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• W2006715676 abstract "Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis. SSAP contains an extremely potent transcription activation domain that functions 4–5-fold better than VP16 in a variety of mammalian cell lines. We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP. One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1. Functional assays establish the ZFM1 protein as a transcriptional repressor. ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region. The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites. The activity is unique in that activated but not basal transcription levels are affected. Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis. SSAP contains an extremely potent transcription activation domain that functions 4–5-fold better than VP16 in a variety of mammalian cell lines. We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP. One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1. Functional assays establish the ZFM1 protein as a transcriptional repressor. ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region. The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites. The activity is unique in that activated but not basal transcription levels are affected. Different families of histone genes in sea urchins are expressed with distinct temporal patterns during early embryogenesis, making this system ideal for studying mechanisms of temporal gene expression. In recent years, much progress has been made in studying the expression of late H1 histone subtype genes (1Lai Z.C. DeAngelo D.J. DiLiberto M. Childs G. Mol. Cell. Biol. 1989; 9: 2315-2321Crossref PubMed Scopus (20) Google Scholar, 2DeAngelo D.J. DeFalco J. Childs G. Mol. Cell. Biol. 1993; 13: 1746-1758Crossref PubMed Google Scholar, 3DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar, 4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). The late H1-β gene is transcribed at low levels until it is transcriptionally activated at the mid-blastula stage, and reaches its peak level of expression in 24-h late-blastula stage embryos. The correct temporal activation of late H1 gene expression is determined by an enhancer element located 220–280 base pairs upstream in its promoter region (1Lai Z.C. DeAngelo D.J. DiLiberto M. Childs G. Mol. Cell. Biol. 1989; 9: 2315-2321Crossref PubMed Scopus (20) Google Scholar). Stage-specific activator protein (SSAP) 1The abbreviations used are: SSAP, stage-specific activator protein; PCR, polymerase chain reaction; 3-AT, 3-aminotriazole; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; TK, thymidine kinase; TBP, TATA-binding protein; TF, transcription factor; hnRNP, heterogeneous nuclear ribonucleoprotein. is a 43-kDa polypeptide that can specifically bind to this enhancer (2DeAngelo D.J. DeFalco J. Childs G. Mol. Cell. Biol. 1993; 13: 1746-1758Crossref PubMed Google Scholar). Early in development, SSAP is present as a monomer; however, it undergoes a posttranslational modification at about 12 h after fertilization, and dimerization coincides with the activation of late H1 gene (2DeAngelo D.J. DeFalco J. Childs G. Mol. Cell. Biol. 1993; 13: 1746-1758Crossref PubMed Google Scholar). Synthetic SSAP mRNA injected into zygotes transactivates reporter genes containing SSAP binding sites (3DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar). Significantly, this transactivation also occurs in a temporal-specific manner. The DNA binding activity of SSAP maps to its N-terminal 180 amino acids (3DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar). This domain contains two RNA recognition motifs, which recognize both double-stranded and single-stranded DNA in a sequence-specific manner (3DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar). In addition to this novel DNA binding domain, SSAP contains an extremely potent transcription activation domain consisting of amino acids 181–404 (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). This activation domain consists of a central glycine/glutamine-rich sequence and a C-terminal region rich in serine/threonine and basic amino acids (referred to as the GQC domain). In a variety of mammalian cell lines, Gal4-GQC fusion protein can transactivate the expression of Gal4-responsive reporter genes as much as 4–5-fold better than Gal4-VP16 (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). This activity requires the presence of both the central G/Q-rich domain as well as the C-terminal domain. The GQ region alone cannot activate transcription, whereas the C-terminal region has minimal activity (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). The activation domain of a transcription factor may promote transcription at several different steps during the formation of a preinitiation complex by recruiting basal transcription factors or during promoter clearance and elongation (5Choy B. Green M.R. Nature. 1993; 366: 531-536Crossref PubMed Scopus (237) Google Scholar, 6Narayan S. Widen S.G. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 12755-12763Abstract Full Text PDF PubMed Google Scholar, 7Yankulov K. Blau J. Purton T. Roberts S. Bentley D.L. Cell. 1994; 77: 749-759Abstract Full Text PDF PubMed Scopus (209) Google Scholar). To do this, the activators must interact with multiple targets in the transcriptional apparatus. These targets include not only basal factors in the transcription machinery, but also various co-activators or adaptors, which facilitate interactions between activator and basal transcription factors or RNA polymerase II (8Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 9Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (350) Google Scholar). In vitrobinding assays have shown that the GQC domain can physically interact with several basal transcription factors of RNA polymerase II, including TBP, TFIIB, TFIIF74, and dTAFII110 (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). On the other hand, the GQC domain has self-squelching activity and the ability to squelch VP16- and E1A-driven reporter genes. This suggests that the activation domains of SSAP, VP16, and E1a share some common targets necessary for maximal transcription. It is believed that these targets may not be basal transcription factors, instead, they may be specific adaptors or coactivator proteins (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). Although the ability of the activation domain of SSAP to drive such high levels of transcription may be explained by stronger association with common targets, it is also possible that it may interact with unique protein targets not shared by VP16 or E1a. It has been well documented that different classes of activation domains may interact with distinct coactivators or adaptors to stimulate transcription (8Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar). For example, the glutamine-rich activation domain of SP1 binds specifically to TAFII110 (10Hoey T. Weinzierl R.O. Gill G. Chen J.L. Dynlacht B.D. Tjian R. Cell. 1993; 72: 247-260Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 11Gill G. Pascal E. Tseng Z.H. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (473) Google Scholar), whereas the acidic activation domain of VP16 cannot stimulate transcription without interaction with TAFII40 (12Goodrich J.A. Hoey T. Thut C.J. Admon A. Tjian R. Cell. 1993; 75: 519-530Abstract Full Text PDF PubMed Scopus (351) Google Scholar). Similarly, a group of transcription factors require p300/CBP coactivator family members for maximal activity, whereas other transcription factors act independently of this regulatory network (13Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (492) Google Scholar, 14Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 15Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2407) Google Scholar). In this paper, a two-hybrid screening technique is employed to identify human protein(s) that can interact with the activation domain of SSAP. The rationale for this screen comes from the extremely potent activation domain used as a “bait” in this study. We believed that the GQC activation domain would interact with interesting transcriptional mediators. This paper describes the properties of one SSAP-interacting protein, ZFM1. ZFM1 was previously cloned as a nuclear protein at a locus linked to multiple endocrine neoplasia type 1 (16Toda T. Iida A. Miwa T. Nakamura Y. Imai T. Hum. Mol. Genet. 1994; 3: 465-470Crossref PubMed Scopus (48) Google Scholar). It was also identified as a presplicing factor SF1 (17Arning S. Gruter P. Bilbe G. Kramer A. RNA. 1996; 2: 794-810PubMed Google Scholar). Here, we show that ZFM1 functions as a transcription repressor. To generate pBTM116-GQC as bait for two-hybrid screening, an EcoRI fragment derived from pSG424-GQC (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar), which contains the entire GQC domain (amino acids 181–404), was ligated into pBTM116 vector cut with EcoRI. The resulting construct was sequenced to verify that the GQC domain was cloned in the correct orientation and in frame with the LexA DNA binding domain. To create pGAD424-ZFM1-E for yeast two-hybrid assays, two primers (5′-GGG GTC GAC CAG ACC ACA TGG CGA CCG GAG CGA AC-3′) and (5′-CCC GTC GAC TCA CTT GTC ATC GTC GTC CTT GTA GTC CCA ATG GGC GCG GAA AGT-3′) were used in a PCR reaction to amplify ZFM1-E from pBluescript SK phagemid DNA. This PCR product was cut by SalI and ligated into the SalI site of pGAD424 to fuse ZFM1-E in frame with the Gal4 activation domain. To generate pCR3.1-ZFM1-A expression construct for functional assays, two PCR fragments that correspond to N-terminal (between nucleotide 383–1607) and C-terminal (between nucleotide 836 and 2251) regions of ZFM1-A, respectively, were created. The N-terminal piece was amplified using template ZFM1-E in pBluescript SK and two primers (5′-GGG GTC GAC CCG CCA CCA TGG CGA CCG GAG CGA AC-3′ and 5′-CAT GCC CAC CTG TCA GGC-3′), and the C-terminal piece was amplified using template CE9 and two primers (5′-TTT GTG GGG CTG CTC ATC-3′ and 5′-GTC GAC TCA CTT GTC ATC GTC GTC CTT GTA GTC TGG CTC GGG CCA TCG C-3′). A unique EcoRI site at nucleotide position 1489 of ZFM1-A was utilized to join these fragments in the correct reading frame. The two non-overlapping portions were joined together and ligated to pCR3.1 vector (Invitrogen) in a three-piece ligation reaction. In the resulting plasmid, pCR3.1-ZFM1-A, the expression of full-length ZFM1-A in mammalian cells is under the control of cytomegalovirus promoter, and there is a FLAG-tag in frame with the C-terminal amino acid of ZFM1-A. To make pGAD424-ZFM1-A and pSG424-ZFM1-A, ZFM1-A fragment was released from pCR3.1-ZFM1-A by SalI digestion and then cloned into the SalI site of pGAD424 and pSG424, respectively. To create pCR3.1-ZFM1-(1–320), this region of ZFM1-A was amplified by PCR using two primers (5′-GGG GTC GAC CCG CCA CCA TGG CGA CCG GAG CGA AC-3′ and 5′-GAC GCG TCG ACT CAC TTG TCA TCG TCG TCC TTG TAG TCC AGT TCA GCC ATG AGG GA-3′). This PCR fragment is cloned in pCR3.1 and the expression of ZFM1-(1–320) with a C-terminal FLAG-tag is under the control of cytomegalovirus promoter in the resulting plasmid. pGAD424-ZFM1-(1–320) and pSG424-ZFM1-(1–320) were created in a manner similar to that for ZFM1-A on these vectors by utilizing the same SalI site. To clone pGEX-KG-ZFM1-(321–478) for bacterial expression of GST fusion protein, ZFM1-(321–478) was amplified from template pCR3.1-ZFM1-A using primer 5′-GCG GGA TCC GTC TAG AGG GTG AAG CAC CTG TC-3′ and primer 5′-GCG GGA TCC AAG CTT ACA TCA TGC CCA TAG GTG-3′. The resulting DNA fragment was digested with XbaI and HindIII and ligated into XbaI/HindIII-digested pGEX-KG. To generate various deletion constructs of ZFM1 for repression domain mapping, PCR product corresponding to defined region of ZFM1 was amplified using pCR3.1-ZFM1-A DNA as template and two primers, which span this region and contain appropriate cloning sites at the ends. Resulting PCR fragments were digested and ligated into pSG424 to make fusion proteins with Gal4-(1–147). The regions of ZFM1 contained in each of these constructs are designated by the amino acids given in parentheses. The HL60 library, a generous gift from Dr. G. Kalpana, was divided into six different pools with a total of 1.12 × 106 individual clones (18Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (705) Google Scholar). To screen this library, pBTM116-GQC plasmid was first transformed into the yeast L40 strain (19Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). Then the DNA from either an individual library pool or combinations of several pools was transformed using the lithium-acetate method (20Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1775) Google Scholar, 21Hill J. Donald K.A. Griffiths D.E. Donald K.A. Nucleic Acids Res. 1991; 19: 5791Crossref PubMed Scopus (460) Google Scholar), followed by 16 h of incubation in selective medium lacking Ura, Trp, and Leu at 30 °C to allow the expression of the HIS3 reporter gene. A fraction of the transformants were then plated on selective medium lacking Ura, Trp, Leu, Lys, and His but with 50 mm 3-aminotriazole (3-AT). A total of 2.85 × 107 transformants were screened from these six pools of HL60 library in three sets of experiments. After the plates were incubated at 30 °C for 3–5 days, 38 His+ colonies were chosen for further study. All of them quickly turned dark blue when they were tested for β-galactosidase activity using a filter assay (22Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). Plasmid DNA was recovered from most of these His+lacZ+ colonies and electrotransformed into Escherichia coli XL1 blue for propogation. The false positives were then eliminated using genetic tests, in which each positive plasmid is retransformed into L40 either alone, with pBTM116, with pBTM116-GQC, with pBTM116-Lamin or with another irrelevant bait. A plasmid was considered as true positive if it could activate the expression of LacZ reporter in L40 only when it was cotransformed with pBTM116-GQC but not in other combinations. Of the 38 His+lacZ+ colonies, 18 were true positives. The cDNA inserts from most of these plasmids were sequenced and used in GenBank™ BLAST searches. ZFM1-(321–484) fragment was amplified from its parental yeast clone by PCR and labeled as a probe by random primer labeling (Amersham Life Science). This probe was used to screen a λ-ZAP HeLa cell cDNA library. The phage from positive clones were then converted to pBluescript SK phagemid by in vivo excision. The cDNA inserts on the phagemid were analyzed by restriction enzyme digestion and sequenced. All GST fusion proteins, GST-ZFM1-(321–478), GST-GQC, and GST alone were expressed in E. coli BL(21) by inducing with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside and coupled to glutathione-Sepharose beads according to the manufacturer's instructions and Ref. 4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar. In vitro translated GQC domain and ZFM1-E were synthesized using a coupled transcription-translation system (TNT lysate, Promega). GST pull-down experiments were conducted according to Defalco and Childs (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). HepG2 cells are maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium, supplemented with 10% fetal bovine serum. The cells were transfected using Lipofectin method as described by the manufacturer (Life Technologies, Inc.). In a typical transfection experiment, DNA mixtures contain 2 μg of CAT reporter plasmid and 1–2 μg of pGK-β-gal control plasmid. pCL-neo (Promega) is used as carrier to make sure that each transfection mixture contained the same amount of total DNA. Cells were harvested 48 h after transfection and lysed by the freeze-thaw method (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). Transfection efficiencies were normalized among all the samples according to β-galactosidase activity expressed from control plasmid. Normalized extracts were used in CAT assays (23La Teana A. Brandi A. Falconi M. Spurio R. Pon C.L. Gualerzi C.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10907-10911Crossref PubMed Scopus (243) Google Scholar). The expression of CAT activity in a given sample was quantitated using a PhosphorImager (Molecular Dynamics). Each transfection for a given experiment was repeated at least three times. To check the expression of transfected ZFM1 in HepG2 cells, normalized extract from freeze-thaw transfected cells used for CAT assays were resolved on 10% SDS-PAGE. The expression of transfected ZFM1 protein was detected using M2 antibody, which recognizes the FLAG-tag on its C terminus. To identify protein(s) that interact with the GQC activation domain, we employed a modified version of the yeast two-hybrid system. The GQC transcription activation domain of SSAP from amino acids 181 to 404 (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar) was fused in frame with the LexA DNA binding domain in pBTM116, and the resulting fusion protein was used as bait. When LexA-GQC was transformed into the yeast L40 strain, the resulting transformants can grow on plates lacking histidine. β-Galactosidase filter assays yield light blue colonies due to the GQC-mediated activation of His3 and LacZ reporters containing upstream LexA-binding sites. Therefore, this bait alone can function as a weak transcriptional activator in yeast (TableI). The GQC domain functions as an extremely potent activation domain in mammalian cells (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar) and in sea urchins (3DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar), but it was questionable if it would function in yeast due to its amino acid content. Yeast do not support “standard” glutamine-rich activators (24Ponticelli A.S. Pardee T.S. Struhl K. Mol. Cell. Biol. 1995; 15: 983-988Crossref PubMed Scopus (53) Google Scholar), and most yeast activation domains are acidic (25Struhl K. Annu. Rev. Genet. 1995; 29: 651-674Crossref PubMed Scopus (136) Google Scholar). The GQC activation domain has a region rich in glycine and glutamine (GQ) and a C-terminal region (C) rich in serine, threonine, and basic amino acids (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). We also made two constructs that individually express LexA fusions to either the GQ or the C regions. As expected from the result in mammalian cells, neither LexA-GQ nor LexA-C can produce β-Galactosidase activity by driving LacZ gene expression in yeast. For this reason, we decided to use the intact activation domain as bait and to inhibit growth of the parent strain used for our screen by addition of the competitive inhibitor of the histidine synthetase 3-AT to the medium. The background growth of LexA-GQC transformants can be eliminated by adding as little as 5 mm 3-AT in the medium. Therefore, we modified the original two-hybrid selection technique by screening library on plates containing 50 or 100 mm of 3-AT to suppress growth of cells not containing true interacting proteins.Table IQuantitation of ZFM1-GQC interactions in the yeast two-hybrid systemLexA-DB fusionGal4-AC fusionβ-Galactosidase activityunitsLexA-GQCAC-ZFM1-(321–484)301 ± 85LexA-GQCAC-ZFM1-A324 ± 124LexA-GQCAC-ZFM1-E185 ± 78LexA-GQCAC-ZFM1-(1–320)46 ± 24LexA-GQC40 ± 8LexA-GQCAC31 ± 10LexAAC-ZFM1-(321–484)2.2 ± 0.7LexAAC-ZFM1-A4.9 ± 1.8LexAAC-ZFM1-E3.7 ± 0.9LexAAC-ZFM1-(1–320)12 ± 1.7 Open table in a new tab A human cDNA library derived from an undifferentiated leukemia cell line (HL60) was screened using this LexA-GQC bait. From 2.85 × 107 yeast transformants, 38 colonies grew on His− plates with 50 mm 3-AT. These colonies quickly turn dark blue using the β-galactosidase filter assay. Twenty of the 38 turned out to be false positives in genetic tests. The remaining 18 are true positives because their ability to activate LacZ and HIS3 genes depends solely on the presence of LexA-GQC. We also tested the interaction between these 18 fusion proteins and GAL4-GQC fusion protein in a different yeast strain, HF7c, which possesses a upstream activating sequence with Gal4 binding sites upstream of the LacZ gene. All 18 positives still interact with the GQC bait but not with plasmids encoding either Gal4 DNA binding domain alone or irrelevant Gal4 fusion proteins. Each fusion protein was also tested for interaction with LexA-GQ and LexA-C alone. There is detectable but weak interaction between the positives with the C domain, but none of them interacts with the GQ domain alone (data not shown). The plasmids from most of these positives were successfully isolated and sequenced. Nine of the 18 positives have identical inserts that encode a fragment corresponding to part of the ZFM1-A protein (GenBank™ accession no. D26120) (16Toda T. Iida A. Miwa T. Nakamura Y. Imai T. Hum. Mol. Genet. 1994; 3: 465-470Crossref PubMed Scopus (48) Google Scholar), between amino acids 321 and 484 (ZFM1-(321–484)), which is the subject of this paper. ZFM1 was first described by Toda et al. (16Toda T. Iida A. Miwa T. Nakamura Y. Imai T. Hum. Mol. Genet. 1994; 3: 465-470Crossref PubMed Scopus (48) Google Scholar) as a nuclear protein at the locus tightly linked to a putative tumor suppressor gene for multiple endocrine neoplasia type 1. ZFM1 was subsequently isolated by Kramer and colleagues (17Arning S. Gruter P. Bilbe G. Kramer A. RNA. 1996; 2: 794-810PubMed Google Scholar) as splicing factor SF1 potentially functioning in pre-spliceosome assembly. Both groups reported that there are multiple splicing variants of ZFM1 in different tissues or cell types. At the same time, we tried to obtain cDNAs of ZFM1 for functional studies by screening a HeLa cell cDNA library using ZFM1-(321–484) as a probe. A number of overlapping cDNA clones were isolated. Interestingly, we isolated a new splicing variant not reported previously. This ZFM1 variant, designated as ZFM1-E, lacks 434 nucleotides between nucleotide position 1785 and 2218 of ZFM1-A and contains an open reading frame that differs from other reported ZFM1 isoforms by deleting part of the proline-rich domain but retaining the same C-terminal tail as ZFM1-B (Fig. 1). The exon-intron junction sites at positions of both 1785 and 2218 show a good match to the consensus splice site sequence. We constructed a clone encoding the full-length ZFM1-A variant by joining an N-terminal fragment amplified from ZFM1-E and a C-terminal fragment amplified from CE9 (Fig. 1). Like ZFM1-(321–484), both ZFM1-E and ZFM1-A can interact with the GQC domain in a yeast two-hybrid assay (Table I). We used GST pull-down assays to confirm the direct association of ZFM1 and GQC. ZFM1-(321–478), which covers most of the region of ZFM1-A that binds to GQC in yeast was fused in frame with glutathione S-transferase to express the GST-ZFM1-(321–478) chimeric protein in bacteria. Radiolabeled in vitro translated GQC was incubated with glutathione-Sepharose beads coupled with either GST-ZFM1-(321–478) or GST alone. After extensive washing, proteins retained on the beads are eluted and separated by SDS-PAGE. The GQC domain specifically associated with GST-ZFM1-(321–478) but not with GST alone (Fig. 2 A). In parallel, we did the reverse experiment. In vitro translated radiolabeled ZFM1-E was incubated with GST-GQC protein. As in the previous experiment, ZFM1-E can bind to GST-GQC but not to GST alone (Fig. 2 B). The GQC domain can function as a potent transcriptional activation domain in a variety of mammalian cell lines (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). To assess the consequences of an interaction between GQC and ZFM1, we asked whether overexpression of ZFM1 can modulate the transcriptional activity of the GQC domain. When pCR3.1-ZFM1-A is transfected into HepG2 cells, we detected a 70–80-kDa protein band that corresponds to the size predicted as ZFM1-A in Western blots using M2 antibody. Moreover, the level of expression of tagged ZFM1-A correlates well with the amount of DNA transfected (Fig.3 C). The open reading frame of ZFM1 contains a potential nuclear localization signal near its N terminus (16Toda T. Iida A. Miwa T. Nakamura Y. Imai T. Hum. Mol. Genet. 1994; 3: 465-470Crossref PubMed Scopus (48) Google Scholar). We confirmed the nuclear localization of ZFM1 protein by immunostaining and Western blot analysis of the nuclear and cytoplasmic fractions of HepG2 cell extract (data not shown). When increasing amounts of pCR3.1-ZFM1-A are cotransfected into HepG2 cells along with pSG424-GQC and G5E1BCAT (a CAT reporter with five Gal4 DNA binding sites upstream in its promoter), we observed that the reporter expression is repressed by expression of ZFM1-A in a dose-dependent manner (Fig. 3 A). At the highest levels of ZFM1-A, transcription driven by Gal4-GQC is repressed by 4–5-fold compared with cells containing endogenous levels of ZFM1 variants. In Fig. 3, we used a level of Gal4-GQC that is within the linear range of a titration curve of Gal4-GQC activator (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). Higher levels of Gal4-GQC exhibit self-squelching, whereas lower levels give linear decreases in activation levels (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). We observe very similar 4–5-fold repression when different amount of Gal4-GQC (from 10 ng to 5 μg) are cotransfected with increasing amounts of pCR3.1-ZFM1-A (data not shown). The reporter gene used in this experiment, G5E1BCAT, has a minimal TATA-containing promoter. Without GAL4-GQC-activated transcription, we asked if the minimal activity of the reporter alone could be repressed by overexpression of ZFM1-A. The observation that the expression of the reporter alone cannot be repressed similarly indicates that repression of transcription from this reporter requires the presence of the Gal4-GQC protein (Fig. 3 B). The GQC activation domain represents a class of transcriptional activation domain rich in glycine, glutamine, serine, and threonine (4DeFalco J. Childs G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5802-5807Crossref PubMed Scopus (11) Google Scholar). EWS, which is frequently involved in translocations in Ewing's Sarcoma tumors, also contains a potent transcriptional activation domain with similar amino acid composition (26Crozat A. Aman P. Mandahl N. Ron D. Nature. 1993; 363: 640-644Crossref PubMed Scopus (757) Google Scholar). We observed that ZFM1-A can also interact directly with this domain of EWS. Similarly, overexpression of ZFM1-A can repress transactivation of reporter genes driven by Gal4-EWS in transient transfection experiments. 2D. Zhang and G. Childs, manuscript in preparation. We speculate that ZFM1 will act on a variety of cellular activators containing" @default.
• W2006715676 created "2016-06-24" @default.
• W2006715676 creator A5030611214 @default.
• W2006715676 creator A5078453161 @default.
• W2006715676 date "1998-03-01" @default.
• W2006715676 modified "2023-09-29" @default.
• W2006715676 title "Human ZFM1 Protein Is a Transcriptional Repressor That Interacts with the Transcription Activation Domain of Stage-specific Activator Protein" @default.
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