Proc. Natl. Acad. Sci. USAVol. 91, pp. 4514-4518, May 1994Biochemistry

The Kruppel-associated box-A (KRAB-A) domain of zinc fingerproteins mediates transcriptional repression

(transcription factors/Kid-1/ZNF2/kidney)

RALPH WITZGALL, EILEEN O'LEARY, ALEXANDER LEAF, DILEK ONALDI, AND JOSEPH V. BONVENTRE*Medical Services, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129; and Department of Medicine, Harvard Medical School,Boston, MA 02115

Contributed by Alexander Leaf, February 3, 1994

ABSTRACT We have previously reported the cloning,sequencing, and partial characterization of Kid-i, a zincfinger-encoding cDNA from the rat kidney. The Kid-i proteinand approximately one-third of all other zinc finger proteinscontain a highly conserved region of -75 amino acids at theirNH2 terminus named Krtippel-associated box (KRAB), whichis subdivided into A and B domains. The evolutionary conser-vation, wide distribution, and genomic organization of theKRAB domains suggest an important role of this region in thetranscriptional regulatory function of zinc finger proteins. Thefunctional significance of the KRAB domain was evaluated bystudying transcriptional activities of yeast GAL4-rat Kid-ifusion proteins containing various regions of the non-zinc-finger domain of Kid-i. Transcriptional repressor activity ofGAL4-Kid-1 fusion proteins maps to the KRAB-A domain.The KRAB-A domain of another zinc finger protein, ZNF2,also has repressor activity. Sitedirected mutagenesis of con-served amino acids in this motif results in decreased repressoractivity. Thus, we have established a functional sign ifnce forthe KRAB-A domain, a consensus sequence common in zincfinger proteins.

A limited number of structural motifs have been described foreukaryotic transcription factors. A common motif is the zincfinger structure, which is highly conserved and found in manydifferent species (1). Though the crystal structure of a co-crystal between the three zinc fingers of zif-268 (Egr-i) andtheir binding site has been elucidated (2), little is known aboutthe interaction between zinc fingers and DNA and how theseprotein-DNA interactions are involved in the regulation oftranscription.Approximately one-third of all zinc finger proteins contain

an evolutionarily conserved region ofabout 75 amino acids attheir NH2 terminus-the Krippel-associated box (KRAB), asequence motif of hitherto unknown function. Similar toregions found in a subset of homeodomain proteins (thepaired box and POU domain), the KRAB domain is rich incharged amino acids (3). Because of the potential a-helicalstructure of KRAB domains, it has been proposed that thisdomain mediates protein-protein interactions and functionsas a transcriptional regulatory domain (3), but this hypothesishas not been previously tested.

Kid-i is a rat zinc finger gene that is expressed primarily inthe kidney (4). Kid-i mRNA levels are regulated in renalontogeny and during repair after ischemic and folic acidinsults to the kidney (4). Kid-i encodes a 68-kDa protein with13 zinc fingers. When COS or LLC-PK1 cells are transfectedwith fusion constructs encoding the non-zinc-finger region ofrat Kid-i and the yeast GAL4 DNA-binding domain, chlor-amphenicol acetyltransferase (CAT) activity from cotrans-fected reporter constructs containing GAL4 binding sites and

either a minimal promoter or a simian virus 40 (SV40)enhancer is strongly repressed (4). We designed experimentsto test the hypothesis that the transcriptional repressoractivity resided in the KRAB domain of Kid-i (Fig. 1). Ourmutagenesis studies with the Kid-i protein define theKRAB-A domain as a strong transcriptional repressor motif.

METHODSExpression Plasmids. Plasmids encoding chimeric proteins

ofthe DNA-binding domain (amino acids 1-147) ofGAL4 andvarious parts of the non-zinc-finger region of Kid-i (desig-nated Kid-iN) were constructed from pBXGi/Kid-lN insense orientation (4) (designated pBXGi/Kid-lN,s), encod-ing a GAL4-Kid-i fusion protein containing the entire non-zinc-finger region of Kid-i (Fig. 2). pBXGi/Kid-i,AB wasmade by vector self-ligation after cutting pBXGi/Kid-lN,swith BamHI. To construct pBXGi/Kid-i,A(-) lacking theKid-i KRAB-A domain, pBXGi/Kid-lN,s was digested withEcoRI and filled in by using the Klenow fragment. The insertwas subsequently removed by digestion with Xba I, and thevector fragment was isolated. Insert was prepared by digest-ing pBXG1/Kid-lN,s with Nco I, filling in the overhang withthe Klenow fragment, and releasing the insert with Xba I. Toconstruct pBXG1/Kid-1,A and pBXG1/Kid-1,B, fragmentscontaining the Kid-i KRAB-A- or B-encoding sequences(designated Kid-iA and Kid-iB) were PCR-amplified frompBXGi/Kid-i,AB with primers containing an EcoRI site orXba I site. PCR products were digested with EcoRI and XbaI and cloned into the EcoRI/Xba I-cut pBXGi vector. Toconstruct pBXG1/ZNF2,A, the exon encoding the KRAB-Adomain of ZNF2 (designated ZNF2,A) was PCR-amplifiedfrom human genomic DNA with primers containing EcoRIand Xba I sites and subcloned into EcoRI/Xba I-cut pBXGi.pCDM8/Kid-l,f', in which Kid-l,t signifies coding region forthe full-length Kid-i protein, was engineered by ligatingBstXI-adapters to a Kid-i cDNA fragment extending fromnucleotide 312 to 2123 [as numbered in the cDNA (4), wherethe putative start codon is at 312 and the stop codon is at 2040]and ligating it into BstXI-cut pCDM8. PCR products weresequenced in their entirety by the chain-termination method.Cloning sites of all constructs were sequenced (5).

Site-Directed Mutagenesis. A PCR-based strategy (6) wasused to construct mutants with single amino acid changes inthe KRAB-A domain of Kid-i (Kid-1,A). Two PCR reactionswere run with pBXG1/Kid-1,A as a template. In the firstreaction, one primer, "F," complementary to the 3'-end ofGAL4, was paired with a primer containing the desired

Abbreviations: KRAB, Krfippel-associated box; SV40, simian virus40; CAT, chloramphenicol acetyltransferase. PBS, phosphate-buffered saline; PCR, polymerase chain reaction.*To whom reprint requests should be addressed at: MassachusettsGeneral Hospital East, Suite 4002, 149 13th Street, Charlestown,MA 02129.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Nati. Acad. Sci. USA 91 (1994) 4515

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FIG. 1. Comparisons of KRAB-A regions of various zinc finger proteins. The second exon of Kid-1 encodes amino acids 12 through 53. Aconsensus sequence is presented at the bottom of the alignment with capital letters indicating at least 90%o identity among the various proteinsand with small letters indicating at least 25% identity but less than 90%o. When more than one amino acid is common, the less frequent one islisted on the second line of the consensus sequence.

mutation. For the second reaction, the primer "R," comple-mentary to the 3' end of Kid-1,A was paired with a primeroverlapping the region of the mutation. The products fromthese two PCR reactions overlapped over a stretch of at leastnine nucleotides and were used in a third PCR reaction withprimers "F" and "R," which in 50%o of the cases yieldeda full-length Kid-1,A fragment with the desired nucleotidechange(s). PCR products were digested with EcoRI and XbaI and cloned into the EcoRI/Xba I-cut pBXG1 vector. Theabsence of additional mutations was verified by sequencingthe entire PCR product. Mutations are named by listing thewild-type amino acid first, followed by the position in Kid-i(Fig. 1) and the amino acid to which it is changed.

Transfection Protocols and CAT Assays. COS cells wereplated 2 days prior to transfection at a density of 2.5 x 105 cellsper 100-mm dish. For transfections, cells were exposed to 20 pgof total DNA in 5 ml ofDMEM/10i NuSerum (CollaborativeBiomedical Products, Bedford, MA)/400 pg ofDEAE-dextranper ml/0.1 mM chloroquine. One microgram of a luciferase-expressing plasmid, poLucSV/T1 (7), was included in allcotransfection experiments to normalize transfection efficien-cies. Three to 4 hr after the addition of DNA, medium wasremoved, and cells were shocked for 2 min at room temperaturewith 10%o dimethyl sulfoxide in phosphate-buffered saline(PBS). After shock treatment, cells were washed once withPBS, and new medium was added.

Forty-eight hours after transfection, cells were washedtwice with PBS, scraped with a rubber policeman into amicrocentrifuge tube, and pelleted. The cell pellet was re-suspended in 200 ,u of 0.25 M Tris chloride (pH 7.8) andsubsequently broken up by subjecting it to a freeze/thawcycle three times in a dry ice/ethanol bath and 37°C waterbath. The supernatant was assayed for CAT and luciferaseactivities according to standard protocols (4). CAT activity isexpressed as the ratio of monoacetylated [14C]chlorampheni-col/(monoacetylated plus nonacetylated) [14C]chlorampheni-col and normalized to luciferase activity.

Protein Gels and Western Blots. SDS/PAGE gels andWestern immunoblots were performed according to standardprotocols (8). An anti-GAL4 antibody (obtained from S. A.Johnston and K. Melcher, University ofTexas SouthwesternMedical Center) was used at a 1:1000 dilution. Immunecomplexes were detected with the Renaissance light-de-tection kit from DuPont.

RESULTSTo define the region in the Kid-i protein that is able tomediate its repressor effect on transcription, we generated aset of expression plasmids encoding chimeras of the DNA-binding region of GAL4 with various regions of the NH2terminus of Kid-i (Fig. 2). A plasmid that encoded GAL4-(1-147) and only the KRAB-A and -B domains (pBXG1/Kid-1,AB) induced a very similar level of transcriptional repres-sion of CAT activity of the cotransfected pGSSV-BCATreporter plasmid as the sense construct encoding the full-length non-zinc-finger region of Kid-i (Kid-lN,s) (Fig. 3).The mean residual CAT activities in cells expressing Kid-1N,s or Kid-1,AB, were 16.7 ± 1.9o and 6.6 ± 1.1%,respectively, when compared to cells transfected with anantisense construct of Kid-iN (pBXG1/Kid-1N,as), whichserved as a negative control (Fig. 3). CAT activity in cellscotransfected with the antisense expression plasmid pBXG1/Kid-lN,as was equal to the activity obtained from cellstransfected with pBXG1, which encoded only the DNAbinding region of GAL4 (data not shown). Transfection ofpBXG1/Kid-1,A resulted in repression of transcription to 15+ 1.5% of antisense CAT activity, a degree of repressionalmost identical to that seen with the plasmid encodingKid-lN,s. Conversely, when the KRAB-A region was de-leted from the NH2 terminus of Kid-i [pBXG1/Kid-1,A(-)]or when the construct encoded only the KRAB-B domain(pBXG1/Kid-1,B), transcriptional repression was almostcompletely absent. To demonstrate that a lack of repressor

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4516 Biochemistry: Witzgall et al.

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activity of pBXG1/Kid-lN,as or pBXG1 was not simply dueto lack of expression ofthe protein, we transfected COS cellswith 3 pg of either pBXGi/Kid-lN,s or pBXG1/Kid-1,A or15 ,ug of pBXG1 or pBXG1/Kid-lN,as. CAT activities were21% (pBXG1/Kid-1N,s), 31% (pBXG1/Kid-1,A), and 108%(pBXG1) of the CAT activity in cells transfected withpBXG1/Kid-lN,as. Expression of proteins encoded by theseplasmids was confirmed by Western blot analysis with ananti-GAL4 antibody (Fig. 4).

Similar to our previous observations with the pBXG1/Kid-1N,s plasmid (4), the pBXG1/Kid-1,A construct repressedtranscription in a dose-dependent fashion (Fig. 5) with vir-tually identical efficiency as the full-length non-zinc-fingerregion construct (4). The plasmid, pBXG1/ZNF2,A, encod-ing a fusion protein of GAL4-(1-147) with the KRAB-Aregion ofZNF2, was as effective a repressor as pBXG1/Kid-1N,s (Fig. 6). This indicates that the repressor activity of theKRAB-A domain is not specific to Kid-1.To establish that binding to the GAL4 site on the reporter

was necessary for transcriptional inhibition, the cDNA en-coding the full-length Kid-1 protein (i.e., including all 13 zincfingers) was subcloned into the expression vector pCDM8,yielding the construct pCDM8/Kid-l,e. The binding site forthe Kid-i protein is undefined, but likely distinct from theGAL4 binding site. Cotransfection of pCDM8/Kid-l,( withpG5SV-BCAT did not result in a reduction of CAT activity(Fig. 7).

Point mutations in the KRAB-A region of Kid-i were gen-erated by PCR to evaluate the importance of individual aminoacids, which were highly conserved in all KRAB-A domains forwhich sequence information was available (Fig. 8). Each ofthemutants repressed transcription from the CAT gene to a lesserextent than the wild-type KRAB-A domain. In the case of the

Kid-lA Kid-1 BKRAB-A KRAB-E

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FIG. 3. Thin-layer chromatography assays of CAT activities incells transfected with the pBXG1 expression constructs encodingfusion proteins of the DNA-binding region of GAL4 with Kid-lN,s,Kid-1Nas, Kid-lAB, Kid-1,A(-), Kid-1,A, and Kid-1,B. Expres-sion of fusion proteins containing Kid-lN,s, Kid-1,AB, or Kid-1,Aresults in marked repression of CAT activity, when compared withthe CAT activity observed in cells transfected with the controlantisense construct (pBXG1/Kid-1N,as). In contrast, when theKRAB-A domain is missing from the NH2 terminus of Kid-i [Kid-1,A(-) or Kid-1,B], CAT activity is similar to that observed in thepresence of the control antisense plasmid. (Lower Right) Graphproviding quantitative CAT activity, with CAT activity obtained withthe antisense pBXG1/Kid-lNbas plasmid taken as 100%. The num-bers above the standard error bars indicate the number of indepen-dent experiments.

Phe-16, Val-19, and Leu-31 mutations, no difference was seenbetween a conservative (Phe -+ Ala, Val Ala, or Leu -* Ala)and a nonconservative (Phe -+ Asp, Val Glu, or Leu to Glu)

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FIG. 4. Western blot with GAL4 antibodies. COS cells weretransfected with 3 pg ofeither pBXG1/Kid-lN,s or pBXG1/Kid-1,Aor with 15 pg ofpBXG1 or pBXG1/Kid-1N,as. Pellets obtained aftersubjecting the transfected cells to freeze/thaw cycles were resus-pended in 200 1d of ix PBS and sonicated; 50 jud of 5x SDS samplebuffer containing 625 mM Tris HCl, 12.5% SDS, 50%1b glycerol,12.5% 2-mercaptoethanol, and 0.05% bromophenol blue was added,and the samples were boiled for 5 min. Aliquots of the supernatantsand pellets corresponding to equal amounts of luciferase activitywere run on a 13% gel and transferred to polyvinylidene difluoridemembranes (Immobilon P, Millipore). GAL4 and GAL4 fusionproteins were detected with an anti-GAL4 antibody. Some of thesmaller proteins leaked out into the supernatant during the freeze/thaw process. Arrows point to the fusion proteins. The GAL4/Kid-1N,s band is above an unrelated band similar in size.

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Proc. Natl. Acad. Sci. USA 91 (1994)

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Proc. Natl. Acad. Sci. USA 91 (1994) 4517

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FIG. 5. Quantitation of CAT activities from thin-layer chroma-tography assays of cells transfected with various amounts ofpBXGl/Kid-1,A and 3 pg of the pG5SV-BCAT reporter plasmid. There is adose-dependent inhibition of CAT activity with increasing amountsofpBXG1/Kid-l,A transfected. CAT activities were unchanged withequivalent amounts of transfected plasmid encoding GAL4/Kid-lNantisense (pBXG1/Kid-lN,as; data not shown).

mutation. For the Tyr-39 mutations, a difference was seenbetween an alanine-substituted mutant and a mutant with anacidic residue. The loss of repressor activity was most pro-nounced for Tyr-39 -+ Asp. Tyr-39 -* Ala and Glu45 -* Ala

showed the strongest repressor effects among all mutantsexamined. While there was some variation in expression, eachof the mutants was expressed at a level similar to that of thewild-type KRAB-A domain as determined by Western blotswith anti-GAL4 antibodies (Fig. 8).

DISCUSSIONOur data provide functional evidence for the importance ofthe KRAB-A domain, a highly conserved motif found inmany zinc finger proteins. It has been estimated that approx-imately one-third of the hundreds of zinc finger genes in themammalian genome are members of the KRAB family (3).With this widespread conservation of the KRAB domain, itis possible that transcriptional repressor activity is associatedwith many members of the zinc finger family.

100% 14.8% 8.8%

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FIG. 6. Thin-layer chromatography assays of CAT activities incells transfected with either pBXG1/Kid-lN,s or pBXG1/Kid-1N,asor an expression construct encoding the GAL4 DNA-binding domainand the KRAB-A domain of ZNF2. The KRAB-A domain from thehuman zinc finger protein ZNF2 represses transcription to an extentsimilar to that seen with Kid-lN,s.

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FIG. 7. Thin-layer chromatography assays of CAT activities incells transfected with an expression construct encoding the full-length Kid-i protein in pCDM8 (pCDM8/Kid-1,e) or with Kid-1N,asor Kid-1,A in pBXG1. pCDM8/Kid-l,t, which encodes Kid-1,ewithout a GAL4 DNA-binding domain, has no effect on CAT activitywhen cotransfected with pG5SV-BCAT. The pCDM8 vector is verysimilar in design to the pBXG1 vector in that it can replicateextrachromosomally to very high copy numbers in COS cells becauseof its SV40 origin of replication.

At present we do not understand how the KRAB-A domainrepresses transcription. Our experiments with pCDM8/Kid-1,( suggest that the KRAB-A domain has to be bound(indirectly through a DNA-binding domain) to DNA to beable to repress transcription. It does not suffice to overex-press a KRAB-A-containing protein like Kid-i to represstranscription, arguing against a "squelching effect" byKRAB proteins. This lack of "squelching effect" infersspecificity, since there are potentially large numbers of zincfinger proteins of the KRAB family expressed in a given cellwith the specificity conferred by the DNA-binding charac-teristics of the specific zinc finger protein. In addition, therepressor effect of the GAL4/Kid-1,A construct cannot beattributed to "competition" with other factors for binding atthe GAL4 binding sites because there are no known mam-malian transcription factors that can bind to GAL4 bindingsites, and a GAL4/Kid-1,A(-) mutant had only a minorrepressor effect. We very likely can also rule out a purely"steric model" of repression in which the KRAB-A domain

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FIG. 8. CAT activities and Western blot of GAL4-(1-147)-Kid-1,A fusion proteins with point mutations in the KRAB-A domain.CAT activities are shown as the percentage of the CAT activity fromcells transfected with pBXGl/Kid-lN,as ("anti"); n = 5-10 for eachconstruct. Western blots were performed as described in Fig. 4. Onlythe pellet fractions are shown. The mutants were all expressed, withmost expressed at levels greater than or equal to levels of wild-typeKid-lN,s (wt).

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4518 Biochemistry: Witzgall et al.

prevents the interaction of a trans-acting domain with thebasal transcription factors because the GALA binding sites inour reporter construct lie upstream of the SV40 enhancer andnot between the SV40 enhancer and the TATA box andbecause larger proteins such as GAL4/Kid-1,A(-) are onlyweak repressors. The finding that GAL4/Kid-lN,s is alsoable to repress transcription from a basal promoter (4) arguesagainst the hypothesis that the KRAB-A domain "masks"the activator region ofa positively acting transcription factor.At present, we suggest the following two models for repres-sion by the KRAB-A domain. Binding of a KRAB-A-containing protein may lead to a change in local "chromatinstructure" and thus impair binding of other transcriptionfactors to their binding sites. It is also possible that theKRAB-A domain, either alone or together with anotherinteracting protein, prevents "assembly" of the basal tran-scription factors or "locks" them in an inactive state andtherefore inhibits transcription.Each of the point mutations we generated showed a loss of

repressor activity although to various extents, emphasizingthe importance of the absolutely conserved residues in theKRAB-A domain. Glu-45 -* Ala and Tyr-39 -- Ala were themutants with the highest remaining repressor activity. Inthree offour residues examined (Phe-16, Val-19, and Leu-31),no difference was detected between a conservative and anonconservative mutation, whereas in the- other case (Tyr-39), a conservative mutation affected the repressor activity toa lesser extent than an acidic residue substitution. Among allthe mutants, the Tyr-39 -* Asp mutant repressed CATactivity least. The introduction of an acidic amino acid forTyr-39 may result in the loss of interaction with the naturalpartner of the KRAB-A domain.The KRAB-A domain is encoded by a single exon in the rat

Kid-] gene (9) and the human ZNF2 (10) and ZNF4S (11)genes. A corresponding arrangement can be assumed for thehuman ZNF43 gene, where one alternatively spliced mRNAspecies has been identified that lacks the KRAB-A region(12). Hence, by alternative splicing, cells may produce zincfinger proteins with or without the KRAB-A transcriptionalrepressor domain.While there are at least four well-defined types of tran-

scriptional activation domains, serine/threonine-rich, acidic,proline-rich, and glutamine-rich (13), to our knowledge theonly other well-defined motif that has been postulated tomediate transcriptional repressor activity is an alanine-richdomain found in four transcriptional repressors from Dro-sophila: Krippel (14), engrailed (15), even-skipped (16), andAEF-1 (14). In other cases where a repressor domain has

been delineated, such as Egr-1 (17), SRF (18), and E4BP4(19), no obvious consensus sequence motifs have been iden-tified. Our data indicate that the KRAB-A domain representsa widely distributed transcriptional repressor motif.

We thank Karen Dellovo for artwork and Xia-Mei Liu for technicalassistance. We thank Dr. Ptashne for the pBXG1 and pG5SV-BCATvectors. This work was supported by National Institutes of HealthGrants DK 39773 and DK 38452. Parts of these data were presentedin the 26th Annual Meeting of the American Society of Nephrologyand have been published (20). Protein sequence alignments wereconstructed with the PILEUP program of the Genetics ComputerGroup of the University of Wisconsin.

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6. Aiyar, A. & Leis, J. (1993) BioTechniques 11, 366-368.7. Shelley, C. S. & Arnaout, M. A. (1991) Proc. Natl. Acad. Sci.

USA 88, 10525-10529.8. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,

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