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• W1968651256 abstract "Lung Krüppel-like factor (LKLF/Krüppel-like factor 2), a member of the Krüppel-like factor family of transcription factors, is expressed predominately in the lungs, with low levels of expression in other organs such as heart, spleen, skeletal muscle, and testis. LKLF is essential during pulmonary development and single-positive T-cell development and is indispensable during mouse embryogenesis. In this study, we performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor. Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain. The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as viral protein 16, VP16. This occurs either when the inhibitory domain is fused directly to VP16 or when the inhibitory domain is independently bound to DNA by GAL4 DNA-binding domain independent of the VP16 activator. Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting that the inhibitory domain binds a cofactor that prevents LKLF from transactivating. A yeast-two hybrid screen identified WWP1, an E3 ubiquitin ligase that binds specifically to the LKLF inhibitory domain but not to other transcription factors. In mammalian cells, WWP1 functions as a cofactor by binding LKLF and suppressing transactivation. These data demonstrate that LKLF contains multiple domains that either potentiate or inhibit the ability of this factor to function as an activator of transcription; moreover, regulation of LKLF transactivation is attenuated by an E3 ubiquitin ligase, WWP1. Lung Krüppel-like factor (LKLF/Krüppel-like factor 2), a member of the Krüppel-like factor family of transcription factors, is expressed predominately in the lungs, with low levels of expression in other organs such as heart, spleen, skeletal muscle, and testis. LKLF is essential during pulmonary development and single-positive T-cell development and is indispensable during mouse embryogenesis. In this study, we performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor. Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain. The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as viral protein 16, VP16. This occurs either when the inhibitory domain is fused directly to VP16 or when the inhibitory domain is independently bound to DNA by GAL4 DNA-binding domain independent of the VP16 activator. Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting that the inhibitory domain binds a cofactor that prevents LKLF from transactivating. A yeast-two hybrid screen identified WWP1, an E3 ubiquitin ligase that binds specifically to the LKLF inhibitory domain but not to other transcription factors. In mammalian cells, WWP1 functions as a cofactor by binding LKLF and suppressing transactivation. These data demonstrate that LKLF contains multiple domains that either potentiate or inhibit the ability of this factor to function as an activator of transcription; moreover, regulation of LKLF transactivation is attenuated by an E3 ubiquitin ligase, WWP1. lung Krüppel-like factor Krüppel-like factor DNA-binding domain erythroid Krüppel-like factor gut-enriched Krüppel-like factor polymerase chain reaction hemagglutinin chloramphenicol acetyltransferase amino acid(s) 3-amino 1,2,4,-triazole viral protein 16 activation domain inhibitory domain Lung Krüppel-like factor (LKLF/KLF2)1 is a member of a multigene family of transcription factors called the KLF family. LKLF is expressed predominantly in fetal and adult lungs, with limited expression in other organs (1Anderson K.P. Kern C.B. Crable S.C. Lingrel J.B. Mol. Cell. Biol. 1995; 15: 5957-5965Crossref PubMed Scopus (227) Google Scholar, 2Wani M.A. Conkright M.D. Jeffries S. Hughes M.J. Lingrel J.B. Genomics. 1999; 60: 78-86Crossref PubMed Scopus (32) Google Scholar). Analysis of chimeric mice derived from LKLF−/− embryonic stem cells demonstrated that LKLF is essential for late stages of normal lung development (3Wani M.A. Wert S.E. Lingrel J.B. J. Biol. Chem. 1999; 274: 21180-21185Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In addition to pulmonary development, the programming of the quiescent state of single-positive (CD4+ or CD8+) T cells and the late-stage survival of these cells in the peripheral lymphoid organs and blood are also dependent on LKLF (4Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar,5Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 278: 788-789Crossref PubMed Google Scholar). Early in T-cell development, single-positive thymocytes are produced and survive in the thymus without LKLF. However, the mature circulating cells undergo apoptosis in LKLF−/− mice, resulting in severely reduced numbers of peripheral T cells (4Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 277: 1986-1990Crossref PubMed Scopus (342) Google Scholar, 5Kuo C.T. Veselits M.L. Leiden J.M. Science. 1997; 278: 788-789Crossref PubMed Google Scholar). Targeted disruption of LKLF through gene targeting in embryonic stem cells results in embryonic lethality (6Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (303) Google Scholar, 7Wani M.A. Means Jr., R.T. Lingrel J.B. Transgenic Res. 1998; 7: 229-238Crossref PubMed Scopus (112) Google Scholar). LKLF homozygous null mice die in utero between 12.5 and 14.5 days of gestation due to severe hemorrhage. Defects in the blood vessel morphology, an abnormally thin tunica media, endothelial cell necrosis, and decreased deposition of the extracellular matrix surrounding the vessels collectively contribute to hemorrhage in LKLF−/− embryos (6Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (303) Google Scholar, 7Wani M.A. Means Jr., R.T. Lingrel J.B. Transgenic Res. 1998; 7: 229-238Crossref PubMed Scopus (112) Google Scholar). Analogous to its role in other organs, LKLF does not appear to be an important regulator of the initiation or early stages of blood vessel morphogenesis; rather, it is active in the late stages of development including the cell-mediated assembly and stabilization of the blood vessel wall (6Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (303) Google Scholar). Whereas the mechanism by which LKLF regulates blood vessel integrity is unknown, it has been suggested that LKLF may regulate a signaling pathway responsible for endothelial cell differentiation or survival required for the formation of the mature blood vessel wall (6Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (303) Google Scholar). LKLF was initially described as a lung-specific transcription factor (1Anderson K.P. Kern C.B. Crable S.C. Lingrel J.B. Mol. Cell. Biol. 1995; 15: 5957-5965Crossref PubMed Scopus (227) Google Scholar). However, it is now clear that LKLF plays a pivotal role in blood vessel formation and T-cell activation in addition to pulmonary development. A unifying theme in these apparently diverse roles is that LKLF is essential for late stages of development but is not required for the initial steps. Despite the large amount of information about the biological role of LKLF, no specific target gene(s) or mechanisms of LKLF regulation have yet been identified. Determination of the molecular mechanisms that underlie LKLF function as a regulator of transcriptional pathways is paramount in providing additional insight into its roles in pulmonary function, maintenance of single-positive T cells, and embryogenesis. One approach to deciphering the molecular mechanisms of transcriptional activation by this factor is to study its functional domains. Transcription factors are commonly composed of two distinct separable domains, an activation domain and a DNA-binding domain. Recent studies of two Krüppel-like factors, the erythroid (EKLF/KLF1) and gut-enriched (GKLF/EZF/KLF4) Krüppel-like factors, demonstrate that for transcriptional activation, only a small subdomain of the activation domain is required (8Chen X. Bieker J.J. EMBO J. 1996; 15: 5888-5896Crossref PubMed Scopus (59) Google Scholar, 9de Geiman T.-T.H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar, 10Yet S.F. McA'Nulty M.M. Folta S.C. Yen H.W. Yoshizumi M. Hsieh C.M. Layne M.D. Chin M.T. Wang H. Perrella M.A. Jain M.K. Lee M.E. J. Biol. Chem. 1998; 273: 1026-1031Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). This is in contrast to intestinal-enriched Krüppel-like factor, which requires the entire activation domain (11Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (140) Google Scholar). LKLF, EKLF, and GKLF comprise a subfamily in the KLF family. All these transcription factors contain an inhibitory subdomain located adjacent to the zinc fingers (8Chen X. Bieker J.J. EMBO J. 1996; 15: 5888-5896Crossref PubMed Scopus (59) Google Scholar, 9de Geiman T.-T.H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar, 10Yet S.F. McA'Nulty M.M. Folta S.C. Yen H.W. Yoshizumi M. Hsieh C.M. Layne M.D. Chin M.T. Wang H. Perrella M.A. Jain M.K. Lee M.E. J. Biol. Chem. 1998; 273: 1026-1031Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Deletion of the inhibitory subdomains in EKLF and GKLF allows these factors to function as more potent activators of transcription. Despite the similarity in size, function, and physical location of these inhibitory subdomains, no obvious conservation in the amino acid sequence has been detected. Thus, activation domains are beginning to emerge as complex multifunctional domains with discrete subdomains that are likely involved in the regulation of transcription factors. By studying the biochemical attributes and configurations of the activation domains present in transcriptional activators we can better understand the mechanisms governing gene regulation and, ultimately, the role of tissue-specific transcriptional activators. Our current studies demonstrate that LKLF contains a modular activation domain that can be separated into inhibitory and transactivating subdomains. The inhibitory domain of LKLF binds specifically to an E3 ubiquitin ligase that is able to attenuate transactivation by LKLF. The interactions between these two proteins likely represent one method by which this transcription factor is regulated. Full-length LKLF cDNA fragment was subcloned into expression vector pM (12Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar) that contained the coding region of the GAL4 DNA-binding domain (DBD) to create a fusion protein between GAL4 DBD and LKLF. Six GAL4-LKLF mutants (GAL4-LKLF 1–267, GAL4-LKLF 1–257, GAL4-LKLF 1–141, GAL4-LKLF 1–110, GAL4-LKLF 1–88, and GAL4-LKLF 1–57) were then created by serial deletions utilizing the restriction enzyme site (BssHI, NotI,RsrII, XbaIII, SacII, andBstXI, respectively) in the coding region of LKLF and a restriction site in the polylinker. After deletion of the 3′ end of LKLF cDNA, the staggered ends generated from the restriction digests were blunted with T4 polymerase before ligation. Stop codons in the vector prevented any read through. Three additional LKLF internal deletion mutants (GAL4-LKLF Δ110–257, GAL4-LKLF Δ88–267, and GAL4-LKLF Δ57–215) were constructed in pGEM3 (Promega, Madison, WI) and then subcloned into pM vector. The internal deletion mutants were created utilizing either internal restriction sites or PCR to amplify the zinc fingers, adding a silent mutation to create a unique restriction site. GAL4-LKLF Δ57–215 was created utilizing two internal NarI sites, and GAL4-LKLF Δ110–257 was created utilizing two XbaIII restriction sites. PCR was used to create GAL4-LKLF Δ88–267 by amplifying the zinc finger domain to introduce a second SacII restriction site by silent mutation at the 5′ end (5′-TCC-CGC-GGC-CAA-ACA-TAC-TTG-CAG-C-3′ and 5′-CTC-AAG-CTT-GCA-GTG-TGT-TTG-CAA-GGG-3′). This allowed a deletion from the SacII site at amino acid 88 to the beginning of the zinc finger. Six additional LKLF-GAL4 DNA-binding domain expression constructs were created to produce fusion proteins that contained the GAL4 DBD on the carboxyl terminus rather than the amino terminus of the LKLF activation domain (LKLF 1–57-GAL4, LKLF 1–88-GAL4, LKLF 1–110-GAL4, LKLF 1–141-GAL4, LKLF 1–257-GAL4, and LKLF 1–267-GAL4). Each of these constructs was created utilizing an internal restriction site (listed above) joined to PCR amplification of the GAL4 DBD (5′-NNC-TCT-CCA-TGG-GAT-TGG-ACA-TGA-AGC-TAC-TGT-CTT-CT-3′, 5′-NNA-TCC-CCG-CGG-ACA-TGA-AGC-TAC-TGT-CTT-CT-3′, 5′-NNC-ACG-GCC-GCA-TGA-AGC-TAC-TGT-CTT-CT-3′, 5′-NNC-ACG-GAC-CGA-TGA-AGC-TAC-TGT-CTT-CT-3′, 5′-NNA-AAC-GCG-GCC-GCA-TGA-AGC-TAC-TGT-CTT-CT-3′, 5′-NNA-AGC-GCG-CCA-TGA-AGC-TAC-TGT-CTT-CT-3′, and 5′-NNN-AAG-CTT- TTA-CGA-TAC-AGT-CAA-CTG-TC-3′). LKLF inhibitory domain fused to the GAL4 DBD (GAL4-LKLF111–267) and deletion mutations of the inhibitory domain (GAL4-LKLF111–200, GAL4-LKLF111–150, GAL4-LKLF150–200, GAL4-LKLF150–267, and GAL4-LKLF200–267) were created by PCR amplification (5′-NNN-GGA-TCC-TTC-CTC-CTT-GCG-CCT-3′, 5′-NNN-GGA- TCC-GCC-CCA-GGA-GCG-ACA-3′, 5′-NNN-GGA-TCC-GGC-GCC- CTC-GAG-CTT-3′, 5′-NNN-AAG-CTT-GCT-GGG-GCC-GGG-ACC-3′, 5′-NNN-AAG-CTT-GGC-GGC-GCG-CTT-GCG-3′, 5′-NNN-AAG-CTT-GGC-GCC-CTC-GAG-CTT-3′, and 5′-NNN-GGA-TCC-AGC-TTC-GGC-GGT-3′). Lex A-VP16-LKLF 111–267 was created by the addition of ApaI restriction sites to each cDNA coding for LKLF amino acids 111–267 by PCR amplification (5′-NNN-TGG-GCC-CCC-CTC-CTT-GCG-CCT-CCC-3′ and 5′-GGG-GGC-CCA-GGC-GGC-GCG-CTT-CCG-GGG-3′). This region was cloned into the ApaI of site Lex A-VP16 expression vector and was confirmed by sequence analysis. Full-length WWP1 and E6AP were kindly provided by Drs. Emery Bresnick and Allen Weissman, respectively. An expression plasmid for carboxyl-terminal hemagglutin (HA) LKLF was generated by PCR. Monkey kidney fibroblasts (COS) or mouse lung adenoma (LA4) cells were plated at a density of 1 × 105cells/35-mm tissue culture dish. The following day, cells were transfected by Fugene 6 (Roche Molecular Biochemicals) with 0.25 µg of reporter HS2β-CAT (1Anderson K.P. Kern C.B. Crable S.C. Lingrel J.B. Mol. Cell. Biol. 1995; 15: 5957-5965Crossref PubMed Scopus (227) Google Scholar), pG5-CAT (12Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar), L8-CAT (13Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar), G5L8-CAT (13Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar), or 5CACCC-LUC plasmid; 0.25 µg of pSV2LUC or CMV-βGal control plasmid; and 1.0 µg of test vector (GAL4-LKLF, LKLF-GAL4 deletion mutant, Lex A-VP16, GAL4-VP16, Lex A-VP16-LKLF111–267, and LKLF-HA of WWP1-FLAG as described in each figure). Vector DNA was added as needed to keep the total DNA constant. The cells were harvested 48 h after transfection. The luciferase and CAT activity was determined by disrupting the cells by three cycles of freeze-thaw lysis in 0.25m Tris (pH 7.5) to make crude protein extract. An aliquot of protein extract was used for analysis of luciferase activity (Promega). The remaining extract was heat-inactivated at 65 °C for 10 min. Extract amounts were normalized for transfection efficiencies, and CAT assays were performed at 37 °C for 1 h. The thin-layer chromatography plates were exposed to a PhosphorImager plate (Molecular Dynamics, Sunnyvale, CA) for quantitation. Normalized values for the CAT activity are based on the percentage conversion of [14C]chloramphenicol substrate to the acetylated forms and corrected for transfection efficiency with luciferase activity. LKLF-GAL4 fusion protein and deletion mutants were transfected into COS cells for 48 h as described above. Cells were lysed with Laemmli buffer (0.0625m Tris, pH 6.8, 2% SDS, 6 m urea, 0.150m dithiothreitol, and 0.005% bromophenol blue), and each sample was separated by electrophoresis on a 9% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to Hybond-P (Amersham Pharmacia Biotech) polyvinylidene difluoride transfer membrane. After blocking, 0.1 µg/ml rabbit polyclonal antibody raised against GAL4 DBD protein or Lex A DBD (Santa Cruz Biotechnology) was incubated with the membrane. The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit (Amersham Pharmacia Biotech), was used at a 1:10,000 dilution. Protein-antibody interaction was visualized by chemiluminescence detection using the ECL Western blotting analysis system (Amersham Pharmacia Biotech). The inhibitory domains of LKLF (aa 111–267) and EKLF (aa 197–292) were cloned by PCR in frame to the GAL4 DBD in pAS-1-CYH2. Likewise, full-length LKLF and EKLF were also subcloned into the pAS-1-CYH2 yeast vector. The GAL4 DBD-GATA 5 (aa 133–265) and GAL4 DBD-GATA 6 (aa 208–351) constructs were gifts from Jeff Molkentin. The library consisted of rat lung cDNA fused to the activation domain of GAL4 in the pGAD10 vector (CLONTECH). A yeast two-hybrid screen was conducted with the inhibitory domain (aa 111–267) of LKLF fused to Gal4 DBD following the manufacturer's protocols (CLONTECH) except where noted. TheSaccharomyces cerevisiae strain AH109 (14James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) (MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-MEL1) was used in all the two-hybrid assays. Approximately 1.5 × 106transformants were screened. The yeast were grown on synthetic dropout, a minimal medium, with appropriate amino acid omissions not only for plasmid selection but also for selection of protein-protein interactions. Tryptophan and leucine were selective markers for the co-transformed pAS-1-CYH2 and pGAD10 plasmids. Histidine and adenine select for protein-protein interactions between the LKLF inhibitory domain and the GAL4 activation domain fusion protein. The pGAD10 plasmid was recovered from the triple positive (His+, Ade+, and Mel1+) clones by growing in leucine-deficient liquid synthetic dropout media for 48 h. Yeast were collected from 1 ml of liquid culture by centrifugation at 10,000 × g for 1 min. The supernatant was decanted, and the cell pellet was resuspended in the residual media in the microcentrifuge tube by vortexing. Next, 50 units of lyticase was added to the yeast, and the cells were digested for 2 h at 37 °C. The remaining plasmid isolation was conducted following the standard Qiagen miniprep protocol for Escherichia coli. Isolation of the yeast plasmid DNA contains a mixture of pAS-1-CYH2 and pGAD10 vectors, and the use of HB101 E. coli, a Leu-2-deficient strain, allows the selection of the pAS-1-CYH2 vector from the pGAD10 vector. Isolated plasmids were transformed into HB101 E. coli and grown on M9 leucine-deficient selection medium with ampicillin. Isolated pGAD10 vector was then subjected to double-stranded nucleotide sequence analysis (Applied Biosystems). To eliminate the possibility that the interaction between WWP1 was fortuitous, an interaction between WWP1 and other transcription factors was examined. GAL4-WWP1 was co-transformed with pAS-1-CYH alone or with pAS-1-CYH containing cDNA for the inhibitory domain of EKLF, full-length EKLF, GATA 5, or GATA 6. In addition, WWP1 was also transformed by itself. LKLF tagged with the HA epitope and WWP1 tagged with the FLAG epitope were transiently transfected into COS cells as described above. After co-transfection with WWP1 and LKLF, the cells were lysed with a hypotonic buffer containing Nonidet P-40. The cell lysates were split and incubated with either a polyclonal antibody against the HA antigen (Santa Cruz Biotechnology) or preimmune serum. The protein-antibody complex was then precipitated by the addition of protein G-Sepharose (Zymed Laboratories Inc.). After washing the absorbed beads in co-immunoprecipitation buffer, the precipitants were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (Millipore). Finally, Western blot analysis using a monoclonal antibody (M2) against the FLAG antigen (Sigma) fused to WWP1 was used to evaluate the association of the two proteins during the precipitation. Previous studies have indicated that some members of the Krüppel-like family of transcription factors consist of multiple domains that function in transcriptional activation, inhibition of activation, protein-protein interaction, DNA binding, and transcriptional repression (8Chen X. Bieker J.J. EMBO J. 1996; 15: 5888-5896Crossref PubMed Scopus (59) Google Scholar, 9de Geiman T.-T.H. Johnson J.M. Yang V.W. Nucleic Acids Res. 2000; 28: 1106-1113Crossref PubMed Google Scholar, 10Yet S.F. McA'Nulty M.M. Folta S.C. Yen H.W. Yoshizumi M. Hsieh C.M. Layne M.D. Chin M.T. Wang H. Perrella M.A. Jain M.K. Lee M.E. J. Biol. Chem. 1998; 273: 1026-1031Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 15Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar). To identify the functional domains of LKLF, a series of plasmids containing various portions of LKLF cDNA joined to the DBD of yeast transcription factor GAL4 were constructed (Fig. 1, A andD). Because GAL4 fusion proteins are of yeast origin, they have the advantage of little or no background interference in mammalian cells. In addition, GAL4 DBD directs proteins to the nucleus, alleviating the concern that deletion mutants might disrupt the natural nuclear localization signal of a protein. Both serial deletions and internal deletions were created and assayed for the ability to transactivate pG5-CAT, a reporter construct containing five GAL4-binding sites in front of the E1b minimal promoter (Fig. 1,A and D). Full-length LKLF (aa1–354) fused to the GAL4 DBD transactivated the reporter gene only modestly. However, because additional portions of the carboxyl terminus of LKLF were deleted, transcriptional activation increased. Removal of amino acids 111–354 resulted in a 25-fold increase in transcriptional activation (Fig. 1 B). Further deletions of the activation domain resulted in a slight attenuation in transactivation. Equivalent results are observed when these experiments are conducted in a mouse lung adenoma cell line (LA4) expressing endogenous LKLF (data not shown). Western blot analysis of cell lysates was performed to demonstrate that the full-length protein and each mutant protein were made in the cell, ruling out the possibility that the inability of LKLF or LKLF deletion mutants to transactivate was the result of the protein not being synthesized. (Fig. 1 C). Amino acids 1–110 are able to function as a potent activator of transcription and transactivate significantly better than full-length LKLF, suggesting that an inhibitory domain is present outside this region of the protein. To determine whether the inhibitory domain is contained within the Cys2-His2 zinc fingers, internal deletion constructs were engineered that maintained this protein feature (Fig. 1 D). The activity of the activating subdomain (aa 1–110) to enhance reporter gene expression is unaffected by the presence of the zinc fingers. This demonstrates that the zinc fingers do not contain the inhibitory subdomain (Fig. 1 E) and further localizes this regulatory region to amino acids 111–267. A Western blot analysis once again demonstrated the presence of full-length LKLF and each mutant protein in the cells (Fig.1 F). Whereas the natural DNA-binding domain of LKLF is located on the carboxyl terminus of the activation domain, the GAL4 DNA binding domain was attached to the amino-terminal portion. To rule out any positional effects from this configuration, additional LKLF deletion constructs were engineered to contain the GAL4 DBD on the carboxyl terminus of the activation domain. These deletion constructs were assayed for the ability to activate transcription from a reporter gene. In agreement with the above-mentioned results, removal of amino acids 111–267 increased transcriptional activation, although the total fold activation is slightly lower. Collectively, these data demonstrate that LKLF contains a potent transactivation domain in the first 110 amino acids. In addition, LKLF contains a region between amino acids 111–267 that is responsible for suppressing transactivation and represents a regulatory region in the protein. Deletion of amino acids 111–267 from LKLF allows this factor to function as a stronger activator of transcription, suggesting that this region functions as a regulator of transcriptional activation. To reinforce the deletion analysis and provide a positive assay for this function, we show that this LKLF inhibitory domain can also directly suppress other transactivators. LKLF inhibitory domain was fused to the heterologous protein Lex A-VP16 (Lex A-VP16-LKLF 111–267) and compared with unmodified Lex A-VP16 for the ability to transactivate a reporter gene. The LKLF inhibitory domain almost completely suppressed transactivation by VP16 (Fig. 2 A). Western blot analysis confirmed that both Lex A-VP16 and Lex A-VP16-LKLF 111–267 proteins were being made in the cell (Fig. 2 B). These results indicate that the LKLF inhibitory domain is not only capable of inhibiting transactivation by a strong transcriptional activator such as VP16 but is a modular protein region that can function independent of other regions of LKLF. The ability of the inhibitory domain to suppress transactivation may occur through an intramolecular or intermolecular mechanism. To examine the possibility that the LKLF inhibitory domain functions through an intermolecular mechanism, several chimeric proteins were generated that consist of the GAL4 DNA-binding domain fused to various regions of LKLF. The ability of these GAL4 fusion proteins to modulate transcriptional activation by the heterologous protein Lex A-VP16 when both factors are simultaneously bound to the promoter region was examined. In all, three GAL4-LKLF heterologous proteins were generated with either the inhibitory domain (GAL4-LKLF 111–267), the activation domain (GAL4-LKLF 1–110), or both domains (GAL4-LKLF 1–267) fused to the GAL4 DBD. Each of the three GAL4-LKLF chimeric proteins or the control GAL4 DBD alone was examined for the ability to modulate transactivation of Lex A-VP16. For these studies we utilized an artificial CAT reporter system in which five GAL4-binding sites are present adjacent to eight Lex A-binding sites (L8G5-CAT) (Fig.3 A) (13Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (580) Google Scholar). In this system, Lex A-VP16 and a GAL4 DBD fusion protein are able to bind and occupy their respective response elements simultaneously. As expected, GAL4 DBD bound to the promoter in the absence of Lex A-VP16 but was unable to transactivate the CAT reporter gene, demonstrating that GAL4 DBD by itself does not have any transactivating capabilities (Fig.3 B). The addition of Lex A-VP16 even in the presence of GAL4 DBD resulted in strong transactivation (Fig. 3 B). When GAL4 DBD was replaced with GAL4 DBD fused to LKLF inhibitory domain, transactivation by Lex A-VP16 was attenuated 6-fold (Fig.3 B). This demonstrates that the LKLF inhibitory domain is able to regulate transactivation by an intermolecular mechanism. Furthermore, the inhibition of the Lex A-VP16 transactivation by the inhibitory domains cannot be attributed to steric hindrance or to a physical block of transactivation. This is demonstrated with the analysis of Lex A-VP16 activation in the presence of GAL4 DBD fused to the LKLF activation domain (GAL4-LKLF 1–110). In this situation, transactivation was potentiated rather than suppressed (Fig.3 B). A similar result is observed when both domains are fused to GAL4 DBD, although, as one might expect, the potentiation was not as high as that of activator alone (Fig. 3 B). Each of the GAL4 DBD fusion proteins and GAL4 DBD fused to VP16 was also tested for the ability to transactivate in the absence of Lex A-VP16. Essentially, this recapitulates the data from the earlier experiment but also demonstrates that the inhibitory domain had no transactivation potential associated with it (Fig. 3 B). In addition, the LKLF activation domain and VP16 are able to transactivate the reporter gene with comparable efficiency (Fig. 3 B). In addition, the three GAL4-LKLF fusion constructs were tested for the ability to inhibit Lex A-VP16 when the GAL4 binding sites are absent from the promoter (L8-CAT) (Fig. 3 C). In this situation, Lex A-VP 16 was able to occupy the promoter; however, because the GAL4-binding sites are absent, the GAL4 DBD fusion protein was unable to bind the promoter. Neither GAL4" @default.
• W1968651256 created "2016-06-24" @default.
• W1968651256 creator A5001114808 @default.
• W1968651256 creator A5002369147 @default.
• W1968651256 creator A5039622330 @default.
• W1968651256 date "2001-08-01" @default.
• W1968651256 modified "2023-10-16" @default.
• W1968651256 title "Lung Krüppel-like Factor Contains an Autoinhibitory Domain That Regulates Its Transcriptional Activation by Binding WWP1, an E3 Ubiquitin Ligase" @default.
• W1968651256 cites W1903792504 @default.
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