The PR Domain of the Rb-binding Zinc Finger Protein RIZ1 Is a Protein Binding Interface and Is Related to the SET Domain Functioning in Chromatin-mediated Gene Expression

Abstract

The PR domain, first noted as thePRDI-BF1-RIZ1 homologous region, defines a sub-class of zinc finger genes that appear to function as negative regulators of tumorigenesis. This family includes the MDS1-EVI1 gene inactivated in myeloid leukemia, the PRDI-BF1/BLIMP1transcription repressor of c-myc involved in driving B-cell differentiation, and the RIZ gene, which encodes proteins capable of binding to the retinoblastoma tumor suppressor protein (Rb). The PR domain of MDS1-EVI1 is disrupted by translocations linked to myeloid leukemia, resulting in the activation of the PR-minus oncogenic product EVI1. Remarkably similar to MDS1-EVI1,RIZ gene also normally produces two protein products of different length, and the smaller protein RIZ2 lacks the PR domain of RIZ1 but is otherwise identical to RIZ1. These observations raise considerable interest to determine the function of PR. We show here that RIZ1 PR domain mediates protein-protein interaction. Recombinant fusion proteins of PR can bind to in vitro translated RIZ1 and RIZ2 proteins. The binding can be disrupted by amino acid substitutions at conserved residues of PR, suggesting that binding is specific. Of the three conserved exons of PR, the first two appear dispensable for binding, whereas the third exon is required. A region in the carboxyl terminus of RIZ proteins was mapped to be necessary and sufficient for PR binding. We also found that the PR domain shares significant sequence identity to the SET domain present in chromosomal proteins that function in modulating gene expression from yeast to mammals. Our data suggest that the PR domain is a derivative of SET domain and may function as protein binding interface in the regulation of chromatin-mediated gene expression. The PR domain, first noted as thePRDI-BF1-RIZ1 homologous region, defines a sub-class of zinc finger genes that appear to function as negative regulators of tumorigenesis. This family includes the MDS1-EVI1 gene inactivated in myeloid leukemia, the PRDI-BF1/BLIMP1transcription repressor of c-myc involved in driving B-cell differentiation, and the RIZ gene, which encodes proteins capable of binding to the retinoblastoma tumor suppressor protein (Rb). The PR domain of MDS1-EVI1 is disrupted by translocations linked to myeloid leukemia, resulting in the activation of the PR-minus oncogenic product EVI1. Remarkably similar to MDS1-EVI1,RIZ gene also normally produces two protein products of different length, and the smaller protein RIZ2 lacks the PR domain of RIZ1 but is otherwise identical to RIZ1. These observations raise considerable interest to determine the function of PR. We show here that RIZ1 PR domain mediates protein-protein interaction. Recombinant fusion proteins of PR can bind to in vitro translated RIZ1 and RIZ2 proteins. The binding can be disrupted by amino acid substitutions at conserved residues of PR, suggesting that binding is specific. Of the three conserved exons of PR, the first two appear dispensable for binding, whereas the third exon is required. A region in the carboxyl terminus of RIZ proteins was mapped to be necessary and sufficient for PR binding. We also found that the PR domain shares significant sequence identity to the SET domain present in chromosomal proteins that function in modulating gene expression from yeast to mammals. Our data suggest that the PR domain is a derivative of SET domain and may function as protein binding interface in the regulation of chromatin-mediated gene expression. The Krüppel-like family of zinc finger genes is estimated to consist of hundreds of human genes (1Bellefroid E.J. Lecocq P.J. Benhida A. Poncelet D.A. Belayew A. Martial J.A. DNA. 1989; 8: 377-387Crossref PubMed Scopus (195) Google Scholar). This family is characterized by the Cys2-His2 zinc fingers and can be further divided into different subsets based on other structural features that include the seven-amino acid “H/C link” between adjacent fingers (2Rosenberg U.B. Schroder C. Preiss A. Keinlin A. Cote S. Riede I. Jackle H. Nature. 1986; 319: 336-339Crossref Scopus (323) Google Scholar) and several conserved amino-terminal modules (3Bellefroid E.J. Poncelet D.A. Lecocq P.J. Revelant O. Martial J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3608-3612Crossref PubMed Scopus (343) Google Scholar, 4Rosati M. Marino M. Franze A. Tramontano A. Grimaldi G. Nucleic Acids Res. 1991; 19: 5661-5667Crossref PubMed Scopus (75) Google Scholar, 5Knochel W. Poting A. Koster M. el Baradi T. Nietfeld W. Bouwmeester T. Pieler T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6097-6100Crossref PubMed Scopus (73) Google Scholar, 6Numoto M. Niwa O. Kaplan J. Wong K.K. Merrell K. Kamiya K. Yanagihara K. Calame K. Nucleic Acids Res. 1993; 21: 3767-3775Crossref PubMed Scopus (103) Google Scholar, 7Zollman S. Godt D. Prive G.G. Couderc J.L. Laski F.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10717-10721Crossref PubMed Scopus (363) Google Scholar). The PR domain is a newly recognized amino-terminal module and was first noted for the homologous 100-aa 1The abbreviations used are: aa, amino acids; EST, Expressed Sequence Tag; GST, glutathione S-transferase; PCR, polymerase chain reaction. 1The abbreviations used are: aa, amino acids; EST, Expressed Sequence Tag; GST, glutathione S-transferase; PCR, polymerase chain reaction. region shared between RIZ, which encodes Rb-binding proteins and the PRDI-BF1/BLIMP1 transcription repressor, which promotes B-lymphocyte maturation (8Buyse I.M. Shao G. Huang S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4467-4471Crossref PubMed Scopus (174) Google Scholar, 9Huang S. Cell. 1994; 78: 9Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 10Keller A.D. Maniatis T. Genes Dev. 1991; 5: 868-879Crossref PubMed Scopus (263) Google Scholar, 11Turner Jr., C.A. Mack D.H. Davis M.M. Cell. 1994; 77: 297-306Abstract Full Text PDF PubMed Scopus (628) Google Scholar). A PR domain was later found in theMDS1-EVI1 myeloid leukemia gene (12Fears S. Mathieu C. Zeleznik-Le N. Huang S. Rowley J.D. Nucifora G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1642-1647Crossref PubMed Scopus (187) Google Scholar). The PR domain is evolutionally conserved, and PR-containing open reading frames were found in the Caenorhabditis elegans andDrosophila genome or EST data bases. 2S. Huang, unpublished observation. 2S. Huang, unpublished observation. RIZ gene normally produces two different products, RIZ1 and RIZ2, which are both widely expressed (13Liu L. Shao G. Steele-Perkins G. Huang S. J. Biol. Chem. 1997; 272: 2984-2991Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). An internal promoter generates RIZ2, which is identical to RIZ1, except that it lacks the RIZ1 PR domain. This is remarkably similar to MDS1-EVI1. An internal promoter within MDS1-EVI1 gene generates theEVI1 myeloid-transforming gene product, which lacks PR but is otherwise identical to MDS1-EVI1 (14Bartholomew C. Ihle J.N. Mol. Cell. Biol. 1991; 11: 1820-1828Crossref PubMed Scopus (86) Google Scholar). The PR domain ofMDS1-EVI1 is a common target of inactivation by viral insertions and chromosomal translocations in leukemogenesis (15Morishita K. Parker D.S. Mucenski M.L. Jenkins N.A. Copeland N.G. Ihle J.N. Cell. 1988; 54: 831-840Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 16Morishita K. Parganas E. William C.L. Whittaker M.H. Drabkin H. Oval J. Taetle R. Valentine M.B. Ihle J.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3937-3941Crossref PubMed Scopus (288) Google Scholar, 17Nucifora G. Begy C.R. Kobayashi H. Roulston D. Claxton D. Pedersen-Bjergaard J. Parganas E. Ihle J.N. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4004-4008Crossref PubMed Scopus (253) Google Scholar), suggesting that the PR domain plays an important negative role in tumorigenesis. Consistently, other PR-containing gene products also have characteristics of a negative regulator of tumorigenesis. TheBLIMP1 gene maps to a tumor suppressor locus 6q21 (18Mock B.A. Liu L. Le Paslier D. Huang S. Genomics. 1996; 37: 24-28Crossref PubMed Scopus (43) Google Scholar) and is a transcriptional repressor of the c-myc oncogene, which is commonly overexpressed in a variety of human cancers (19Lin Y. Wong K.-k. Calame K. Science. 1997; 276: 596-598Crossref PubMed Scopus (345) Google Scholar). Forced expression of BLIMP1 causes differentiation or apoptosis, depending on cell types (11Turner Jr., C.A. Mack D.H. Davis M.M. Cell. 1994; 77: 297-306Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 19Lin Y. Wong K.-k. Calame K. Science. 1997; 276: 596-598Crossref PubMed Scopus (345) Google Scholar). A tumor suppressor role for RIZ is suggested by its chromosomal location at the distal short arm of human chromosome 1 next to the marker D1S228 on 1p36.23 (20Buyse I.M. Takahashi E. Huang S. Genomics. 1996; 34: 119-121Crossref PubMed Scopus (44) Google Scholar, 21Muraosa Y. Takahashi K. Yoshizawa M. Shibahara S. Eur. J. Biochem. 1996; 235: 471-479Crossref PubMed Scopus (42) Google Scholar), which commonly undergoes deletions, rearrangements, or loss of heterozygosity in a broad spectrum of human tumors, including those of breast (22Nagai H. Negrini M. Carter S.L. Gillum D.R. Rosenberg A.L. Schwartz G.F. Croce C.M. Cancer Res. 1995; 55: 1752-1757PubMed Google Scholar,23Genuardi M. Tsihira H. Anderson D.E. Saunders G.F. Am. J. Hum. Genet. 1989; 45: 73-82PubMed Google Scholar), liver (24Simon D. Knowles B.B. Weith A. Oncogene. 1991; 6: 765-770PubMed Google Scholar), colon (25Bardi G. Pandis N. Fenger C. Kronborg O. Bomme L. Heim S. Cancer Res. 1993; 53: 1895-1898PubMed Google Scholar), and neurocrest tissues (26Dracopoli N.C. Harnett P. Bale S.J. Stanger B.Z. Tucker M.A. Housman D.E. Kefford R.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4614-4618Crossref PubMed Scopus (210) Google Scholar, 27Fong C.T. White P.S. Peterson K. Sapienza C. Cavenee W.K. Kern S.E. Vogelstein B. Cantor A.B. Look A.T. Brodeur G.M. Cancer Res. 1992; 52: 1780-1785PubMed Google Scholar). We have recently established that RIZ1, but not RIZ2, expression was commonly decreased or lost in breast cancer, and forced RIZ1 expression caused cell cycle arrest in G2/M and/or apoptosis. 3L. He, J. X. Yu, L. Liu, I. M. Buyse, M.-S. Wang, Q. Yang, A. Nakagawara, G. M. Brodeur, Y. E. Shi, and S. Huang, submitted for publication. 3L. He, J. X. Yu, L. Liu, I. M. Buyse, M.-S. Wang, Q. Yang, A. Nakagawara, G. M. Brodeur, Y. E. Shi, and S. Huang, submitted for publication. Recent investigation shows that the PR domain of RIZ1 has neither a transcriptional repressor nor activator function (28Xie M. Shao G. Buyse I.M. Huang S. J. Biol. Chem. 1997; 272: 26360-26366Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), although full-length RIZ gene products function in both transcription activation (21Muraosa Y. Takahashi K. Yoshizawa M. Shibahara S. Eur. J. Biochem. 1996; 235: 471-479Crossref PubMed Scopus (42) Google Scholar) and repression (28Xie M. Shao G. Buyse I.M. Huang S. J. Biol. Chem. 1997; 272: 26360-26366Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Both RIZ1 and RIZ2 bind to GC-rich Sp-1-like DNA elements and repress transcription of the simian virus 40 early promoter, but RIZ1 is a more potent repressor than RIZ2, suggesting that the PR domain of RIZ1 modulates transcription (28Xie M. Shao G. Buyse I.M. Huang S. J. Biol. Chem. 1997; 272: 26360-26366Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). MDS1-EVI1 is recently shown to act as a transcriptional activator, whereas EVI1 as a repressor; the activation domain of MDS1-EVI1 maps to the PR domain (47Soderholm J. Kobayashi H. Mathieu C. Rowley J.D. Nucifora G. Leukemia. 1997; 11: 352-358Crossref PubMed Scopus (88) Google Scholar). Although these studies shed light on understanding PR domain function, no biochemical activities have been described for this important domain. Conserved motifs found in transcription factors generally function in binding to DNA and/or to polypeptides. We have generated purified recombinant fusion proteins of RIZ1 PR domain and used these proteins to examine whether this domain serves as a polypeptide binding interface. We found that PR domain polypeptides interact with in vitro translated RIZ1 or RIZ2 proteins. The binding appears to be specific, as it can be disrupted by specific mutations in the PR domain. A region in the carboxyl terminus of RIZ proteins was mapped to be necessary and sufficient for PR binding. The results demonstrate that the PR domain of RIZ1 mediates protein-protein interaction. We also found that the PR domain shares significant sequence identity to the SET domain present in chromosomal proteins that function in modulating gene expression from yeast to mammals. Our data suggest that the PR domain is a derivative of SET domain and may function as the protein binding interface in the regulation of chromatin-mediated gene expression. The RIZ1 PR domain was expressed as a GST fusion protein by using the pGEX-KG vector as described previously (8Buyse I.M. Shao G. Huang S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4467-4471Crossref PubMed Scopus (174) Google Scholar). A HincII-StuI PR domain fragment corresponding to residues 37–214 of rat RIZ1 cDNA was subcloned into the blunted NcoI site in pGEX-KG to generate pGST-RIZ1-PRB. domain in a fragment was using as a with and The fragment was into the and of pGEX-KG to generate acid substitutions were into the PR domain to generate using the from The two at in 1 were to The used were and of exon or of PR domain, an was into the site in the to generate exon or was generated by the PR domain in with an fragment exon The used for was from tissues of a for an of exon Steele-Perkins and S. Huang, unpublished The used were and exon or A was generated by a fragment of into and in pGEX-KG using and and for RIZ1 and RIZ2 products by in vitro have been described previously (13Liu L. Shao G. Steele-Perkins G. Huang S. J. Biol. Chem. 1997; 272: 2984-2991Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). of in products from was RIZ2 expression This was from by the of a fragment of rat RIZ1 This the for RIZ1 but of into generated full-length RIZ2 protein at similar as that from generate in vitro product of the PR domain, a fragment of was into the and in a with was into site to generate was generated to the of RIZ1 protein by of the amino-terminal region from NcoI to in was to to by the to fragment into was generated to the region by of the amino-terminal region from NcoI to in The were used to the to generate vitro GST fusion proteins of the region human RIZ1 cDNA was using and The fragment was into the and in pGEX-KG This region of human RIZ1 protein to of rat RIZ1 of the recombinant proteins from was with and for at was and recombinant GST fusion protein was purified as described R. J.A. K. in Scholar). was used to GST fusion proteins from E. cell the were with in the purified proteins or cell were first with in vitro products by the The was with and are the 1 1 of and proteins to the were by The were by with or The sequence of the PR domain of RIZ1 was used as a to the protein data of for The for was which is more to J. W. Nucleic Acids Res. 1997; PubMed Scopus Google Scholar). RIZ gene and the exon encodes the of RIZ1 protein 1 The PR domain is by three exons exon in PR a conserved of or in 1 B. The PR domain is in the amino-terminal acid region of RIZ1 of exons 1 This sequence is in RIZ2 protein 1 This region is to RIZ1 specific activities of this amino-terminal region and in the PR domain, two different GST fusion proteins were in E. and purified protein RIZ1 residues of the amino-terminal acid sequence of of exon and exons This protein be used to examine the function of the amino-terminal region including PR and its protein the PR domain sequence as by with other PR domain genes and of of exon and exons This protein be used to examine the function of PR The conserved motifs of transcription factors are to We whether PR interact with RIZ1 GST fusion proteins as shown in were to The were with in vitro translated RIZ1 of RIZ1 protein was was for or or whereas no binding was found for the GST These results suggest that the amino-terminal acid region of RIZ1 in protein-protein between RIZ1 to RIZ1 was for the PR domain. in vitro translated RIZ2 product also to or The binding of PR to RIZ2 to be with binding to RIZ1, that PR is is not for binding of RIZ proteins. that the PR not interact with in vitro PR domain product was for binding to of protein was which is than or The results suggest a of PR. is not for the binding in the protein binding of PR, and mutations were into the protein the role of conserved exon in PR exon were and expressed the binding and the role of of the conserved amino acid amino acid substitutions were into conserved in exon two residues at to These proteins as shown in used for binding to in vitro translated RIZ1 shown in of exon disrupted binding but of exon and not binding and The protein with substitutions of for at residues in exon also disrupted binding, further suggesting a for exon in binding. These data the of the PR domain in RIZ protein-protein further the PR binding of RIZ of RIZ1 were generated by in vitro transcription and of RIZ proteins were for binding to The to PR of RIZ was whereas the amino-terminal binding of The binding of PR to the amino-terminal of RIZ1 is with the described and in B. the PR binding was in the of RIZ1, of were generated by in vitro transcription and A fragment residues to not bind to but a fragment residues to the PR binding This the PR binding to within the acid region of this region by was of residues by or to by not binding, but of residues by disrupted binding. The results suggest that the region from amino acid to of rat RIZ1 is for binding to PR. whether this region is also sufficient for binding to PR, a GST fusion protein of this region of human RIZ1, to rat RIZ1 protein residues was expressed in E. and purified This fusion protein was for binding to in PR binding of PR PR to but not to GST protein was The results show that RIZ1 PR binding is at the region between amino and the of PR domain, the data for protein that may to the PR domain. is a recently to protein and to function for proteins J. W. Nucleic Acids Res. 1997; PubMed Scopus Google Scholar). an to generate a from this a which is as a in a This can be to for homologous that not have been by the We used the PR domain sequence of RIZ1 as and of the protein data of PR genes as as two C. elegans open reading frames and were with the of the The next of significant were found for the SET domain present in and proteins with from to The SET domain is a conserved sequence present in chromosomal proteins that function in modulating gene activities from yeast to B. A. R. G. G. J. 1994; PubMed Scopus Google Scholar, T. G. R. G. Cell. Mol. Sci. 54: PubMed Scopus Google Scholar). is important to that the shared residues between PR and SET are also the conserved residues in domain, suggesting may a common function of the two conserved residues for RIZ1 PR protein binding as shown in is also conserved in the SET domain. the between PR and which is identical in amino are PR genes are and SET SET are found at the carboxyl of whereas PR are at the amino PR are with zinc finger whereas SET have been found in a variety of proteins including the domain, zinc and binding motifs T. G. R. G. Cell. Mol. Sci. 54: PubMed Scopus Google Scholar). PR domain is not found in yeast genome and is a derivative of SET domain in RIZ1 is identical to RIZ2, except that it has an residues at the amino These residues may specific function to RIZ1 The PR domain the within the RIZ1 amino-terminal We found that at function of this region is to as a protein binding interface. GST fusion proteins of the RIZ1 amino terminus from 37–214 similar activities as the PR from residues suggesting that the PR domain is the protein binding in the amino-terminal The protein binding function of RIZ1 PR is specific, as by of function by is to that of exon or not PR binding to RIZ both exons conserved exon is as or within this exon disrupted binding. The a of PR domain is that an conserved of PR may activities that are of other activities may also from of these The of a PR binding to a region of RIZ1 the of a in this This appears not conserved PR as data not proteins with significant the the that PR be in or of may on other protein-protein of PR domain with the carboxyl domain as an in full-length RIZ1 but not in between RIZ1 or between RIZ1 and RIZ2 be by this binding is also that both PR and the PR binding of RIZ1 may interact with other as proteins. this of in an important PR domain is to the SET domain is a SET domain genes have to and an important role in cancer, and gene and in chromosome T. G. R. G. Cell. Mol. Sci. 54: PubMed Scopus Google Scholar). are regulators with activities in both and The of the family include the suppressor gene B. A. R. G. G. J. 1994; PubMed Scopus Google Scholar), the of Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar), and the J.A. 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We suggest that SET and PR may function to involved in gene activation or gene or inactivation of SET or PR domain in a gene may the of the gene the other of the gene as DNA binding and transcriptional activation or regulation may a between a product and a PR-minus product of a PR gene, which may the of these products in tumorigenesis. with the that PR domain family is a derivative of SET domain PR genes with SET The MDS1-EVI1 gene is involved in C. Davis K. Ihle J.N. Mucenski M.L. Dev. 1997; PubMed Scopus Google Scholar) and human leukemia similar to has been found in tumors, suggesting it may act as a negative regulator of R. Negrini M. K. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). PR genes are to repress a shared by SET genes that function in gene studies are to generate more in the biochemical and of these although may also be PR and SET are two the Rb-binding protein RIZ1 is to function in regulation further a role for in chromatin-mediated gene expression. other Rb-binding proteins are to function in including human Cell. 92: Full Text Full Text PDF PubMed Scopus Google Scholar, A. J.L. T. Nature. PubMed Scopus Google Scholar, L. R. I. P. S. Le D. A. Nature. PubMed Scopus Google Scholar) and are of D. S. D. G. K. P. Nature. 1996; PubMed Scopus Google Scholar, C. A. D. J. G. P. 1996; Google Scholar). of this three have been SET domain function in protein-protein C. E. Proc. Natl. Acad. Sci. U. S. A. X. M. L. Genet. and L. Hum. Mol. Genet. The SET domain of is shown to interact with and proteins of the involved in and the family of protein The SET domain is shown to bind to the gene product, a of the family involved in We and for and for the We for