 6 unrelated nucleo-tides [(G4N6G4N12)2]. Again, Zac antiserum completely pre-vented this shift.

Additional experiments with solely the Zac DNA-bindingdomain obtained from nuclear extracts, purified from Esche-richia coli or produced by in vitro translation failed due toeither undetectable expression levels, strongly reduced DNA-binding activity, or the formation of multiple nonspecific com-plexes. Because the Zac-DNA complex on the repeat elementmigrated near the one on a single G4N6G4 site and because theDNA fragments tested were of equal size, we suggest thatdimerization of native Zac led to the formation of complexeswith enhanced mobility. Moreover, we propose that the aber-rant migration of this Zac-DNA complex could point to dis-tinct conformations of native Zac assembled on a half-siteversus the repeat motif and that subtle changes in Zac DNA-binding domain structure might be translated to other Zacregions, resulting in more global conformational changes thatcontrol transcriptional activities.

We then sought to detect Zac dimerization in vivo by coim-munoprecipitation experiments with cellular extracts obtainedfrom cotransfection of HA- or vesicular stomatitis virus Gprotein epitope-tagged Zac. p53 which was similarly taggedserved as a positive control in these studies. Immunoblots fromZac or p53 cotransfections evidenced no Zac immunoreactiv-ity, whereas p53 could be readily detected (data not shown).Based on these results, Zac dimerization appeared to be tooweak to persist under these experimental conditions.

Therefore, to study Zac interaction in more detail, we em-ployed the GST pulldown assay. These experiments clearly


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demonstrated that in vitro-translated Zac bound efficiently to aGST-Zac fusion protein. Remarkably, deletion of the zinc fin-ger domain completely abolished retention (Fig. 5B, lanes 3 to4), whereas absence of Zac transactivation domain (Zac�LPR)or of the whole C terminus (Zac�C) maintained binding toGST-Zac. In the reverse experiment, GST-Zac lacking the zincfinger domain (GST-Zac�ZF) bound none of these proteins,and none of them bound to GST alone. Moreover, a GSTfusion protein containing solely zinc fingers 1 to 7 (GST-Zac-ZF) efficiently retained Zac (Fig. 5C, lanes 1 to 2). Zac bindingresisted the inclusion of ethidium bromide (100 �g ml1) inthe incubation step, demonstrating that it was not due to thepresence of bacterial DNA (data not shown). Therefore, theZac zinc finger domain was necessary and sufficient for dimer-ization.

To narrow the interacting region within the zinc finger do-main, successive N-terminal deletions of Zac were tested witha GST fusion protein containing the entire zinc finger domain.Retention of Zac was reduced by one-third and a strikingninefold for deletion of zinc fingers 1 and 2, respectively (Fig.5C, lanes 3 to 6). Further downstream truncations graduallydeclined in GST-Zac binding, which was completely abolishedupon deletion of zinc finger 6, indicative of a second, albeitweak interaction interface. In turn, a GST fusion protein con-taining solely zinc fingers 1 and 2 (GST-Zac-ZF1-2) efficientlyretained full-length Zac, demonstrating that this region wasnecessary and sufficient for binding (Fig. 5E, lanes 1 to 2).

This finding raised the question of which structural domainsof the second zinc finger participate in dimerization. Aminoacids of zinc fingers 1 and 2 in relation to the structure of aprototypical C2H2 zinc finger are shown in Fig. 5D. Additionalrefined analysis of zinc finger 2 demonstrated that Zac bindingrequired amino acids 28 to 40, whereas the putative �2 strandand the �-helix of zinc finger 2 were dispensable (Fig. 5E).Within this interactive region, mainly the first H-C link and �1strand, but less so the amino acid loop connecting the cysteineligands, were required for Zac binding.

To evaluate these findings for Zac interaction in vivo, weemployed the mammalian two-hybrid system and cotrans-fected the Zac zinc finger domain fused to Gal4 (Gal4-ZF)with a plasmid containing the zinc finger domain fused to thetransactivation domain of VP16 (ZF/VP). This hybrid dose-dependently increased transactivation, whereas VP16 alonewas inactive (Fig. 5F, left). In the reverse experiment, Gal4-ZFbut not Gal4 alone restored transactivation by ZF/VP (datanot shown). These findings were further strengthened by elec-trophoretic mobility shift assay studies with Zac constructscontaining N-terminal truncations of zinc finger 2 and the

FIG. 5. Cooperative DNA binding and dimerization. (A) Zacdimerization enhances DNA binding. Fragments containing one ortwo (G4N6G4) sites were separated by 6 (N6) or 12 (N12) unrelatednucleotides, as noted at the top. In case of N6, fragments were of equalsize. Electrophoretic mobility shift assays were done in the absence() or presence (�) of Zac antiserum (�-Zac). DNA-binding activity(DB%) for (G4N6G4N6)2 and (G4N6G4N12)2 was set to 100% to cal-culate cooperativity (CO). Arrows mark Zac-DNA complexes. Noteenhanced migration of the dimer DNA complex. (B) Zac dimerizesthrough its zinc finger domains. In the GST pulldown assay, equalamounts of in vitro-translated Zac, Zac�ZF, Zac�LPR, and Zac�Cwere incubated with GST-Zac, GST-Zac�ZF, and GST alone. Lanes, 10% of the input; lanes �, fraction of the input (100%) bound byeach GST protein (BD%). (C) Mapping of zinc fingers participating indimerization. Mutants with deletions C-terminal to each zinc fingerwere incubated with GST-Zac-ZF containing zinc fingers 1 to 7. Se-quences upstream of zinc finger 2 are necessary for binding. (D) Top,scheme of prototypical C2H2 zinc finger structure; �1 and �2 strandand �-helix are outlined. Bottom, Zac sequence of zinc fingers 1 and 2;amino acids are numbered. (E) Fine mapping of interactive subregions

of zinc finger 2. Refined N-terminal deletions of zinc finger 2 (numbersas above) were incubated with GST-Zac-ZF1-2 containing solely zincfingers 1 and 2. The first H-C link and the �1 strand of ZF2 areimportant to binding. (F) Zac dimerization in vivo depends on zincfinger 2. In the mammalian two-hybrid system, G-ZF (10 ng) contain-ing the Zac zinc finger domain fused to Gal4 was cotransfected withincreasing doses of the hybrid ZF/VP, which contains the zinc fingerdomain fused to the VP16 transactivator. The transactivator VP aloneserved as a control (left). To map the interactive subregions of zincfinger 2, the hybrids ZF/VP, ZF�31/VP, ZF�34/VP, and VP alone(200 ng each) were cotransfected with G-ZF (right).


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repeat element (G4N6G4)2. We noted in the absence of thefirst H-C link (Zac�31) a twofold impairment in DNA binding,whereas absence of the �1 strand of zinc finger 2 abolished anyDNA binding, precluding detailed analysis at this step (datanot shown).

Lastly, we evaluated the importance of these amino acids forZac dimerization by means of the two-hybrid assay. Theseexperiments showed substantial reductions in transactivationupon deletion of the first H-C link and the �1 strand (Fig. 5F,right). In sum, our results from electrophoretic mobility shiftassays, GST pulldown experiments, and the two-hybrid systemgave strong and concurrent evidence for Zac dimerizationthrough zinc finger 2.

Zac differentially regulates the cytokeratin gene family andtransactivates through the palindromic and repeat element.To address the importance of our present findings for theregulation of endogenous genes, we used tetracycline-regu-lated Zac expression (24). Zac protein levels reached a plateau12 h after tetracycline removal, and RNAs from cells grown foran additional 12 h were probed with a cDNA expression array.These results were compared to signals obtained from cellskept for 24 h with tetracycline (A. Hoffmann and D. Spengler,unpublished observations). Zac regulated the family of cyto-keratin (CK) genes in a differential manner by inducing CK4,CK6, and CK14 gene expression and repressing CK2, CK7,CK12, and CK19 gene expression (Fig. 6A).

We analyzed CK14 gene regulation in more detail by reversetranscription-PCR experiments, which we performed at differ-ent time points following tetracycline removal. Upregulation ofCK14 expression occurred as early as 6 h after tetracyclineremoval, compatible with a direct transcriptional effect of Zac,and peaked at 24 h (Fig. 6B). In contrast, a tetracycline-regu-lated cell clone containing Zac lacking the zinc finger domain(Zac�ZF) showed no significant change in CK14 mRNA levelsdespite similar steady-state levels of Zac and Zac�ZF proteins(data not shown). We observed faint immunostaining for CK14protein in the repressed state of both cell clones, whereasrobust immunoreactivity resulted from wild-type Zac expres-sion in the absence of tetracycline (Fig. 6C).

The human CK14 promoter contains several G- and C-richsequences matching both classes of Zac DNA elements, whichwere designated M1 to M6 (Fig. 6D). Two imperfect repeatmotifs (M2 and M3) and one incomplete palindromic site (M5)localized to a SmaI-BstXI fragment present in the distal CK14promoter region (Fig. 6D, lower scheme). To study regulationby Zac, we transferred this fragment in front of a TATAelement. Despite somewhat lower constitutive activity com-pared to the intact CK14 promoter, expression levels of thisconstruct still largely exceeded that of the minimal promoter(�5,000-fold). Cotransfection of Zac, however, furtherstrongly activated (eightfold) this promoter plasmid but notthe parent vector (Fig. 6E). Moreover, a Zac cDNA containingsolely zinc fingers 6 and 7 (Zac�ZF1-5) conferred significanttransactivation, albeit less than that obtained with wild-typeZac, whereas Zac proteins containing a broken zinc finger 7(ZacZF7mt) or lacking the transactivation domain (Zac�LPR)were inactive in CK14 promoter regulation.

These findings led us to analyze Zac DNA binding andtransactivation in the context of the CK14 promoter and tocompare the results with those described in the previous sec-

tion. To this end, we first studied the behavior of the individualmotifs, then identified mutations that abolished their activities,and finally analyzed the effect of these mutations in the contextof the distal CK14 promoter. Oligonucleotides encoding motifsM1 to M6 were inserted separately into the minimal reporterplasmid, and DNA binding and transactivation were studied asdescribed in the previous sections. Zac bound with similar oreven higher affinity to the imperfect repeat motifs M1, M2, andM4 compared to the perfect repeat, whereas motif M3, whichcontained an interrupted central G cluster in one half-site,showed clearly reduced complex formation (Fig. 6F, left). Ad-dition of the Zac antibody abolished these Zac-DNA com-plexes and demonstrated their specificity.

Importantly, Zac transactivation measured for the imperfectrepeat motifs M1, M2, and M4 was consistently less than thatobtained for the perfect repeat motif concurrent with the dif-ferential use of zinc finger contacts for DNA binding andtransactivation (Fig. 6F, bottom). Moreover, we studied theimperfect palindromic motifs M5 and M6, which both showedspecific, albeit reduced Zac DNA binding and transactivation(Fig. 6F, right). In further agreement with our previous results,transactivation of these imperfect palindromes was orientationdependent.

To identify mutations that could abolish Zac transactivationby the motifs M2, M3, and M5, we schematically comparedthem to the respective perfect repeat and palindromic motifsas depicted in Fig. 6G. The repeat motif M2 contains thesequence G3N1G4, which confers moderate transactivation inthe context of a repeat motif, in one half-site, whereas thesecond half-site corresponds to the sequence G2N6G4, which isinactive in this respect. Substitution of one G residue in theoutward cluster of the functional half-site led to the motifM2mt, largely deprived of transactivation despite unchangedZac DNA binding (Fig. 6G). In contrast, motif M3 showedconcomitant reductions in DNA binding and transactivationdue to disruption of one of the central G clusters which regu-late Zac dimerization. We reasoned that additional disruptionof the outward G clusters (M3mt) should interfere with theresidual transactivation but not with DNA binding, which wasin fact the case (Fig. 6G). Furthermore, insertion of one nu-cleotide at the center of the imperfect palindrome (M5mt)completely prevented Zac DNA binding and transactivation(Fig. 6G).

Having identified mutations that interfered with transactiva-tion of the isolated motifs, we studied their effects within thecontext of the distal CK14 promoter fragment. We stepwisemutated either both repeat motifs or the palindrome or bothclasses of motifs. The corresponding distal CK14 promoterconstructs were cotransfected with either native Zac, a dele-tion of the N-terminal five zinc fingers (Zac�ZF1-5), or abroken zinc finger 7 (ZacZF7mt). In the presence of the mu-tated repeat motifs, transactivation by wild-type Zac was re-duced by 60% compared to the intact promoter plasmid,whereas that of Zac�ZF1-5 was unchanged (Fig. 6H). In con-trast, the mutated palindrome (M5mt) additionally preventedtransactivation by Zac�ZF1-5 (Fig. 6H). Because wild-typeZac similarly transactivated both mutated promoter plasmids,albeit at a reduced level compared to the intact promoter,transactivation apparently occurred alternatively through therepeat and palindromic binding sites. Lastly, a CK14 fragment


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FIG. 6. Zac differentially regulates the cytokeratin gene family and transactivates through the palindromic and repeat elements. (A) Zacdifferentially regulates cytokeratin (CK) gene expression. Zac induces CK4, CK6, and CK14 gene expression but represses CK2, CK7, CK12, andCK19 gene expression (fold regulation). (B) Time course of CK14 gene induction. CK14 gene expression was measured by reverse transcription-PCR in Zac- and Zac�ZF-expressing cell clones at the indicated times (hours) after tetracycline removal. (C) Zac expression enhances CK14immunoreactivity. Immunostaining for CK14 protein (brown) in Zac- and Zac�ZF-expressing cells grown with (�) or without () tetracycline for24 h. Counterstaining was done with toluidine blue. (D) Scheme of CK14 promoter sequences matching the palindromic or repeat ZacDNA-binding site. Elements represented in the distal SmaI-BstXI fragment are shown in black, and the locations of the different classes of motifswithin the intact promoter are schematically indicated. (E) Zac transactivates the distal CK14 promoter plasmid. Cotransfection of Zac andZac�ZF1-5 dose-dependently transactivated the SmaI-BstXI fragment derived from the CK14 promoter in front of a TATA element but not thevector alone. In contrast, Zac cDNAs encoding a broken zinc finger 7 (ZacZF7mt) or lacking the transactivation domain (Zac�LPR) were inactive.(F) Zac differentially binds and transactivates motifs from the CK14 promoter. Zac DNA binding of the perfect repeat (rep) was set to 100% andcompared to that of the imperfect repeat motifs M1 to M4 (left). Similarly, the perfect palindrome (pal) was compared to the incompletepalindromic motifs M5 and M6 (right). Half-maximal transactivation of the corresponding reporter plasmids is given at the bottom. (G) MutatedCK14 promoter motifs distinguish Zac DNA binding (DB%) from transactivation (TA%). Structural scheme of motifs M2, M3, and M5. Colorcode: green, orange, and red symbolize nucleotides predicted to promote or to interfere slightly or strongly with transactivation, respectively.Actual DNA binding (DB%) and transactivation (TA%) were measured as above and are indicated. (H) Mutated repeat and palindromic elementsin the distal CK14 promoter selectively interfere with Zac transactivation. CK14 promoter fragments containing wild-type () or mutatedsequences (�) of M2, M3, and M5 as shown above were cotransfected with Zac, Zac�ZF1-5, and ZacZF7mt (50 ng of each).


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containing mutations in both classes of binding sites failed tosignificantly confer Zac transactivation, as was the case for aDNA-binding-defective Zac protein (Fig. 6H).

Taken together, these results strongly support Zac’s role astranscription factor regulating gene repression and transacti-vation and demonstrate the presence of the palindromic andrepeat DNA elements in the context of a complex promoter ofa newly identified Zac target gene. Moreover, Zac DNA bind-ing and transactivation of a natural gene confirmed the criticalrole of the zinc finger domain in communicating with thetransactivation function.

DISCUSSION

We report here that Zac contains transactivation and repres-sor activities which are differentially controlled by DNA bind-ing (Fig. 7). Moreover, we demonstrate for Zac a role as atranscription factor which confers transactivation through tworelated DNA elements in a differential manner (Fig. 7).

Zac transactivation. We used the heterologous Gal4 fusionsystem to map two adjacent structurally distinct domains whichconferred synergistically potent transactivation. The zinc fingerdomain strongly silenced transactivation of the full-length Zacfusion protein, although it did not interfere with DNA binding.This could indicate that the native Zac zinc finger domainexerts an inhibitory effect on cognate transcriptional activationfunction through conformational restraints and/or heterolo-gous proteins (15). Our results on differential control of Zactranscriptional activities support such a possibility, and addi-tional studies are needed to resolve this issue. Importantly,however, separate expression of each of the candidate domainsshowed transactivation. Moreover, the absence of either orboth of them partly and completely, respectively, preventedZac transactivation either in the heterologous Gal4 system oron the palindromic DNA element.

The Zac proline repeat domain contains a characteristicstructural motif which consists of proline in conjunction withan acidic residue. Acidic residues are crucial to the interactionof acidic activators with their target(s), though they do notdepend on the precise structural complementarity usually as-sociated with the formation of specific protein-DNA com-plexes (9). The amino acid triplet PLE behaved as an auton-omous unit, which conferred synergistic and strongenhancement of transactivation upon concatenation. Wecoined the term PLE minidomain to emphasize the distinctionof the general concept of the modular nature of transactivationdomains. The PLE minidomain was independent of the orderof prolines but strictly required the negatively charged glu-tamic acid. Recent evidence suggests that acidic and hydro-phobic residues play an important role in acidic transactivationdomains, though the position of these residues appears to begenerally unimportant (26). The PLE minidomain exemplifiedboth principles at the level of a simple triplet motif.

Differential control of transcriptional activities. It has beenincreasingly recognized that although some factors are pureactivators or pure repressors, transcriptional regulators mayalso be modified in an allosteric manner by response elementsthemselves to generate the pattern of regulation that is appro-priate to an individual gene (reviewed in references 14 and 15).Zac bound with high affinity to a single half-site of the direct

repeat element and potently repressed constitutively activepromoters containing this site. Because a Zac protein lackingits C-terminal repressor domain remained transcriptionally in-active when bound under this condition (A. Hoffmann and D.Spengler, unpublished observations), we additionally proposethat Zac transactivation was not simply inhibited by repressionbut was actively concealed. In this regard, the nature of theDNA sequence to which Zac bound seemed to regulate thetranscriptional response, as has been reported previously forsteroid and thyroid hormone receptors (15). Distinct Zac con-formations could promote selective interactions with the tran-

FIG. 7. Molecular bar code of Zac DNA binding and transactiva-tion. (A) Zac binds as a monomer to the palindrome G4C4 to trans-activate in an orientation-dependent manner. All key residues withinthe �-helix of zinc fingers (ZF) 6 and 7 (except position 1 of zincfinger 6) participate in DNA binding (DB) and transactivation (TA).The function of each key residue is shown in color, as defined in theinset. (B) Zac binding to one half-site of the direct repeat elementconfers repression instead of transactivation. DNA binding occurs bymultiple zinc finger contacts, including zinc fingers 2 to 4, 6, and 7.(C) Zac binding to the direct repeat element (G4N6G4)2 promotesdimerization through zinc finger 2 (open arrow) and transactivation.Multiple zinc fingers, including 2 to 4, 6, and 7, contribute to DNAbinding. Amino acid base contacts of key residues from the recognitionhelix of zinc fingers 6 and 7 involve DNA-dependent and DNA-inde-pendent interactions. The latter, as highlighted by position 6 of zincfinger 7, are essential to transactivation. Dots mark the positions of thecorresponding positive control phenotypes. (D) Zac binding to thereverse repeat element (G2N6G4)2 depends on dimerization throughzinc finger 2 (solid arrow). Transactivation is largely concealed, whichcorrelates with a transition in the amino acid base contacts of keyresidues within the �-helices of zinc fingers 6 and 7. Note that theamino acid base contacts of zinc fingers 2 to 4 are conserved betweenC and D, arguing against a direct role in regulation of transactivation.


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scriptional machinery, leading to either repression or transac-tivation. Since deletion of the repressor domain did not restoretransactivation of half-site-bound Zac and since dimerizationwas not sufficient for transactivation (see below), various con-formational transitions could exist depending on each DNAelement, and further studies are necessary to resolve this issue.

We identified here two related high-affinity DNA-bindingsites that operated through different mechanisms to controlZac transcriptional activities. Zinc fingers 6 and 7 were neces-sary and sufficient to recognize the palindromic DNA elementand to confer transactivation. Single-amino-acid base contactsinvolved all potential key residues of zinc fingers 6 and 7 andwere indistinguishably required for DNA binding and transac-tivation, in contrast to their differential regulatory role on therepeat elements (Fig. 7). These data, together with the orien-tation-dependent transactivation, strongly support the ideathat Zac binds as a monomer to the palindromic DNA-bindingsite.

Zac dimerization on the different repeat elements conferredtransactivation, which strictly correlated with the pattern ofamino acid base contacts by key residues within the �-helicesof zinc finger 6 and, more importantly, zinc finger 7. Position 6within the �-helix of zinc finger 7 played a key role in distin-guishing contacts necessary for DNA binding from those nec-essary for transactivation (Fig. 7). In the case of the reverserepeats (G3N6G4)2 and (G2N6G4)2, we observed a transition inthe function of these amino acid residues which tightly corre-lated with a parallel decrease in transactivation. The positivecontrol phenotype of these residues was most evidently dis-closed by the direct repeat element (G4N6G4)2 and suggestedthat a surface of the DNA-binding domain may be directlyinvolved in transcriptional regulation, by regulating either pro-tein-protein contacts, DNA structure, or a combination ofthese mechanisms. These positive control residues play a crit-ical role in interpreting and signaling the information providedby the response element at which Zac binds to its transactiva-tion domain.

In the well-characterized C2H2 zinc finger motif, the recog-nition helix binds in an antiparallel manner to DNA, with theN terminus directed into the major groove and the C terminusdirected toward the surrounding environment. The positivecontrol subregion itself or a region affected by these changescould define a site that interacts with other proteins in tran-scriptional regulation. To our knowledge, this mode of regu-lation is unprecedented among transactivators, since key resi-dues within the recognition helix alternate between basecontacts necessary for transactivation and DNA binding, andspecific zinc finger dimer conformations may preclude or en-hance recruitment of factors necessary to transactivation. Insupport of this view, specific dimer configurations of the DNA-binding domain of the transcription factor Oct-1 have recentlybeen reported to differentially synergies with the coactivatorOBF-1 (Oct-binding factor 1) (25). These observationsstrongly emphasize the connection between DNA sequence,protein conformation, multimerization state, and transcrip-tional activity, although the dimer interface appeared to playno direct role in Zac transcriptional activation (see below).

Importantly, we demonstrated that Zac differentially regu-lates gene expression in vivo, as exemplified by the family ofcytokeratin genes. We identified both classes of DNA-binding

sites within the distal CK14 promoter which specifically con-ferred transactivation by Zac and further supported the differ-ential role of Zac zinc finger contacts in DNA binding andtransactivation in the regulation of a natural promoter. Nu-merous reports employed the common DNA-binding site se-lection assay to identify DNA elements for transcription fac-tors from distinct families. In many cases, in vitro selection ledto the isolation of optimal binding sites, whereas naturallyoccurring response elements displayed nucleotide divergencethat lowered binding affinity and/or transcriptional activities.For instance, all steroid receptor-specific elements character-ized from natural promoters are of lower affinity than thecanonical binding site (18). Therefore, not surprisingly, theZac DNA-binding sites identified in vivo in the CK14 genepromoter conferred less transactivation than the in vitro-se-lected optimized sites due to differences in symmetry. Impor-tantly, however, mutation of the structural core of these nat-ural Zac elements was poorly tolerated, if at all (Fig. 6G).

Zac dimerization and transcriptional regulation. Zac inter-acted through amino acid residues localized in the first H-Clink and the �1 strand of zinc finger 2. Protein-protein inter-actions can be mediated via C2H2 zinc fingers (17), and geneticselection experiments to search libraries for peptides identifiedsequences as short as six residues to support in vivo dimeriza-tion on DNA (31). Relatively weak interactions that are notsufficient to give stable dimers in solution can still dramaticallystabilize the corresponding protein-DNA complexes (29). Un-der these conditions, both the peptide and the surface that itrecognizes are present at high local concentrations when theseproteins bind to adjacent DNA sites. Concordant with thisview, our coimmunoprecipitation experiments showed no Zacinteraction. More importantly, we obtained concurrent evi-dence for Zac dimerization by electrophoretic mobility shiftassays, GST pulldown experiments, and the two-hybrid system.

Zac dimerization was dispensable for DNA binding to thehalf-site element G4N6G42, which conferred transcriptional re-pression. In contrast, cooperative DNA binding at the directrepeat element involved dimerization and conferred potenttransactivation. Moreover, Zac dimerization was prerequisitefor DNA binding to the reverse repeat elements (G2N6G4)2

and (G3N6G4)2, although it was not sufficient to confer robusttransactivation. Based on these findings, we conclude that ZacDNA binding and dimerization mutually depended on eachother to unlock transactivation. Additional analysis of the re-verse and direct repeat elements with Zac proteins containing�-helical mutations within zinc fingers 2, 3, and 4 showedsimilar and mostly parallel reductions in DNA binding andtransactivation (A. Hoffmann and D. Spengler, unpublishedobservations). Therefore, the zinc fingers engaged in Zacdimerization appeared to play no direct role in transcriptionalregulation, in contrast to zinc fingers 6 and 7 (Fig. 7).

DNA binding distinguishes between Zac biological activi-ties. Our results strongly support a role for Zac as a transcrip-tion factor in addition to recent reports pointing to a role ascoactivator and corepressor (11, 12, 23). Numerous nuclearreceptor-interacting proteins have been identified in the pastfew years (22) and raised questions about the definition of acoactivator. A real coactivator first must interact directlyand/or indirectly with the activation domain of a nuclear re-ceptor in an agonist-dependent manner, leading to enhance-


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ment of the receptor activation function, and second must notenhance the basal transcriptional activity on its own, althoughit contains an autonomous activation function. Our presentwork can potentially shed new light on Zac’s regulatory func-tion for nuclear receptors (12), and additional investigations tounderstand the mode of these interactions in greater detail arewarranted. Regardless of whether Zac is eventually found tohave a dual role as a transcription factor and coactivator, ourpresent report emphasizes the possibility that the identificationof Zac-dependent target genes could advance our insight intoits role in proliferation and metabolism and may help to elu-cidate the consequences of altered Zac/ZAC expression inneoplasia and diabetes.

The existence of two classes of Zac DNA-binding sites wasanticipated from our recent studies (5). Here, we identifiedZAC�2, an alternatively spliced variant of human ZAC lackingthe sequence encoding the two N-terminal zinc fingers. Inter-estingly, although both proteins were equally potent in anti-proliferation, their activities ensued from a differential regula-tion of apoptosis versus cell cycle progression, since ZAC�2was more efficient at induction of cell cycle arrest than ZAC,whereas the reverse was the case for the induction of apopto-sis. We obtained similar data for tetracycline-regulated expres-sion of Zac and Zac�ZF1-5 proteins (A. Hoffmann and D.Spengler, unpublished observations). Based on these results,we had previously predicted (5) the possibility of ZAC DNAbinding to elements requiring zinc finger 1 and/or 2, as illus-trated now by the repeat motifs.

Collectively, we propose that Zac binding to genes contain-ing the repeat element in their regulatory region preferablyinduces apoptosis, whereas those containing the palindromicelement preferably control G1 arrest (Fig. 8). It is noteworthy

that the ratio of ZAC to ZAC�2 mRNA varies among indi-viduals (5), and further studies will be necessary to determinewhether this heterogeneity is due to polymorphic variationsamong human populations or whether it is linked to differentphysiological conditions between individuals at the time thesamples were collected. In either case, alternative splicingcould serve to distinguish between ZAC antiproliferative ac-tivities due to differential DNA binding and might contributeto ZAC-related pathologies.

ACKNOWLEDGMENTS

The human K14 promoter fragment (U11076) was a kindly gift fromJ. E. Kudlow (University of Alabama at Birmingham).

This work was supported by grants from the MPIP and the DFG(SP386/4-1) to D.S. and the CNRS to L.J.

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Transcriptional Activities of the Zinc Finger Protein Zac Are