Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press KAP-1, a novel corepressor for the highly conserved KRAB repression domain Josh R. Friedman, 1 William J. Fredericks, 1 David E. Jensen, 1 David W. Speicher, I Xiao-Pei Huang, ~ Eric G. Neilson, 2 and Frank J. Rauscher III 1'3 JThe Wistar Institute, Philadelphia, Pennsylvania 19104 USA; 2The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 USA The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING.B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal eorepressor for the large family of KRAB domain-containing transcription factors. [Key Words: Corepressor; RING finger; KRAB domain; zinc-linger] Received April 23, 1996; revised version accepted July 9~ 1996. Transcriptional regulation of gcnc expression is medi- 1995; Schrciber-Agus et al. 1995), and N-CoR or SMRT ated primarily by DNA sequence-specific transcription interaction with the nuclear hormone receptor family factors, which are generally composed of a DNA-binding iChen and Evans 1995; Horlein ctal. 19951. A common domain and one or more separable effector domains that theme among these examples is that mutations in the may activate or repress transcriptional initiation (for re- DNA-bound repression domain that abolish interaction view, see Tjian and Maniatis 1994; Zawcl and Reinberg with the corepressor molecule also eliminate the repres- 1995). Activation domains may function by directly in- sion function. teracting with components of the basal transcriptional The KRAB [K___mppel-associated box} is a repression do- machinery, thereby nucleating, stabilizing, and/or facil- main [Fig. 1) that is encoded by many transcription fac- itating the assembly and initiation of the RNA pol II tors. It was originally identified in humans as a con- transcription complex. Alternatively, activation do- served amino acid sequence motif at the amino termini mains may function through a novel class of intermedi- of proteins that contain multiple TFIIIA/Krfippel-class ary molecules variously termed adaptors or coactivators Cys2-Hisz (C2H2} zinc fingers in their carboxyl termini (for review, see Guarente 1995}. These intermediary mol- (Bellefroid et al. 1991]. The KRAB domain has now been ecules must be tethered to the DNA template via pro- identified in frog, rodent, and human zinc-finger proteins tein-protein interactions with the effector domain of the fAltaba et al. 1987; Thiesen et al. 1991; Constantinou- DNA-bound transcription factor, thus serving to bridge Deltas et al. 1992; Witzgalt et al. 1994; Vissing et al. the activation domain to the ultimate downstream tar- 1995}. Between 300 and 700 human genes encode C2H~ get. zinc-finger proteins (Klug and Schwabe 1995), and one- In contrast to our knowledge of activators, less is third of these contain KRAB domains [Bellefroid et al. known regarding the mechanisms utilized by repression 19911. To date -65 unique KRAB domain sequences domains. Not surprisingly, repression domains may con- have been isolated. The KRAB domain homology (Fig. 1) tact components of the basal transcription machinery consists of -75 amino acids, is divided into A and B (Fondell et al. 1993, 1996; Baniahmad et al. 1995; Sauer boxes {on the basis of common intron-exon boundaries), et al. 1995; Um et al. 1995} or molecules with properties and is predicted to fold into two charged amphipathic suggestive of corepressor function. Emerging models of helices (Bellefroid et al. 1991}. repressor-eoreprcssor interaction include hairy-groucho The KRAB domain is a potent, DNA binding-depen- in Drosophila (Paroush et al. 1994; Fisher et al. 1996), dent repression domain (Margolin et al. 1994; Witzgall et SSN6/TUP1-MCM1/MATot2 in yeast (Keleher et al. al. 1994; Pengue et al. 1995; Vissing et al. 1995). The 1992; Cooper et al. 1994; Tzamarias and Struhl 1994}, minimal KRAB repression module is -45 amino acids, MAD/MAX-roSIN-3 in mammalian cells (Ayer et al. and substitutions for conserved residues within this do- main abolish repression. To date, 10 KRAB domains have been demonstrated to be potent repressors, suggest- aCorresponding author. ing that this is a common property of independently en- GENES & DEVELOPMENT 10:2067-2078 © 1996 by Cold Spring HarborLaboratory Press ISSN 0890-9369/96 $5.00 2067 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Friedman et al. Figure 1. Comparison of the KRAB domains A Box B Box from various zinc-finger proteins. The amino acid sequences of 16 KRAB domains (retrieved helix helix from sequence data bases and referenced in the Consensus: VTF DVAV F EEW LD QR LYR VMLENY LVSL G KPDL LEQ W text) are aligned. Each gene encodes the indicated L I D Q V VAFV EV P KRAB domain at its amino terminus; the super- ZNF7 2EVVTFGDVAVHFSREEWQCLDPGQRALYREVMLENHSSVAGLAGFLVFKPELISRLEQGEEPW script numeral at the left of each sequence indi- ZNFg0 2GPLEFRDVAIEFSLEEWHCLDTAQQNLYRDVMLENYRHLVFL.GIVVTKPDLITCLEQGKKPF cates the position of the amino acid with respect zNrs5 2GPLTFRDVAIEFSLKEWQCLDTAQRNLYRNVMLENYRNLVFL.GITVSKPDLITCLEQGKEAW ZNFI41 2ELLTFRDVAIEFSPEEWKCLDPDQQNLYRDVMLENYRNLVSL.GVAISNPDLVTCLEQRKEPY to the initiator methionine. The zinc fingers en- ZNF45 6EAVTFKDVAVVFSEEELQLLDLAQRKLYRDVMLENFRNVVSV.GHQST.PDGLPQLEREEKLW coded by each gene are not shown. The KRAB NK10 12ESVTFKDVAVNFTQEEWHHVGPAQRSLYRDVMLENYNHLVSL.GYQVSKPEVIFKLEQGEEPW ZNFI0 12TLVTFKDVFVDFTREEWKLLDTAQQI~ENYKNLVSL.GYQLTKPDVILRLEKGEEPW domain consensus sequence is presented at the ZNFIV7 12NSVTFQEVAVDFSQEEWGLLDPAQKNLYKDVMLE.FRNLASV.GYQLCRHSLISKVDQEQLKT top and consensus residues (indicating >90% ZNF91 12GLLTFRDVAIEFSPEEWQCLDTAQQNLYR/qVMLENYKNLAFL.GIALSKPDLITYLEQGKEPW identity) are in bold throughout. Capital, lightface KID-1 12VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKPKVISLLQQGEDPW ZNFI57 24GSVSFEDVAVDFTRQEWHRLDPAQRTMHKDVMLETYSNLASV.GLCVAKPEMIFKLERGEELW letters in the consensus indicate the next most ZNFI40 4GSVTFRDVAIDFSQEEWKWLQPAQRDLYRCVMLENYGHLVSL.GLSISKPDVVSLLEQGE.PW frequent residue in that position. The substitu- ZNF133 IMAFRDVAVDFTQDEWRLLSPAQRTLYREVMLENYSNLVSL.GISFSKPELITQLEQGKETW KRK-I 26CSVSFEDVAVDFTWEEWQALDAAQRTLCRDVMLETYSSLVSL.GHCMAKPELIFNLEQGLGPR tion mutations DV--* AA and MLE--~ KKK in EEK-I 2EAVTFGDVAVAFSREEWQCLDSGQRALYKEVMLENHSSVAGLAGFLVFKPELISRLEQGQEPW the KOX1 KRAB domain dramatically reduce re- KOXl 12TLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSL.GYQLTKPDVILRLEKGEEPW pression activity (Margolin et al. 1994). A dot in- $$ $$$ dicates a gap introduced to facilitate alignment. Mutations: AA KKK coded KRAB domains. Thus, the KRAB-zinc-finger pro- this study the KRAB domain from the KOX1 protein was tein (KRAB-ZFP) family represents a large, mechanisti- utilized). Typically, squelching refers to the inhibition of cally unexplored class of transcriptional repressor transcriptional activation that is observed in the pres- molecules. ence of excess non-DNA-bound activator domain; this is KRAB-ZFPs are likely to play important regulatory thought to reflect competition for a limiting cofactor re- roles during development. Expression of the KRAB-ZFP quired for activation by the DNA-bound transcription KID1 is temporally and spatially restricted to the devel- factor (Gill and Ptashne 1988). By analogy, squelching of oping mouse kidney (Witzgall et al. 1993). A cluster of repression by the KRAB domain would thus indicate that >40 KRAB--ZFP-encoding genes has been identified on the KRAB domain functions through a titratable core- human chromosome 19p12-p13.1, and several members pressor. As reported previously (Margolin et al. 1994), the of this cluster are differentially expressed during T-cell GAL4-KRAB fusion protein strongly represses transcrip- ontogeny and activation (Bellefroid et al. 1993). Expres- tion from a reporter plasmid containing five GAL4 UAS sion of the KRAB--ZFPs HPF4, HTF10, and HTF34 is binding sites (Fig. 2B, lanes 2,5). However, cotransfection down-regulated during myeloid differentiation (Belle- of GAL4-KRAB domain with a plasmid encoding the froid et al. 1991). Finally, a number of KRAB-ZFPs are KRAB domain alone inhibited repression >50-fold in a candidate genes for human diseases on the basis of chro- dose-dependent manner (lanes 3,4,6,7). Transfection of mosomal location (Tommerup et al. 1993; Crew et al. the KRAB domain plasmid alone had little effect on the 1995). reporter plasmid (lanes 9,10). The mechanism of repression by the KRAB domain is The amino terminus of the WT 1 protein lacks a KRAB not known. In this study, we have identified KRAB-as- domain yet exhibits a potent transcriptional repression sociated protein-1 (KAP-1), a novel protein that binds to function when fused to GAL4 (Madden et al. 1991). To the KRAB domain and functions as a transcriptional determine whether the KRAB repression domain and the corepressor. KAP-1 is thus a potential mediator of repres- WT1 repression domain function via similar titratable sion for the large class of KRAB domain-containing tran- cofactors, we attempted to squelch GAL4-WTI-medi-