COMMD Proteins, a Novel Family of Structural and Functional Homologs of MURR1

MURR1 is a multifunctional protein that inhibits nuclear factor κB (NF-κB), a transcription factor with pleiotropic functions affecting innate and adaptive immunity, apoptosis, cell cycle regulation, and oncogenesis. Here we report the discovery of a new family of proteins with homology to MURR1. These proteins form multimeric complexes and were identified in a biochemical screen for MURR1-associated factors. The family is defined by the presence of a conserved and unique motif termed the COMM (copper metabolism gene MURR1) domain, which functions as an interface for protein-protein interactions. Like MURR1, several of these factors also associate with and inhibit NF-κB. The proteins designated as COMMD or COMM domain containing 1–10 are extensively conserved in multicellular eukaryotic organisms and define a novel family of structural and functional homologs of MURR1. The prototype of this family, MURR1/COMMD1, suppresses NF-κB not by affecting nuclear translocation or binding of NF-κB to cognate motifs; rather, it functions in the nucleus by affecting the association of NF-κB with chromatin. MURR1 is a multifunctional protein that inhibits nuclear factor κB (NF-κB), a transcription factor with pleiotropic functions affecting innate and adaptive immunity, apoptosis, cell cycle regulation, and oncogenesis. Here we report the discovery of a new family of proteins with homology to MURR1. These proteins form multimeric complexes and were identified in a biochemical screen for MURR1-associated factors. The family is defined by the presence of a conserved and unique motif termed the COMM (copper metabolism gene MURR1) domain, which functions as an interface for protein-protein interactions. Like MURR1, several of these factors also associate with and inhibit NF-κB. The proteins designated as COMMD or COMM domain containing 1–10 are extensively conserved in multicellular eukaryotic organisms and define a novel family of structural and functional homologs of MURR1. The prototype of this family, MURR1/COMMD1, suppresses NF-κB not by affecting nuclear translocation or binding of NF-κB to cognate motifs; rather, it functions in the nucleus by affecting the association of NF-κB with chromatin. NF-κB is a dimeric complex formed by members of a highly conserved family of proteins that share a defining motif designated the Rel homology domain (RHD). 1The abbreviations used are: RHD, Rel homology domain; EGFP, enhanced green fluorescence protein (GFP); TAP, tandem affinity purification; GST, glutathione S-transferase; TNF, tumor necrosis factor; MS, mass spectroscopy; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; RNAi, RNA interference; siRNA, small interfering RNA; HIV-1, human immunodeficiency virus-1. Through transcriptional regulation of many gene products, NF-κB participates in a number of biological processes including innate and adaptive immune responses, programmed cell death, cell cycle progression, and oncogenesis (1Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar, 2Silverman N. Maniatis T. 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EMBO J. 1995; 14: 1552-1560Crossref PubMed Scopus (218) Google Scholar, 9Nabel G.J. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1456) Google Scholar, 10Bohnlein E. Lowenthal J.W. Siekevitz M. Ballard D.W. Franza B.R. Greene W.C. Cell. 1988; 53: 827-836Abstract Full Text PDF PubMed Scopus (248) Google Scholar), NF-κB also participates in viral cycle progression. Studies into the regulation of NF-κB activation have largely focused on the role of cytoplasmic sequestration of the NF-κB complex as a mainstay level of control. In most cells NF-κB is localized in the cytoplasm through the interaction of the complex with members of the IκB family (11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). These proteins contain ankyrin repeats that allow their interaction with NF-κB and mask the nuclear localization signal present in the RHD. Phosphorylation of IκB by a multimeric kinase known as the IκB kinase complex targets these proteins for ubiquitination and proteasomal degradation (3Karin M. Yamamoto Y. Wang Q.M. Nat. Rev. Drug Discov. 2004; 3: 17-26Crossref PubMed Scopus (1249) Google Scholar, 12Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar). This allows the translocation of NF-κB to the nucleus where it binds to cognate DNA sequences present in an array of gene promoters. MURR1 is a recently identified factor that has been shown to participate in two apparently distinct activities, regulation of the transcription factor NF-κB and control of copper metabolism (13Greene W.C. Nat. Immunol. 2004; 5: 18-19Crossref PubMed Scopus (13) Google Scholar). Mutations in MURR1 are responsible for copper toxicosis in an inbred canine strain (Bedlington terriers) (14van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (316) Google Scholar), and an interaction between MURR1 and the copper transporter ATP7B (15Tao T.Y. Liu F. Klomp L. Wijmenga C. Gitlin J.D. J. Biol. Chem. 2003; 278: 41593-41596Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) has been recently reported. In addition to its role in copper metabolism in mammals, more recent studies implicate MURR1 in the regulation of the transcription factor NF-κB (13Greene W.C. Nat. Immunol. 2004; 5: 18-19Crossref PubMed Scopus (13) Google Scholar, 16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar). MURR1 was found to be a broad inhibitor of NF-κB, affecting κB-responsive transcription from endogenous and viral promoters including the HIV-1 enhancer (16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar). Through this effect, MURR1 can function as a factor that limits HIV-1 replication in resting CD4+ lymphocytes. Here we report the discovery of a family of proteins structurally and functionally related to MURR1. These factors contain a unique and defining domain termed the COMM (copper metabolism gene MURR1) domain, and thus, these proteins have been named COMM domain-containing or COMMD proteins. Similar to MURR1/COMMD1, several of these factors associate with NF-κB and inhibit its transcriptional activity. In addition, we find that COMMD proteins form heteromeric complexes that are mediated by the COMM domain. The prototype of the family, MURR1/COMMD1, exerts its ability to inhibit κB-mediated transcription without affecting nuclear translocation but through nuclear regulation of NF-κB. We show here that MURR1/COMMD1 is recruited to chromatin of a κB-responsive promoter upon NF-κB activation and negatively regulates the association of RelA to chromatin. Therefore, this work identifies a novel family of factors that regulate NF-κB-mediated transcription by controlling the occupancy of NF-κB on chromatin. Plasmids—The plasmids pEBB, pEBG, pEBB-MURR1-Flag and pEBB-MURR1-GST, pEBB-T7-IκB-αS.D., 2κB-luc, and EGFP-p65 (kindly provided by Dr. Rainer de Martin) have been described previously (17Duckett C.S. Li F. Wang Y. Tomaselli K.J. Thompson C.B. Armstrong R.C. Mol. Cell. Biol. 1998; 18: 608-615Crossref PubMed Scopus (193) Google Scholar, 18Richter B.W.M. Mir S.S. Eiben L.J. Lewis J. Reffey S.B. Frattini A. Tian L. Frank S. Youle R.J. Nelson D.L. Notarangelo L.D. Vezzoni P. Fearnhead H.O. Duckett C.S. Mol. Cell. Biol. 2001; 21: 4292-4301Crossref PubMed Scopus (87) Google Scholar, 19Burstein E. Ganesh L. Dick R.D. van De Sluis B. Wilkinson J.C. Lewis J. Klomp L.W.J. Wijmenga C. Brewer G.J. Nabel G.J. Duckett C.S. EMBO J. 2004; 23: 244-254Crossref PubMed Scopus (183) Google Scholar, 20Duckett C.S. Thompson C.B. Mol. Cell. Biol. PubMed Google Scholar, A. M. B.R. de J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.S. B.W.M. Duckett C.S. 15: Google Scholar). and were by pEBB-MURR1-Flag as with the in was by MURR1 into which was by of the for the tandem affinity as F. B. E. E. M. B. 2001; PubMed Scopus Google Scholar). for COMMD proteins in with and glutathione or were by of the sequences for of these proteins. that the were used as to through and and and and were by the and as (kindly provided by Dr. C.S. N.D. Schmid R.M. A.S. Nabel G.J. Mol. Cell. Biol. 1993; PubMed Google Scholar). and cells and and cells were from cells were in with and and cells were in with and C.S. Thompson C.B. Mol. Cell. Biol. PubMed Google Scholar) was used to plasmids and into of into cells was as by the cells were in and with of and of the were as described previously Reffey S. A.B. Duckett C.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of for cells were in and with a of of of endogenous COMMD was by of cells in with of the cells in were with of and were in The was with and and to a containing of the was and with and for in with the was and with and binding This was to a containing and for The was and with binding with a was were by in the was by for in and to These were to the the of for including and tandem mass to and RNA was from cells to the and was by of RNA in and for and of COMMD were with the of the sequences and are upon was used to the an with of RNA in was and reverse transcription This was by in the In with the and was and for and as an control were from in were from the of the T. S. D. J. M. J.R. S. A. 2004; PubMed Scopus Google Scholar), and and were by and were as previously described E. Ganesh L. Dick R.D. van De Sluis B. Wilkinson J.C. Lewis J. Klomp L.W.J. Wijmenga C. Brewer G.J. Nabel G.J. Duckett C.S. EMBO J. 2004; 23: 244-254Crossref PubMed Scopus (183) Google Scholar). was by which was used as was in by the was a and was by of the affinity to the RelA and were used as and cells were in or and with EGFP-p65 or for nuclear translocation of EGFP-p65 were by cells with a and with were and cells were and cells were in and as of cells with for The of nuclear and have been described previously C.S. N.D. Schmid R.M. A.S. Nabel G.J. Mol. Cell. Biol. 1993; PubMed Google Scholar). a a κB was used as cells were in the was and the cells were in and in a The cells were in of and of were with and the cells were with for The were for and the to the cytoplasmic was The was in of of and the as the was as of the cytoplasmic The was by the addition of of and with for the were for The to the nuclear was cells were with for chromatin cells were in on as previously described F. Mol. Cell. 2004; 16: Full Text Full Text PDF PubMed Scopus (183) Google Scholar). and sequences have been previously described F. C.S. EMBO J. 2004; 23: PubMed Scopus Google Scholar). used in the studies RNA and RelA for MURR1-associated the of MURR1, a biochemical screen for proteins was on the that has been previously described F. B. E. E. M. B. 2001; PubMed Scopus Google Scholar). MURR1 in with the affinity was in and was from cells two containing and The was to and the were identified by tandem mass number of factors were including proteins that upon the presence of a with homology to MURR1 in their and These factors were designated as and of the COMMD the of MURR1 factors in biochemical we an of the for Through this we were to proteins in including MURR1, that contain highly conserved sequences The of these were known as and not been previously of present that the of homology in the of these proteins a previously and highly conserved motif This is to form a We have termed this the copper metabolism gene MURR1 domain. The of homologs of MURR1 identified here the of a new its from its to the in this is not in organisms including In addition, an gene that also in to has been designated the of this for MURR1 homologs A. M. T. Mol. Cell. Biol. PubMed Scopus Google Scholar). In with the gene the COMMD is to these factors on the structural domain that this family of proteins and has been in The is for MURR1 as a to the to this protein family and be used the of have been previously described in was identified as an that is in and in a number of and cell N. S. S. J. F. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The protein to the a role in transcription not been previously is to a gene in a that is for it is is for E. Wang J. Hum. Mol. Genet. 2003; 12: PubMed Scopus Google Scholar). was previously identified as a with to the gene Y. J.M. Mol. Biol. PubMed Scopus Google Scholar). an to was found to be in an to screen for factors in F. S. L. D. M. M. P. J. M. R.B. A. S. A. PubMed Scopus Google Scholar). COMMD Genes found that have been conserved as can be from found in and the In sequences are conserved with their human including and also COMMD of these were identified in eukaryotic organisms or of these were found in and and and of the COMMD can be found in with and to the the presence of a conserved and defining motif in these a of the of COMMD protein is of unique that are members of the human and are human and are conserved the COMM domain are in the not COMMD Genes that COMMD proteins were identified as factors in we the of of human COMMD in this cell this we for of of these including in that was an This the of from DNA are In addition, the for the was into in the this we identified for human COMMD with the and of in these and RNA from cells we were to the for of the COMMD not these not This that cells COMMD a that was also by shown the level of COMMD in was from the of the for more human were T. S. D. J. M. J.R. S. A. 2004; PubMed Scopus Google Scholar). This were and the for most COMMD the of were in were and are in are in human but the of COMMD is the is in the is the in the in the and in the has a of COMMD that and these are not in not with COMMD and were identified by their ability to with Therefore, the ability of the members of the family to with was of the COMMD proteins was to and in proteins were from cell with and the was for endogenous and endogenous also with but to a shown These that can with and with COMMD with the in the by the COMM define the for COMMD a of of were for their ability to and The to of were used as in proteins with The canine copper toxicosis described previously of a of that the (14van De Sluis B. Rothuizen J. Pearson P.L. van Oost B.A. Wijmenga C. Hum. Mol. Genet. 2002; 11: 165-173Crossref PubMed Scopus (316) Google Scholar). This protein was also in with and for its ability to and These proteins were in cells and with The ability of these to an interaction was by the presence of or in the and The of which the COMM domain, was found to be for with and The of the COMM domain in the protein binding in the the protein in with copper toxicosis and of the COMM domain, a in These that the COMM domain that this protein family as an interface for interactions. COMMD κB-mediated the prototype of the family, was recently to inhibit κB-mediated transcription from endogenous and viral promoters (16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar). Therefore, the ability of COMMD proteins to inhibit NF-κB was were with a κB-responsive with of the COMMD and the to was shown in and were of NF-κB and NF-κB in this most COMMD proteins also of κB-mediated the ability of these proteins to control transcription of an endogenous κB-responsive gene was of endogenous and were by the of RNA and the of in to was and The of and was by with present in control with and in a of with of these the of COMMD on transcription was In cells with an with in a in as by a that is to that in cells not COMMD in of with this most Therefore, these factors not share a domain but have functional in the regulation of NF-κB transcriptional activity. COMMD with the NF-κB ability of to inhibit κB-mediated transcription was previously found to on its association with the NF-κB complex (16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar). of endogenous with the of in the of endogenous RelA and The that COMMD proteins can with the NF-κB complex was proteins with in with were by for of endogenous NF-κB and was the for most COMMD proteins were also of NF-κB complexes the of of NF-κB with the level of of the proteins not the of association with NF-κB was between the various COMMD proteins complexes containing and in the of and to with complexes in the of and and associate more with NF-κB were associate with and was the COMMD to also with NF-κB but more with to ability of various of to an interaction with endogenous RelA was In these of various of as in were used for that to the COMM domain and are with Therefore, the interaction between and RelA on the presence of the COMM domain. the interaction between and RelA was by the in RelA that are for this interaction of RelA in with were in cells and with the presence of or its and in the was by shown in the of RelA was for binding to or and The of RelA which to the domain of the not of these the RHD, the the DNA binding domain, the of the in IκB binding and with and the nuclear localization the of RelA were of binding but not or of the of the in which of was to its ability to to of also these not The is highly conserved in NF-κB Nature. 1998; PubMed Scopus Google Scholar). Therefore, the that also to sequences present in the of NF-κB was in and were found to be to by the not of these in with was in cells and from cell with and the presence of was by shown in these present in the of NF-κB were of binding to of In most cells NF-κB is localized in the cytoplasm through with members of the IκB family, which in of the nuclear localization signal and cytoplasmic sequestration of the complex (11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). the translocation of NF-κB from the to the nucleus is a in the activation of the ability of to this was localization of a protein between RelA and was by fluorescence and in nuclear translocation of a that was in cells with a form of and In the of was by that not the nuclear translocation of the NF-κB The translocation of NF-κB complexes into the nucleus and their DNA binding was also by with a control and with κB binding in nuclear a this was by of on the of κB binding in nuclear In addition, of to transcriptional activation of NF-κB, this not to of κB binding by not to κB-responsive and NF-κB to COMMD proteins can with the NF-κB the ability of to inhibit transcriptional activation is of the nuclear translocation of NF-κB. This that the of NF-κB complexes in the nucleus was by this the presence of nuclear was were from cells with an and shown in was present in nuclear and and its in the also in nuclear translocation of RelA and degradation of with of this protein in the nuclear The of the was by for and on the the of on NF-κB nuclear function were by the of RelA to chromatin. ChIP, the of on the of RelA to the κB-responsive promoter were The of with were of control and in endogenous in of the occupancy of RelA on the In addition, was found to be recruited to this κB-responsive of was also to in protein have the on RelA to chromatin. In this cell to was used as the for NF-κB as this in and of RelA to the promoter shown in of in a in the of RelA association with chromatin NF-κB to chromatin in to was also these that the of the complex that is recruited in to is recruited to chromatin and NF-κB has been this that or through the of factors can the affinity of NF-κB for chromatin. In this report we the of a novel and conserved family of homologs of MURR1. These factors are defined by the presence of a unique motif termed the COMM domain. The of this protein family and into the functions of these factors were largely to this We show here that COMMD proteins can with factors are in and were identified as factors in that be present in In addition, complexes that not are to and of the protein interaction recently L. C. A. B. Li Y. B. E. P. M. Y. B. Z. A. M. S. L. E. F. J. N. M. E. N. S. E. Y. A. J. M. T. S. M. J. J. J.M. 2003; PubMed Scopus Google Scholar) an interaction between and Therefore, to proteins are the of complexes be to be We also show that between COMMD proteins are mediated by the COMM domain. COMMD proteins are functionally to the prototype of the family, MURR1/COMMD1, and to and NF-κB. We in the binding by which with RelA and that this is distinct from to NF-κB or binding to that these two are not for the interaction of with NF-κB. This is by the that can also associate with an NF-κB that not associate with S. S. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). These that the of an association between and that the ankyrin repeats of be as a the ankyrin repeats are for binding to the NF-κB (16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar). the association between and or are not by the COMM domain and and that the association of to NF-κB complexes is mediated by a distinct from the of COMMD association to NF-κB not be by COMMD the interaction between and In addition, not the interaction and NF-κB-mediated transcription is largely by the cytoplasmic sequestration of NF-κB complexes through the binding to ankyrin as the (11Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar, T. T. M. Y. Nature. 1993; PubMed Scopus Google Scholar). of the IκB kinase complex in to a of a of to IκB degradation and nuclear translocation on NF-κB. a role for MURR1/COMMD1 in the regulation of degradation has been (16Ganesh L. Burstein E. Guha-Niyogi A. Louder M.K. Mascola J.R. Klomp L.W. Wijmenga C. Duckett C.S. Nabel G.J. Nature. 2003; 426: 853-857Crossref PubMed Scopus (194) Google Scholar), here that this is not to its ability to inhibit NF-κB-mediated nuclear translocation of NF-κB is by we find that can regulate the nuclear function of NF-κB through its to κB-responsive promoters where it the of RelA binding to chromatin. NF-κB can of gene the are to the cell and in NF-κB has been to be to transcription of and factors in various M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar, 6Baldwin A.S. J. Clin. Investig. 2001; 107: 241-246Crossref PubMed Scopus (1199) Google Scholar). The regulation of NF-κB by sequestration of the complex is to for these in gene In this of NF-κB L. De S. 2002; PubMed Scopus Google Scholar) and promoter binding by have been shown to participate in the regulation of promoters in to S. S. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. D. R.C. M. EMBO J. 2004; 23: PubMed Scopus Google Scholar). of regulation that for the and of the are The here of the COMMD family an level that be in this The of COMMD through and the of association to NF-κB complexes that their COMMD proteins unique and of the of of these factors in cell to for the and by which of the of can be in to a of nuclear of NF-κB as and the activity. The transcriptional functions of the in be in to by a factor M. R.G. Nat. Rev. Mol. Biol. 2001; PubMed Scopus Google Scholar), can in its degradation E. Ganesh L. Dick R.D. van De Sluis B. Wilkinson J.C. Lewis J. Klomp L.W.J. Wijmenga C. Brewer G.J. Nabel G.J. Duckett C.S. EMBO J. 2004; 23: 244-254Crossref PubMed Scopus (183) Google Scholar), that the regulation of COMMD proteins be into that are known to NF-κB. We are to B. for We Dr. for and for several plasmids used in We are also to Dr. de provided with with