letter 394 nature genetics • volume 22 • august 1999 RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex Ingolf Bach 1,7 , Concepción Rodriguez-Esteban 2 , Catherine Carrière 1 , Anil Bhushan 3 , Anna Krones 1 , David W. Rose 4,5 , Christopher K. Glass 6 , Bogi Andersen 5 , Juan Carlos Izpisúa Belmonte 2 & Michael G. Rosenfeld 1 1 Howard Hughes Medical Institute, Eukaryotic Regulatory Biology Program, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0648, USA. 2 Gene Expression Laboratory, 3 The Clayton Foundation, The Salk Institute for Biological Sciences, 10010 North Torrey Pines Road, La Jolla, California 92186, USA. 4 Whittier Diabetes Program, 5 Division of Endocrinology and Metabolism, 6 Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0648, USA. 7 Center for Molecular Neurobiology, University of Hamburg, Martinistr. 85, 20246 Hamburg, Germany. Correspondence should be addressed to I.B. (e-mail: ingolf.bach@zmnh.uni-hamburg.de), M.G.R. (e-mail: mrosenfeld@ucsd.edu) or J.C.I.B. (belmonte@salk.edu). LIM domains 1 are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcrip- tional activation events 1–4 . The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2–4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins 4,5 , suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regula- tor via the recruitment of the Sin3A/histone deacetylase core- pressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the for- mation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacety- lase-containing complexes to the LIM domain. m Rlim MENSDSNDKGS-DQSAAQRRSQMDRLDREEAFYQFVNNLSEEDYRLMRDNNLLGTPGESTEEELLRRLQQIKEGPPPQSPDENRAGESSD 89 c RLIM MESSDSSDKGNIDQSEAQRQSQLDRLDREEAFYQFVNNLSEEDYRLMRDNNLLGTPGEITEEELLRRLHQVKEGPPQQNSDENRGAESTE 90 ||.|||.|||. |||.|||:||.|||||||||||||||||||||||||||||||||||.|||||||||:|:|||||.|..||||::||:: . . . . . . . . m Rlim DVTNSDSIIDWLNSVRQTGNTTRSGQRGNQSWRAVSRTNPNSGDFRFSLEINVNRNNGSQTSENESEPSTRRLSVENMESSSQRQMENSA 179 c RLIM DVSNGDSIIDWLNSVRQTGNTTRSGQRGNQSWRAVSRTNPNSGDFRFSLEINVNRNNGNTNPETENEPSAEPSGGEDLEN-SQSDSEIPR 179 m Rlim SESASARPSRAERNSTEAVTEVPTTRAQRRARSRSPEHRRTRARAERSVSPLQPTSEIPRRA------PTLEQSSENEPEGSSRTRHHVT 263 m Rlim LRQQISGPELL--GRGLFAASGSRNPSQGTSSSDTGSNSESSGSGQRPPTIVLDLQVRRVRPGEYRQRDSIASRTRSRSQAPNNTVTYES 351 ||.|:|||||||||||||||||||||||||||||||||||||||||||||||||||||. ..|.|.|||... :.|:.|. || | . . . . . . . . . . . . . . . . . c RLIM SESPSVRQPGSERSTSEELTEEASPRGQRRARSRSPEQRRTRARTDRSRSPINPVSEAPRRSHHNTSSQTFDHSAVNEAEGSSRTRQHVT 269 |||:|.| . .||.::| :|| :. |:||||||||||:|||||| :|| ||:.|.||.|||. |.::|. ||:|||||||:||| . . . NLS . . . . . :|||||. | |||| |:|. ||||:::.||:||: |:.|||:||||||||||||||||||||:|||||||||||| c RLIM LRQHAVGTEIPSENAVLFSALETGPVPQAAGSSETNGASESAAPGQRPPTIVLDLQVRRVRPGEYRQRDSIANRTRSRSQTPNNTVTYES 359 |||:. | |: .||.| : .|:.:||:|:: |||.:.||||||||||||||||||||||||||||.|||||||:||||||||| . . . . . . . . m Rlim ERGGFRRTFSRSERAGVRTYVSTIRIPIRRILNTGLSETTSVAIQTMLRQIMYGFGELSYFMYSDSDSEPSASVSSRNVERVESRNGRGS 441 c RLIM ERGGFRRTFSRSERAGVRTYVSTIRIPIRRILNTGLSETTSVAIQTMLRQIMYGFGELSYFMYSDSDADPSGPTPNQNVDASEPQNG--- 448 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||.:||:. ...||:. |..|| m Rlim SGGGNSSGSSSSSSPSPSSSGESSESSSKMFEGSSEGGSSGPSRRDGRH-RAPVTFDESGSLPFLSLAQFFLLNEDDEDQPRGLTKEQID 53O c RLIM -----GSGSSSNESTD-VSSGEVFEGSN-------EGGSTSGARREGRNTRGSVTFEESGSLPFLSLAQFFLLNEDDDDQPRGLTKEQID 523 . . . . . . . . m Rlim NLAMRSFGENDALKTCSVCITEYTEGNKLRKLPCSHEFHVHCIDRWLSENSTCPICRRAVLSSGNRESVV 600 c RLIM NLAMRNFGETDALKTCSVCITEYTEGNKLRKLPCSHEYHVHCIDRWLSENSTCPICRRAVLASGNRESVV 593 |||||.|||||||||||||||||||||||||||||||:|||||||||||||||||||||||.|||||||| . . . RING-H2 finger . . a b Rlim ALKTCSVCITEYTE----GNKLRKLPCSHEFHVHCIDRWLSE------NSTCPICRRAVL CRZF EYDVCAICLDEYED----GDKLRILPCSHAYHCKCVDPWLTKT-----KKTCPVCKQKVV goliath DSDCCAICIEAYKP----TDTIRILPCKHEFHKNCIDPWLIE------HRTCPMCKLDVL CELG EDATCAICLDNLQN(20)GTTVIVMPCKHRFHYFCLTLWLEA------QQTCPTCRQKVK PSMP MELTCGLCGESIGDQ---NSQLQALPCSHLFHLKCLQTDG--------NRGCPNCKRSSV FAR1 IGEKCLICEESISSTFT-GEKVVESTCSHTSHYNCYLMLFETLYFQGKFPECKICGEKSK PEP3 PGKSCDECGKFLQ-----IKKFIVFPCGHCFHWNCIIRVIL(27)NIIVEKCGLCSDINI PEP5 KNQTCFMCRLTL------DIPVVFFKCGHIYHQHCLNEEEDTLESERKLFKCPKCLVDLE consensus C C C H H C C C RING-H2 finger e c input 20% GST GST Isl1 LIM GST Lhx2 Rlim GST input 10% GST Lmo2 GST Lmk LIM GST CRPLIM GST Pax LIM GST Zyx LIM Rlim GST input 20% GST Lhx3 GST Lhx3 LIM GST Lhx3 ∆LIM Rlim Rlim α-Clim α-Lhx3 Lhx3 Clim1 Rlim* – + – – + – + + – + + – – + + Clim1 f g GST input 10% GST Rlim (1-207) GST Rlim (208-423) GST Rlim (403-600) Clim1 d Rlim (HA) Lhx3 LIM (myc) Lmo2 (myc) – – – + + – + – + Clim1 Lhx3 Rlim Lhx3/Clim1 Lhx3 – + – + + – + + + Rlim αHA + + + Fig. 1 Rlim, a novel LIM domain-binding RING zinc-finger protein. a, Amino acid sequences of mouse and chicken Rlim (m Rlim and c RLIM, respectively) are 75% iden- tical. A putative nuclear localization signal (NLS) is shaded and the RING-H2 zinc finger is boxed. b, Sequence comparison of RING-H2 zinc-finger domains of Rlim, CRFZ, goliath, CELG, PSMP, FAR1, PEP3 and PEP5. c, Co-immunoprecipitation of full-length [ 35 S]Met-labelled Rlim (asterisk) with bacterially expressed Lhx3 and Clim1 using anti-Lhx3 (αLhx3) and anti-Clim (αClim) antibodies. The first lane corresponds to 10% input. d, Co-immunoprecipitations of nuclear extracts from CV1 cells co-trans- fected with HA-tagged Rlim and Myc-tagged LIM domains of Lhx3 or Lmo2 expression plasmids with the anti-HA (αHA) antibody. The western blot was visualized using an anti-Myc antibody. e, GST protein interaction assays between [ 35 S]-labelled Rlim protein and bacterially expressed GST fusion proteins: full-length Lhx3 (GST-Lhx3), Lhx3 mutant proteins containing only the LIM domain (GST-Lhx3LIM) or no LIM domain (GST-Lhx3∆LIM); Lhx2 (GST-Lhx2); Lmo2 (GST-Lmo2); and different LIM domains from Isl-1 (GST-Isl1LIM), LIM kinase 1 (GST-Lmk1LIM), CRP (GST-CRPLIM), paxillin (GST-PaxLIM) and zyxin (GST-ZyxLIM). f, GST protein interaction assays between [ 35 S]- labelled Clim1 and bacterially expressed regions of Rlim fused to GST. g, Inhibition by Rlim of Lhx3/Clim1 interactions in an EMSA. A [ 32 P]-labelled Lhx3/Clim1/DNA com- plex supershift on an Lhx3 DNA binding site was inhibited by addition of 2×Rlim, and completely inhibited with 5×Rlim (data not shown). a b c d e f g © 1999 Nature America Inc. • http://genetics.nature.com © 1999 Nature America Inc. • http://genetics.nature.com