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• W2050793927 abstract "LAP/NF-IL6 is a member of the C/EBP family of transcriptional activators and has been shown to be involved in the regulation of the acute-phase response. We have previously shown that phosphorylation of the liver-enriched transcriptional activator protein (LAP) Ser-105 enhances the activation of LAP-dependent genes. To identify the region which is important for gene activation, a series of LAP mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed. These experiments point to an acidic region located between amino acids 21 and 105 of LAP/NF-IL6 which activates genes independent of the DNA-binding domain and the leucine zipper of LAP/NF-IL6. Computer-assisted predictions reveal two regions, a helical and a hydrophobic region in the transactivation domain, which could be important in mediating the direct interaction with the basal machinery. Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery. Our results demonstrate that a hydrophobic region in the acidic transactivation domain of LAP/NF-IL6 seems to be relevant in mediating gene activation of LAP-dependent genes. LAP/NF-IL6 is a member of the C/EBP family of transcriptional activators and has been shown to be involved in the regulation of the acute-phase response. We have previously shown that phosphorylation of the liver-enriched transcriptional activator protein (LAP) Ser-105 enhances the activation of LAP-dependent genes. To identify the region which is important for gene activation, a series of LAP mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed. These experiments point to an acidic region located between amino acids 21 and 105 of LAP/NF-IL6 which activates genes independent of the DNA-binding domain and the leucine zipper of LAP/NF-IL6. Computer-assisted predictions reveal two regions, a helical and a hydrophobic region in the transactivation domain, which could be important in mediating the direct interaction with the basal machinery. Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery. Our results demonstrate that a hydrophobic region in the acidic transactivation domain of LAP/NF-IL6 seems to be relevant in mediating gene activation of LAP-dependent genes. For many genes cell type-specific expression has been shown to be regulated at the level of transcription. Most genes are controlled by several transcription factors which interact with cis-acting elements in the promoter or in remote enhancer regions. The interaction of transcription factors with their DNA recognition sites can enhance the frequency of transcription initiation. The increase in the rate of transcription is believed to be mediated by direct contact of the transcription factor with the transcription apparatus (basal machinery) after DNA binding(1Johnson P.F. McKnight S.L. Annu. Rev. Biochem. 1989; 58: 799-839Crossref PubMed Scopus (830) Google Scholar, 2Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2210) Google Scholar). The gene, encoding the liver-enriched transcriptional activator protein (LAP)1( 1The abbreviations used are: LAPliver-enriched transcriptional activator proteinLIPliver inhibitory proteinILinterleukinCATchloramphenicol acetyltransferaseCMVcytomegalovirusCREBcAMP-responsive element-binding proteinC/EBPCCAAT/enhancer-binding proteinORFopen reading frameCBPphospho-CREB-binding protein.) has been cloned recently(3Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes & Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (420) Google Scholar). The same or a related protein has also been named NF-IL6(4Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar), IL6-DBP(5Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (459) Google Scholar), AGP/EBP(6Chang C.-J. Chen T.-T. Lei H.-Y. Chen D.-S. Lee S.C. Mol. Cell. Biol. 1990; 10: 6642-6653Crossref PubMed Scopus (200) Google Scholar), C/EBPβ(7Cao Z. Umek R.M. McKnight S.L. Genes & Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar), and CRP2(8Williams S.C. Cantwell C.A. Johnson P.F. Genes & Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (439) Google Scholar). LAP, a member of the C/EBP family, interacts with cis-acting elements in the promoter region of the albumin gene and several other liver-specific genes(9Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). The albumin promoter consists of six cis-acting elements (A-F). The B- and D-sites are especially important as binding sites for transcription factors which determine hepatocyte-specific expression of the albumin gene(10Maire P. Wuarin J. Schibler U. Science. 1989; 244: 343-346Crossref PubMed Scopus (211) Google Scholar). LAP shows strong affinity for the D-site of the albumin promoter and leads to stimulation of transcription in vivo and in vitro(3Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes & Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (420) Google Scholar). liver-enriched transcriptional activator protein liver inhibitory protein interleukin chloramphenicol acetyltransferase cytomegalovirus cAMP-responsive element-binding protein CCAAT/enhancer-binding protein open reading frame phospho-CREB-binding protein. Besides LAP, two other transcription factors, C/EBP-α and DBP, bind to the D-site of the albumin promoter. All three proteins belong to the bZIP family of transcriptional activators(3Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes & Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (420) Google Scholar, 11Landschulz W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes & Dev. 1988; 2: 786-800Crossref PubMed Scopus (630) Google Scholar, 12Mueller C.R. Maire P. Schibler U. Cell. 1990; 61: 279-291Abstract Full Text PDF PubMed Scopus (277) Google Scholar). LAP and C/EBP-α have similar affinities toward the cognate DNA and show more than 71% homology in their DNA-binding domain and the adjacent α helix in the C-terminal part of the protein. The C-terminal part of the protein is responsible for dimerization in vivo and in vitro of the two proteins. Descombes et al.(3Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes & Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (420) Google Scholar) postulated a model in which the interaction of different proteins at promoter sites may determine distinct functions in target gene transcription. According to this model the functional differences would be encoded in the different N-terminal region of the two transcription factors LAP and C/EBP-α. LAP/NF-IL6 has been implicated as a master regulator of the acute-phase response. Earlier data indicated that it might be involved in the induction of several cytokine genes such as IL6, IL8, tumor necrosis factor-α, and granulocyte-colony-stimulating factor, which include LAP recognition sites in their critical cis-acting elements(4Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar, 13Mukaida N. Shiroo M. Matsushima K.J. J. Immunol. 1989; 143: 1366-1371PubMed Google Scholar, 14Nishizawa M. Tsuchiya M. Watanabe-Fukunaga R. Nagata S. J. Biol. Chem. 1990; 265: 5897-5902Abstract Full Text PDF PubMed Google Scholar). Recent data of Tanaka et al.(15Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar), in NF-IL6 deficient mice, raise doubt about the importance of NF-IL6 for induction of some of these genes. Further studies have to show whether the results of Tanaka et al.(15Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji N. Fujiwara H. Suematsu S. Yoshida N. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar) observed in the transgenic mouse model do account for the in vivo situation. Homozygous LAP/NF-IL6-deficient transgenic mice might develop mechanisms during embryonal development, for example by other members of the C/EBP family, which may circumvent the loss of LAP/NF-IL6. Genes of the acute-phase response that share LAP/NF-IL6 recognition sites in their promoters have been named the class I of the acute phase genes(16Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (490) Google Scholar), in contrast to the acute-phase class II genes, where binding sites for acute phase response factor have been described(17Lütticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pelle Grinni S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (713) Google Scholar, 18Akira S. Nishio Y. Inoue M. Wang X.-J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Abstract Full Text PDF PubMed Scopus (877) Google Scholar). In response to environmental signals, modulation of target gene expression can be achieved through posttranscriptional modifications of transcriptional activators(19Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1120) Google Scholar). Posttranscriptional mechanisms have also been postulated to be important in the activation of genes with LAP recognition sites in their promoter. An IL6-dependent pathway leads to increased binding and transactivation of LAP (NF-IL6) (5). A direct pathway which may lead to the phosphorylation of LAP during IL6 induction has not been shown yet; however, LAP is a phosphoprotein in vivo. Different signal transduction pathways can induce LAP-mediated gene transcription, and cAMP-mediated phosphorylation of LAP is associated with the nuclear translocation of LAP in rat pheochromocytoma PC12 cells(20Metz R. Ziff E. Genes & Dev. 1991; 5: 1754-1766Crossref PubMed Scopus (302) Google Scholar, 21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar, 22Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (308) Google Scholar). We showed previously that stimulation of a protein kinase C pathway in HepG2 hepatoma cells increases the phosphorylation of Ser-105 which is located within the N-terminal region of LAP(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). Next to LAP, a liver inhibitory protein (LIP) also exists in the liver and is translated from the same LAP mRNA, lacking the 145 N-terminal amino acids of LAP. The resulting truncated protein is found to bind the cognate DNA with higher affinity than LAP. As the N-terminal transactivation domain is deleted in LIP, binding results in a strong competitional inhibition of LAP mediated gene transcription(9Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). Additional LIP has been shown to antagonize the effect of LAP in blocking hepatoma cell proliferation(23Buck M. Turler H. Chojkier M. EMBO J. 1994; 13: 851-860Crossref PubMed Scopus (108) Google Scholar). Therefore, besides activating liver-specific gene transcription, the N-terminal part of the protein seems to be important in mediating the LAP-dependent cell cycle arrest in hepatoma cells. Here we demonstrate that an 84-amino acid-long domain in the N-terminal domain of LAP is sufficient to mediate activation of a LAP-dependent reporter gene construct. Deletion analysis and domain swapping show that the effect on transactivation mediated by this domain is independent of the basic DNA-binding domain and of the leucine zipper region. HepG2 cells (ATCC) were cultured in minimal essential medium supplemented with 10% fetal calf serum. DNA transfection into HepG2 cells was carried out as described previously(2Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2210) Google Scholar), with the exception of a calcium phosphate precipitation for only 5 h, followed by a 15% glycerol shock for 1 min. In experiments with the LAP deletion proteins, LAP-responsive CAT-reporter constructs, the D- or CRP-CAT vectors, were cotransfected. CAT assays were performed 48 h after transfection using thin layer chromatography for the separation of the reaction products(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). Quantification of the CAT results was carried out with a Fuji Imager. In transfection experiments with the LAP/GAL4 fusion proteins the GAL4-responsive reporter 5 ´ GAL4-Luc was cotransfected. For luciferase activity cell extracts were prepared and measured as described before(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). The pBS-LAP Δ21 construct (24Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Crossref PubMed Scopus (129) Google Scholar) was used for plasmid constructions. For site-directed mutagenesis the cDNA of pBS LAP Δ21 was used to prepare single stranded DNA in the Escherichia coli strain JM 109 using the helper phage M13 KO 7, according to Sambrook et al.(25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Site-directed mutagenesis was performed by using the mutagenesis kit from Amersham, according to the supplier's instructions. To obtain the 5′ deletion of LAP, restriction sites were introduced into the open reading frame (ORF) of LAP: LAP Δ 1-41, 5′- CGC GGC GCG GAC CGC CTT GGC CC −3′ (RsrII site); LAP Δ 1-63, 5′-CAG GTA GGG GCT CAT GAC GAT GGC GCG-3′ (BspHI site); LAP Δ1-75, 5′-GCG GCG AAG TCC TAG GCG GCG GG-3′ (AvrII site); LAP Δ1-104, 5′-CCG TAG TCG GAC CGC TTC TTG CTC G-3′ (RsrII site); LAP Δ1-121, 5′-GGG AAG CAG GCC TGC GGT GCG GC-3′ (StuI site); LAP Δ1-189, 5′-GGC GTC GGC GGG ACC CGG CGT CCC GGG-3′. For LAP Δ1-139 and LAP Δ1-152 natural sites BsiCI and NcoI, respectively, in the ORF were used to obtain the deletions. After introduction of the restriction sites in the ORF of LAP, constructs were further modified to obtain the deletions, while maintaining the ORF of LAP. To obtain the internal deletions of LAP, the same restriction sites were used, and DNA pieces were modified and religated. All the resulting constructs were sequenced and subcloned into a CMV-driven mammalian expression vector as described before(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). To construct the GAL4 fusion proteins the NcoI-EcoRI fragment from the pBS LAP Δ21 construct containing the basic domain and the leucine zipper region of LAP was replaced by the BspHI-EcoRI fragment containing the GAL4 DNA-binding domain (amino acids 1-147) of a modified version of the pSG424 vector as described before(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). The chimeric LAP-GAL4 reading frames were excised by HindIlI-EcoRI digestion and were cloned into the pSG424 vector from which the GAL4 DNA-binding domain was removed. To introduce the point mutations of the acidic residues in the activation domain of LAP the following primers were used: mutation codon 56 and/or 58, 5′-GGC GCG CT G/C GTG CT G/C GCC GAT GGC-3′; mutation codon 94 and/or 95, 5′-GCC GTA GT T/C GT T/C GGC GAA GAG-3′. Nuclear extracts were prepared from HepG2 hepatoma cells using the Dignam C method as described previously(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). For the gel retardation assays 2.5 μg of nuclear extracts were used. The binding reaction was performed for 20 min on ice. For binding assays where the LAP DNA-binding region was maintained an oligonucleotide spanning the D-site of the albumin promoter was used as a 32P-labeled probe. For gel shift experiments using the chimeric LAP-GAL4 proteins an oligonucleotide spanning the GAL4-binding site was used as a 32P-labeled probe. Free DNA and DNA∙protein complexes were resolved on a 6% polyacrylamide gel as described previously (24Trautwein C. van der Geer P. Karin M. Hunter T. Chojkier M. J. Clin. Invest. 1994; 93: 2554-2561Crossref PubMed Scopus (129) Google Scholar) with the difference that the gel was run for 4 h at 300 V to get a better resolution of the different deletion mutants. Nuclear extracts were separated on a 10% SDS-polyacrylamide gel (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and blotted onto a nitrocellulose membrane (Schleicher and Schuell) in 1% SDS, 20% methanol, 400 mM glycine, 50 mM Tris-HCl, pH 8.3, at 4°C for 2 h at 200 mA. LAP Δ21 and the LAP deletions were analyzed by using polyclonal LAP antibodies(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). The antigen-antibody complexes were visualized using the ECL detection system as recommended by the manufacturer (Amersham). Three different ATGs exist in the ORF of LAP. Only the second and the third ATG are efficiently used to translate the two different forms of LAP in the liver; LAP Δ21 and LIP (LAP Δ1-152). In contrast, the full-length LAP is not efficiently translated and has only a minor effect on transactivation of LAP-responsive reporter constructs compared with the LAP Δ21 protein(9Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). Therefore LAP Δ21 was used for deletion analysis to identify the transactivation domain of LAP. LIP has been shown to be a repressor of LAP, as it blocks transactivation of a LAP dependent reporter construct(9Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). From these data we concluded that the transactivation domain should be in the 131 N-terminal amino acids (21-152) of LAP. These 132 N-terminal amino acids were further characterized by the introduction of restriction sites into the ORF of LAP to create a series of 5′ deletion mutants between LAP amino acid 21 and 153 (Fig. 1A). The mutant sequences were cloned into a CMV-driven mammalian expression vector. The effect on transactivation of the different N-terminal deletion mutants was investigated in cotransfection experiments with a LAP-responsive reporter plasmid (CRP-CAT). Interestingly, a deletion of the first 20 amino acid in the LAP construct LAP Δ1-41 had a tremendous effect on the activation of the reporter plasmid. Only around 10% of the CAT activity was detected compared with LAP Δ1-21 construct (Fig. 1A). A deletion of further 22 amino acids in LAP Δ1-63, and the other consecutive 5′ deletions, showed no increased activation of the reporter plasmid compared to the transfection of the reporter construct alone (Fig. 1A). The nuclear expression of all the N-terminal LAP deletion mutants was monitored by Western blot analysis of nuclear extracts (Fig. 1B). All the constructs were well expressed in the nucleus. As deletion mutants could have an effect on the tertiary structure of the artificial protein and resulting in a reduction of DNA binding, gel shift experiments were performed with the same nuclear extracts as shown in the Western blot experiments. All the deletion proteins were found to bind the cognate DNA (data not shown). To exclude that differences in the expression of the proteins may influence the results shown in Fig. 1A, and because we showed that LAP has a strong squelching effect on gene activation(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar), increasing amounts of the LAP expression constructs were cotransfected with a constant amount of the reporter plasmid. However only minor effects on the activation of the reporter plasmid could be observed, when increasing amounts of the LAP deletions LAP Δ1-63, LAP Δ1-75, LAP Δ1-104, LAP Δ1-121, LAP Δ1-139, and LAP Δ1-152 were used as these proteins did not activate the reporter plasmid over background level (data not shown). Like already shown for the LAP Δ1-21 protein(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar), the first LAP Δ1-41 mutation showed a similar kinetic on activation of the reporter construct as found for the wild type protein (data not shown). After we determined the N-terminal boundary of the activation domain of LAP, the following experiments were directed to find the C-terminal boundary of the activation domain. We created a series of internal LAP deletion mutants. Increasingly larger pieces were excised from the internal part of the protein beginning at the third ATG in the ORF. The 145 C-terminal amino acids, which code for the LIP protein and which contain the basic domain and the leucine zipper important for mediating DNA binding, were left unchanged. In the LAP Δ140-152, LAP Δ105-152, and LAP Δ64-152, 13, 47, and 90 amino acids, respectively, were deleted (Fig. 2A). The modified LAP proteins were cotransfected with the LAP-responsive reporter plasmid (CRP-CAT). Only a minor decrease in CAT activity was observed when the smallest deletion LAP Δ140-152 was cotransfected with the specific reporter plasmid. The deletion of further 34 amino acids in the mutant LAP Δ105-152 resulted in a reduction of nearly 40% activity of the wild type. Further excision of 41 amino acids in the LAP Δ64-152 construct resulted in a decline to background activity, detected when the reporter construct was used alone (Fig. 2A). In order to determine the significance of amino acids 105-152, we deleted amino acids 64-104 in the construct LAP Δ64-104 and cotransfected the CMV expression vector with the LAP-responsive reporter plasmid. As shown in Fig. 2A no increase over background activity was observed when the LAP Δ64-104 construct was expressed. Therefore we concluded that the LAP amino acids 64-104 contain sequences which are important for mediating the transactivation of LAP. From our results obtained with the N-terminal and internal LAP deletion mutants we were confident that an important part of the LAP transactivation domain was located between the LAP amino acids 21-104. We speculate that the difference in activity between LAP Δ140-152 and LAP Δ105-152 could be due to changes in the tertiary structure of the mutant proteins, alternatively the amino acids between 105 and 140 are important to stabilize the region between amino acids 21 and 104. To exclude that an additional transactivation domain which may be repressed by other sequences is further 3′ of the third ATG in the LAP ORF, an additional deletion was constructed which leaves only 107 amino acids of the LAP molecule containing the important region for DNA binding (LAP Δ1-189). Like for the LAP Δ1-152 protein, no change in CAT activity was observed. As with the 5′ deletions, the internal deletions were also checked for protein expression by Western blot analysis (Fig. 2B) and DNA binding by gel shift experiments, respectively (data not shown). For all the proteins, the expression in the Western blot analysis was in agreement with the effect on DNA binding in the gel shift experiments, except for the LAP Δ1-189 construct. A dramatic difference was observed between the nuclear expression of the protein and its strong shift in the gel shift experiment when compared with the other deletion proteins in the same experiment. At the present time we have two possible explanations for this discrepancy in the results. Either the polyclonal antibody directed toward the LAP protein shows a much lower affinity for these 107 C-terminal amino acids of the protein, or as shown for the LIP protein, which has a stronger affinity toward the cognate DNA(9Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar), further 3′ deletions have an even more dramatic effect on the DNA binding affinity of the protein. For many other transcription factors it has been shown that the different functional domains could be dissected and that these domains work independently from each other. The GAL4 DNA-binding domain of GAL4 is an ideal candidate; it has been mapped within the first 147 amino acids of the wild type protein. The truncated protein has been shown to translocate to the nucleus and is incapable of activating transcription in vivo. From earlier results we know that a chimeric LAP/GAL4 construct has the capacity to activate a GAL4-responsive reporter construct(21Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar). To further support our results in the mapping of the transactivation domain, we performed domain swapping experiments and tested whether the same effects on transactivation shown for the whole protein could be observed for the deletion series using only the N-terminal domain of LAP. In the following series of experiments the LAP DNA binding and leucine zipper domain corresponding to the coding sequences of the LAP protein were swapped versus the GAL4 DNA-binding domain (GAL4(1-147)). The resulting 5′ deletions of the chimeric proteins are demonstrated in Fig. 3A. As shown for the LAP protein, the chimeric LAP/GAL4 5′ deletions when cotransfected with a GAL4 reporter construct also showed a dramatic drop in LUC activity when the first 20 amino acids were deleted and no increase over background activity was observed when a further 22 amino acids were deleted (Fig. 3A). As demonstrated by gel shift experiments, all the proteins had a comparable affinity in DNA binding (Fig. 3B). Also the internal LAP deletion mutants were used to construct and express the chimeric LAP/GAL4 proteins (Fig. 4A). As shown for the internal LAP deletion mutants also, the chimeric LAP/GAL4 proteins point to a region between amino acids 22 and 104 which is important for transactivation (Fig. 4A). The gel shift experiment showed only minor differences in DNA binding between the different chimeric proteins (Fig. 4B). Interestingly a severalfold increase in activation of the GAL4 LUC reporter construct was observed when the LAP Δ105-152/GAL4 expression vector was cotransfected instead of the LAP Δ1-21/GAL4 expression vector (Fig. 4A). As both proteins have a comparable affinity in gel shift experiments toward the cognate DNA (Fig. 4B), this difference is very likely to be directly mediated by the transactivation domain of the LAP Δ105-152/GAL4 chimeric protein. These findings with the chimeric LAP/GAL4 proteins support our findings that the N-terminal 84 amino acids are responsible in mediating the effect on the expression of LAP-dependent genes. Distinct regions in the transactivation domains of transcription factors are known to mediate the activation of the dependent genes(27Ferreri K. Gill G. Montminy M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 28Gill G. Pascal E. Tseng Z.H. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (473) Google Scholar, 29Roberts S.G.E. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1993; 363: 741-744Crossref PubMed Scopus (180) Google Scholar). A characteristic feature of the transactivation domain of LAP is its acidic nature. It was shown for other transcription factors that acidic regions are important to interact with parts of the general RNA-polymerase machinery and to regulate gene transcription(30Lin Y-S. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-571Crossref PubMed Scopus (261) Google Scholar, 31Lin Y.-S. Green M.R. Cell. 1991; 64: 971-981Abstract Full Text PDF PubMed Scopus (366) Google Scholar). Therefore we analyzed the transactivation domain of LAP between amino acids 21 and 105 for regions which could be important in mediating these protein-protein interactions. Computer-assisted prediction by Kyte and Doolittle (32Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) and by Deléage (33Deléage G. Tinland B. Roux B. Anal. Biochem. 1987; 163: 292-297Crossref PubMed Scopus (17) Google Scholar) for protein structure was performed to determine the possible regions, which could be important for the LAP transactivation domain. Two regions were found in the transactivation domain of LAP which could be interesting in mediating the activation of LAP-dependent genes (Fig. 5). The region between amino acids 38 and 63 was predicted by the algorithms to be helical, whereas the region between amino acids 85 and 95 contains several hydrophobic amino acids. From these computer-assisted predictions we wondered whether acidic amino acids in these two regions are important in mediating the activation of LAP-dependent genes. Therefore acidic amino acids in both regions were selected." @default.
• W2050793927 created "2016-06-24" @default.
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• W2050793927 date "1995-06-01" @default.
• W2050793927 modified "2023-09-28" @default.
• W2050793927 title "Transactivation of LAP/NF-IL6 Is Mediated by an Acidic Domain in the N-terminal Part of the Protein" @default.
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