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Differential Role of the Proline-rich Domain of Nuclear Factor 1-C Splice Variants in DNA Binding and Transactivation

2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês

10.1074/jbc.m200418200

1083-351X

Félix Prado, Guillermo P. Vicent, Carina Cardalda, Miguel Beato,

Plant Molecular Biology Research

We have addressed the functional significance of the existence of several natural splice variants of NF1-C* differing in their COOH-terminal proline-rich transactivation domain (PRD) by studying their specific DNA binding and transactivation in the yeastSaccharomyces cerevisiae. These parameters yielded the intrinsic transactivation potential (ITP), defined as the activation observed with equal amounts of DNA bound protein. Exchange of 83 amino acids at the COOH-terminal end of the PRD by 16 unrelated amino acids, as found in NF1-C2, and splicing out the central region of the PRD, as found in NF1-C7, enhanced DNA binding in vivo and in vitro. However, the ITP of the splice variants NF1-C2 and NF1-C7 was found to be similar to that of the intact NF1-C1. Additional mutations showed that the ITP of NF1-C requires the synergistic action of the PRD and a novel domain encoded in exons 5 and 6. Intriguingly the carboxyl-terminal domain-like motif encoded in exons 9/10 is not essential for transactivation of a reporter with a single NF1 site but is required for activation of a reporter with six NF1 sites in tandem. Our results imply that differential splicing is used to regulate transcription by generating variants with different DNA binding affinities but similar ITPs. We have addressed the functional significance of the existence of several natural splice variants of NF1-C* differing in their COOH-terminal proline-rich transactivation domain (PRD) by studying their specific DNA binding and transactivation in the yeastSaccharomyces cerevisiae. These parameters yielded the intrinsic transactivation potential (ITP), defined as the activation observed with equal amounts of DNA bound protein. Exchange of 83 amino acids at the COOH-terminal end of the PRD by 16 unrelated amino acids, as found in NF1-C2, and splicing out the central region of the PRD, as found in NF1-C7, enhanced DNA binding in vivo and in vitro. However, the ITP of the splice variants NF1-C2 and NF1-C7 was found to be similar to that of the intact NF1-C1. Additional mutations showed that the ITP of NF1-C requires the synergistic action of the PRD and a novel domain encoded in exons 5 and 6. Intriguingly the carboxyl-terminal domain-like motif encoded in exons 9/10 is not essential for transactivation of a reporter with a single NF1 site but is required for activation of a reporter with six NF1 sites in tandem. Our results imply that differential splicing is used to regulate transcription by generating variants with different DNA binding affinities but similar ITPs. Regulation of transcription operates by the combinatorial action of sequence-specific trans-acting factors bound to upstream regulatory regions of promoters and enhancer/silencer regions. The particular linear disposition of these cis-regulatory sequences and their topological organization in chromatin, along with the cellular repertoire of transcription factors results in the formation of gene- and cell-specific constellations of chromatin bound factors. The particular array of proteins on the chromatin target modulates the transcriptional rates by multiple interactions with chromatin remodeling complexes, co-regulators, and components of the basal transcriptional machinery (1.Lemon B. Tjian R. Genes Dev. 2000; 14: 2551-2569Crossref PubMed Scopus (606) Google Scholar). Transcriptional regulators often exhibit a modular structure with independent DNA-binding domains (DBDs) 1The abbreviations used are: DBDDNA-binding domainNF1nuclear factor 1PRDproline-rich transactivation domainITPintrinsic transactivation potentialCTDCOOH-terminal domainMMTVmouse mammary tumor virusGEMSAgel electrophoretic mobility shift assay and regulatory regions. The transactivation domains are classified according to their amino acid composition as either rich in acidic side chains, glutamine, or proline residues (2.Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2209) Google Scholar). Although the precise fashion by which transactivators regulate transcription of a specific gene depends on many different promoter features and cell-specific factors, the mechanisms of action for acidic and proline-rich domains appear to be conserved in eukaryotic organisms from yeast to man (3.Kim T.K. Roeder R.G. J. Biol. Chem. 1993; 268: 20866-20869Abstract Full Text PDF PubMed Google Scholar, 4.Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1179) Google Scholar). DNA-binding domain nuclear factor 1 proline-rich transactivation domain intrinsic transactivation potential COOH-terminal domain mouse mammary tumor virus gel electrophoretic mobility shift assay Most transcription factors are expressed in several variants that can be grouped in large families, whose members show only subtle differences. The expression of these variants is tightly regulated in a cell-type or developmental-stage specific manner. Alternative splicing is the mechanism most widely used to generate this precisely regulated, diversity of transcription factors. However, with few exceptions, the functional significance of transcription factor variants generated by alternative splicing is poorly understood (5.Lopez A.J. Dev. Biol. 1995; 172: 396-411Crossref PubMed Scopus (87) Google Scholar, 6.Graveley B.R. Trends Genet. 2001; 17: 100-107Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar). The prototype of the proline-rich class of activators is nuclear factor 1 (NF1), originally identified through its role in stimulating adenovirus DNA replication (7.Jones K.A. Kadonaga J.T. Rosenfeld P.J. Kelly T.J. Tjian R. Cell. 1987; 48: 79-89Abstract Full Text PDF PubMed Scopus (574) Google Scholar, 8.Nagata K. Guggenheimer R.A. Enomoto T. Lichy J.H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6438-6442Crossref PubMed Scopus (216) Google Scholar). NF1 is expressed in vertebrates from at least four different genes (NF1-A, NF1-B,NF1-C/CTF, and NF1-X), each of them giving rise to different variants by alternative splicing of the COOH terminus (9.Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (492) Google Scholar, 10.Paonessa G. Gounari F. Frank R. Cortese R. EMBO J. 1988; 7: 3115-3123Crossref PubMed Scopus (173) Google Scholar, 11.Rupp R.A.W. Kruse U. Multhaupt G. Gı̂bel U. Beyreuther K. Sippel A.E. Nucleic Acids Res. 1990; 18: 2607-2616Crossref PubMed Scopus (150) Google Scholar, 12.Meisterernst M. Rogge L. Foeckler R. Karaghiosoff M. Winnacker E.L. Biochemistry. 1989; 28: 8191-8200Crossref PubMed Scopus (72) Google Scholar, 13.Kruse U. Sippel A.E. J. Mol. Biol. 1994; 238: 860-865Crossref PubMed Scopus (77) Google Scholar). The variants bind as homo- and heterodimers to the consensus binding site TTGGC(N5)GCCAA (14.Kruse U. Sippel A.E. FEBS Lett. 1994; 348: 46-50Crossref PubMed Scopus (106) Google Scholar). This wide variety of forms is differentially expressed during mouse development and regulates the activity of many genes expressed in multiple organs (Ref.15.Chaudhry A.Z. Lyons G.E. Gronostajski R.M. Dev. Dyn. 1997; 208: 313-325Crossref PubMed Scopus (178) Google Scholar and references therein). Disruption of the NF1-A gene in mice causes multiple developmental defects and perinatal lethality (16.das Neves L. Duchala C.S. Godinho F. Haxhiu M.A. Colmenares C. Macklin W.B. Campbell C.E. Butz K.G. Gronostajski R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11946-11951Crossref PubMed Scopus (185) Google Scholar), suggesting that at least some functions of the various NF1 proteins are not redundant. However, the functional implications of the existence of a large variety of NF1 proteins remain largely unknown. Comparison of the primary structures of different NF1-C variants reveals their modular organization (Fig. 1 A). All variants share a very well conserved NH2 terminus containing the DBD and the dimerization domain. This NH2-terminal half activates Adenovirus 2 DNA replication by interacting with the viral DNA polymerase (17.Armentero M.T. Horwitz M. Mermod N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11537-11541Crossref PubMed Scopus (40) Google Scholar, 18.Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar). The COOH-terminal half can be divided in a central region, which is specific for the products of each of the four genes, and a variable carboxy region, which is specific for each splice variant (13.Kruse U. Sippel A.E. J. Mol. Biol. 1994; 238: 860-865Crossref PubMed Scopus (77) Google Scholar, 18.Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar, 19.Roulet E. Armentero M.T. Krey G. Corthesy B. Dreyer C. Mermod N. Wahli W. Mol. Cell. Biol. 1995; 15: 5552-5562Crossref PubMed Scopus (34) Google Scholar). A functional comparison of NF1-C variants showed different efficiencies of transcriptional activation, indicating that the very COOH-terminal region determines the transcriptional potential of NF1 (20.Wenzelides S. Altmann H. Wendler W. Winnacker E.L. Nucleic Acids Res. 1996; 24: 2416-2421Crossref PubMed Scopus (28) Google Scholar, 21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar). This region contains the proline-rich transactivation domain (PRD), whose importance in the transactivation functions of NF1 has been largely confirmed (3.Kim T.K. Roeder R.G. J. Biol. Chem. 1993; 268: 20866-20869Abstract Full Text PDF PubMed Google Scholar, 18.Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar). A detailed analysis of this region identified a sequence homologous to the COOH-terminal domain (CTD) of the largest subunit of RNA polymerase II as an essential element of the PRD. In addition, a stretch of hydrophobic amino acids contributes strongly to the activity of the CTD-like motif (22.Kim T.K. Roeder R.G. Nucleic Acids Res. 1994; 22: 251Crossref PubMed Scopus (25) Google Scholar, 23.Wendler W. Altmann H. Winnacker E.L. Nucleic Acids Res. 1994; 22: 2601-2603Crossref PubMed Scopus (20) Google Scholar, 24.Xiao H. Lis J.T. Xiao H. Greenblatt J. Friesen J.D. Nucleic Acids Res. 1994; 22: 1966-1973Crossref PubMed Scopus (56) Google Scholar). The very last 20 amino acids at the COOH terminus of NF1-C1 have been reported to interact with the globular domain of histone H3 and may mediate regulation of NF1-C1 activity by transforming growth factor-β (25.Alevizopoulos A. Dusserre Y. Tsai-Pflugfelder M. von der Weid T. Wahli W. Mermod N. Genes Dev. 1995; 9: 3051-3066Crossref PubMed Scopus (120) Google Scholar). However, some variants lacking most of the PRD and the CTD-like motif enhance transcription to a greater extent than the full-length NF1-C1 (20.Wenzelides S. Altmann H. Wendler W. Winnacker E.L. Nucleic Acids Res. 1996; 24: 2416-2421Crossref PubMed Scopus (28) Google Scholar, 21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar). These observations suggested the existence of additional regulatory sequences in NF1-C, and placed a question mark on the role of the variable PRD in transactivation by NF1-C. Here we report studies in Saccharomyces cerevisiae showing that although the absolute transactivation obtained with three natural splice variants of NF1-C (NF1-C1, NF1-C2, and NF1-C7, differing in their PRD) are different, this is largely due to differences in DNA binding. Once corrected for DNA binding activity, the intrinsic transactivation potential (ITP) of the three isoforms is similar and requires the synergistic action of the PRD and an internal region encoded by exons 5 and 6. The CTD-like motif exhibits a reporter-specific behavior. We show that the splice variants NF1-C2 and NF1-C7 bind to DNA with higher affinity than the full-length NF1-C1 and conclude that splicing out part of the PRD regulates transactivation by increasing the DNA affinity of the resulting protein without affecting its ITP once bound to DNA. The yeast strain used in this study was YPH499 (a ade2–101 his3-Δ200 leu2-Δ1 trp1-Δ1 ura3–52 lys2–801). Standard media, such as rich medium YEPD and synthetic complete medium (SC) with bases and amino acids omitted as specified, were prepared as described previously (26.Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Yeast strains were transformed using the lithium acetate method (27.Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) modified according to Schiestl and Gietz (28.Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1775) Google Scholar). All plasmid constructions were performed usingEscherichia coli strain DH5α. pLR-NF1(x6) (21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar) and pSCh105 (29.Candau R. Chávez S. Beato M. J. Steroid Biochem. Mol. Biol. 1996; 57: 19-31Crossref PubMed Scopus (18) Google Scholar) are YEp plasmids based on the URA3 gene containing the NF1(x6)-GAL1(ΔUAS)-lacZ and the MMTVΔ-lacZfusion constructs, respectively. pAA-CTF1, pAA-CTF7 (21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar), and pAA-CTF2 are YEp expression vectors for pig NF1-C1, NF1-C7, and NF1-C2, respectively, derived from pAAH5 (30.Ammerer G. Methods Enzymol. 1994; 101: 192-201Crossref Scopus (252) Google Scholar). pAA-CTF1ΔCT7 is a YEp expression vector for pig NF1-C1ΔCT7, constructed by cloning theEcoRI-XhoI NF1 fragment (made blunt ended) from pEG202-CTF1m(Δ7-2) (23.Wendler W. Altmann H. Winnacker E.L. Nucleic Acids Res. 1994; 22: 2601-2603Crossref PubMed Scopus (20) Google Scholar) into the bluntedHindIII site of pAAH5. pAA-CTF-(1–6), pAA-CTF-(1–7), and pAA-CTF(1-Bg) are YEp expression vectors for pig NF1-C319, NF1-C362, and NF1-C406, respectively. NF1sequences coding for amino acids 1 to 319, 1 to 362, or 1 to 406 were amplified by PCR and inserted at the HindIII site of pAAH5. pBR-CTF1 consists of a SphI-SphINF1-C1 fragment from pAA-CTF1 inserted at theSphI site of pBR322. pAA-CTFsb is a YEp expression plasmid for pig NF1-CΔ234–406. It was constructed in two steps. First, pBR-CTF1 was cut by partial digestion with SacI (at position 687 of the CTF1 ORF)-BglII and religated by using a SacI-BglII linker (pBR-CTFsb); second, theSphI-SphI NF1 fragment from pBR-CTFsb was inserted at the SphI site of pAAH5 (pAA-CTFsb). pGPDCTFbd is a YEp expression vector for pig NF1-C229, based on theLEU2 gene. It was constructed in four steps. First, pBR-CTF1 was cut by partial digestion with SacI (at position 687 of the CTF1 ORF and made blunt ended)-SmaI, and religated (pBR-CTF1mbd); second, the SphI-SphINF1 fragment from pBR-CTF1mbd was inserted at theSphI site of pAAH5 (pAA-CTF1mbd); third, a linker 5′-ATCCTCTAGATAACTAGTTAGTCATCTAGAGTCG-3′ containing a stop codon was cut with XbaI and inserted at the XbaI site of pAA-CTF1mbd by partial digestion (pAA-CTF1bd-(1–4)); fourth, the blunted SspI-SpeI NF1-C229 fragment of pAA-CTFbd-(1–4) was cloned into the SmaI site of p425GPD (31.Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). ptCTF1, ptCTF2, ptCTF7, ptCTFΔCT7, ptCTF-(1–7), ptCTFsb, and ptCTFbd are YEp expression vectors for tagged NF1-C1, NF1-C2, NF1-C7, NF1-CΔCT7, NF1-C362, NF1-CΔ234–406, and NF1-C229, respectively.NF1 sequences were amplified by PCR introducing in the amino-terminal end hemeagglutinin and histidine tags, and inserted at the HindIII site of pAAH5. p415MCTF1 is a YCp plasmid based on the LEU2 gene expressing NF1-C1 under control of theMET25 promoter (32.Prado F. Koop R. Beato M. J. Biol. Chem. 2002; 277: 4911-4917Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Transcription activity was determined by β-galactosidase assays of permeabilized cells (33.Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (874) Google Scholar). Yeast cells were grown overnight in the appropriate selective medium at 30 °C, diluted to an A 660 of 0.1, and incubated for 8 h. Cells were then harvested and assayed for β-galactosidase activity (33.Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (874) Google Scholar). For the preparation of the NF1 DNA probe, the oligonucleotides 5′-cctttggcactgtgccaag-3′ and 5′-cctttggcacagtgccaag-3′ were annealed, gel-purified, and end-labeled by T4 polynucleotide kinase with [γ-32P]ATP. The binding reactions contained the indicated amounts of yeast protein extract, prepared as described previously (34.McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (234) Google Scholar). In a final volume of 20 μl the reactions contained: 12 mm HEPES-NaOH (pH 7.9), 210 mm KCl, 4 mm Tris-HCl (pH 7.9), 1 mm EDTA, 12% glycerol, 4.2 mm β-mercaptoethanol, 3 μg of poly(dI-dC), 3 μg of sheared salmon sperm DNA, and 0.5–1 ng of the labeled probe. Reactions were incubated at 30 °C for 45 min. The protein-DNA complexes were resolved by electrophoresis (4 h at 200 V) on 5% polyacrylamide, 10% glycerol gels (acrylamide to bisacrylamide weight ratio of 37.5:1) in 0.5 × TBE at 4 °C. Quantification of DNA complexes was performed with a PhosphorImager. 1–40 μg of yeast protein extract were run on a 10% SDS-polyacrylamide gel and transferred to nylon membranes. After blocking with TBS containing 0.1% Tween 20 and 5% milk, proteins were detected with antibody against the hemeagglutinine tag and peroxidase-conjugated goat anti-mouse IgG. The blots were washed with TBS, 0.1% Tween 20 and developed by enhanced chemiluminescence (ECL) reactions (Amersham Bioscience). DNase I treatment was performed as described previously (35.Chávez S. Candau R. Truss M. Beato M. Mol. Cell. Biol. 1995; 15: 6987-6998Crossref PubMed Scopus (41) Google Scholar). Briefly, transformants were grown in appropriate selective medium to mid-log phase. Cells were harvested, spheroplasts were prepared and treated with different amounts of DNase I. After DNA extraction, DNase I-cleaved genomic DNA samples were extended for 30 cycles with a radioactively labeled primer, GAL-pLR (5′-agtattagttaaagtggttatgcag-3′), corresponding to the sequence located 96 bp downstream of the 6 NF1 sites in pLR-NF1(x6). Amplified DNA was resolved on 6% polyacrylamide sequencing gels. Quantification was performed by a PhosphoImager. To gain insight into the mechanisms of transcriptional activation by members of the NF1 family we have studied the ITP of three natural NF1-C splice variants (Fig. 1 A). We have chosen the yeastS. cerevisiae for these studies because NF1-C is known to transactivate in this organism and endogenous NF1 homologues are not known. NF1-C1 encompasses all 11 exons of the gene (Fig. 1 A). In NF1-C2, the splicing of exon 9 deletes 83 amino acids of the PRD and creates a new reading frame leading to the COOH-terminal addition of 16 amino acids with a high proline content. NF1-C7 lacks exons 7, 8, and 9 including most of the PRD (Fig. 1 A) (12.Meisterernst M. Rogge L. Foeckler R. Karaghiosoff M. Winnacker E.L. Biochemistry. 1989; 28: 8191-8200Crossref PubMed Scopus (72) Google Scholar, 21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar). We transformed yeast with plasmids expressing each of these variants together with the reporter plasmid pLR-NF1(x6) (21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar). In this plasmid, which we will name NF16GAL1, thelacZ gene is driven by the GAL1 promoter with its regulatory region replaced by six NF1-binding sites. As previously reported, NF1-C7 transactivated this promoter to a much larger extent than NF1-C1 (Fig. 1 A), despite lacking most of the PRD (21.Altmann H. Wendler W. Winnacker E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3901-3905Crossref PubMed Scopus (42) Google Scholar). NF1-C2 transactivated with similar efficiency (48%) as NF1-C1 (Fig. 1 A), confirming previous findings in DrosophilaSchneider cells (9.Santoro C. Mermod N. Andrews P.C. Tjian R. Nature. 1988; 334: 218-224Crossref PubMed Scopus (492) Google Scholar, 18.Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar). We also tested the behavior of the three NF1-C variants in the context of a natural promoter with only one NF1 site, namely a mutant MMTV promoter (MMTVΔ), truncated just upstream of the NF1-binding site to remove the hormone responsive region (29.Candau R. Chávez S. Beato M. J. Steroid Biochem. Mol. Biol. 1996; 57: 19-31Crossref PubMed Scopus (18) Google Scholar). Although the activity on this promoter was lower, the results with the NF1-C variants were qualitatively similar to those obtained with NF16GAL1 (Fig. 1 A). NF1-C7 was by far the best transactivator, while NF1-C2 was very similar to NF1-C1. Thus, independent of the reporter used, NF1-C7 was a better transactivator than NF1-C1 and NF1-C2. Most studies on the transactivation properties of NF1 in yeast have not taken into account possible differences in levels of expression or in DNA binding affinity of the different variants. To incorporate these parameters in our study, we determined the extent of specific DNA binding to calculate the ITP of each variant by comparing amounts of protein yielding equivalent DNA binding activity. To establish the reliability of our measurements, we first expressed NF1-C1under control of the regulated MET25 promoter, whose activity depends on the methionine concentration in the medium. We prepared extracts from cells grown at different methionine concentrations and measured β-galactosidase activity and specific binding to an oligonucleotide with a NF1-binding site by GEMSA. As expected, DNA binding increased with progressive activation of theMET25 promoter (Fig. 2 A). Within the range tested, the NF1-C1 dependent activation of the NF16GAL1 reporter was a linear function of the amount of DNA binding activity (Fig. 2 B, circles). The same is true for the MMTVΔ promoter (data not shown), confirming the validity of our quantitation of DNA binding. Next we compared the DNA binding activity of extracts from cells expressing the three natural variants of NF1-C. While extracts containing NF1-C1 and NF1-C2 showed very similar binding to the NF1 oligonucleotide, extracts containing NF1-C7 showed a 7-fold higher specific DNA binding (Fig. 1 B). The activation values obtained with the three NF1-C variants in these experiments fitted very well in the standard curve constructed with NF1-C1 under the control of the inducible MET25 promoter (Fig. 2 B,squares). Correction of the transactivation activities (Fig. 1 A) by the corresponding DNA binding values of the NF1-C variants yielded their respective ITP, which were very similar on the MMTVΔ reporter (Fig. 1 C, light columns). On the NF16GAL1 reporter, NF1-C2 exhibited a 2-fold lower intrinsic transcriptional potential when compared with NF1-C1 and NF1-C7 (Fig. 1 C, dark columns) (see below). These results demonstrate that the splicing of different regions of the PRD of NF1-C1 generates two proteins, NF1-C2 and NF1-C7, with different DNA binding activity, but with similar ITP. Previous work on NF1 has defined the COOH-terminal PRD as the main transactivation determinant of the protein (3.Kim T.K. Roeder R.G. J. Biol. Chem. 1993; 268: 20866-20869Abstract Full Text PDF PubMed Google Scholar, 18.Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar). However, the results obtained with NF1-C7 show that most of the PRD of NF1-C1 could be spliced out without affecting the ITP. Along the same line, the natural variant NF1-C5, which lacks exons 9 and 10, acts as a strong transactivator in yeast (20.Wenzelides S. Altmann H. Wendler W. Winnacker E.L. Nucleic Acids Res. 1996; 24: 2416-2421Crossref PubMed Scopus (28) Google Scholar). These observations suggest the presence of additional transactivation functions in NF1-C. To address this issue, we have analyzed different deletion mutants of the full-length NF1-C1 for their ability to transactivate the two reporter genes in yeast (Fig. 3 A). As for the natural NF1-C variants, the activities of the mutant proteins have been corrected for their corresponding DNA binding activities (Fig. 3 B) to calculate their ITP once bound to DNA (Fig. 3 C). In previous studies the core PRD has been defined as the region encoded in exons 9–11 (amino acids 407–506). Deletion of this region, as in NF1-C406, led to a dramatic reduction in transactivation of both reporters (Fig. 3 A). This was accompanied by a 5-fold reduction in DNA binding activity (Fig. 3 B). After correction for DNA binding, the ITP of NF1-C406 on the NF16GAL1 and the MMTVΔ reporter was 5 and 15% that of NF1-C1, respectively (Fig. 3 C), confirming that the core PRD is important for transactivation. As exon 8 is also rich in proline (19.5%), we tested the behavior of a deletion of all the proline-rich regions (exons 8–11). This deletion, NF1-C362, showed slightly higher transactivation than NF1-C406 (Fig. 3 A), but a 13-fold higher DNA binding activity compared with NF1-C406 (Fig. 3 B). Thus, the ITP was further reduced 3-fold, to 1.3% of NF1-C1 with the NF16GAL1 reporter and 5% with the MMTVΔ reporter (Fig. 3 C). These results confirm the importance of the proline-rich region, and show that it encompasses sequences encoded in exons 8–11 of NF1-C1. Constructions lacking this region exhibited 3-fold higher residual activity on the MMTV reporter than on the reporter with six NF1-binding sites, suggesting that the PRD is also involved in the synergism between DNA-bound NF1 molecules. Although part of the proline-rich region can be deleted and replaced by different sequences without influencing the ITP, as in NF1-C2 and NF1-C7 (Fig. 1), other regions are obviously essential. It remains to be elucidated what additional features of these regions, besides the high proline content, determine their transactivation properties. If the PRD were the only transactivation function within the COOH-terminal half of NF1-C, a protein with this domain fused to the DBD, as generated by deleting exons 5–7, should display similar activity as the intact protein, provided the DBD has no transactivation function. We confirmed that the NF1-DBD, although able to transactivate to some extent when expressed from a strong promoter (Fig. 3 A), had negligible ITP (Fig. 3 C,C229) after correction for the high DNA binding activity (Fig. 3 B, C229). The internal deletion of exons 5–7 in construct NF1-CΔ234–406 led to a 2–2.5-fold decrease in transactivation (Fig. 3 A) and to a 4-fold increase in DNA binding activity (Fig. 3 B). After appropriate correction this construction showed a 10-fold decrease of ITP compared with intact NF1-C1 (Fig. 3 C). Nevertheless, these values were still 7–10-fold higher than those obtained with the DBD of NF1-C (Fig. 3 C, compare CΔ234–406 andC229). These findings identify a transactivation function in sequences encoded by exons 5–7. Since NF1-C7 shows maximal activity and lacks amino acids 320–472 (Fig. 1), and deletion of exon 7 encoded sequences does not reduce the ITP (compare C319 andC362), the novel transactivation function must be located between amino acids 234–319 in the region encoded by exons 5 and 6. A construction including just the DBD and exons 5–6, NF1-C319, exhibited low but reproducible ITP, in particular with the MMTVΔ reporter (Fig. 3 C) (see below). Thus, our results with natural variants and mutations support the existence of a weak internal transactivation domain in the region encompassing exons 5 and 6. As the sum of the ITPs of the PRD and this internal domain accounts only for 15–20% of the ITP of NF1-C1, the two domains seem to act synergistically in the intact protein. The PRD of NF1 contains a sequence with a striking similarity to the heptapeptide repeats of the CTD of the largest subunit of RNA polymerase II. This CTD-like motif has been claimed to be essential for the transactivation function of the PRD (22.Kim T.K. Roeder R.G. Nucleic Acids Res. 1994; 22: 251Crossref PubMed Scopus (25) Google Scholar, 23.Wendler W. Altmann H. Winnacker E.L. Nucleic Acids Res. 1994; 22: 2601-2603Crossref PubMed Scopus (20) Google Scholar, 24.Xiao H. Lis J.T. Xiao H. Greenblatt J. Friesen J.D. Nucleic Acids Res. 1994; 22: 1966-1973Crossref PubMed Scopus (56) Google Scholar). However, this claim is in conflict with the strong ITP of the natural variants NF1-C2 and NF1-C5, which lack the CTD-like motif (Fig. 1 and Ref. 20.Wenzelides S. Altmann H. Wendler W. Winnacker E.L. Nucleic Acids Res. 1996; 24: 2416-2421Crossref PubMed Scopus (28) Google Scholar). Therefore, we tested a selective deletion of this motif. The resulting protein, NF1-CΔCT7, exhibited a lower transactivation than NF1-C1 (Fig. 3 A) and an almost normal DNA binding activity (Fig. 3 B). These changes result in a 5-fold decrease in its ITP on the NF16GAL1 promoter (Fig. 3 C), in agreement with previous results with similar constructs (23.Wendler W. Altmann H. Winnacker E.L. Nucleic Acids Res. 1994; 22: 2601-2603Crossref PubMed Scopus (20) Google Scholar, 24.Xiao H. Lis J.T. Xiao H. Greenblatt J. Friesen J.D. Nucleic Acids Res. 1994; 22: 1966-1973Crossref PubMed Scopus (56) Google Scholar). However, the ITP of NF1-CΔCT7 was hardly affected when tested on the MMTVΔ promoter, which contains a single NF1-binding site (Fig. 3 C). Except C229, all constructions lacking