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• W2009456747 abstract "Inositol-phosphorylceramide synthase 1 (Ipc1) is a fungal-specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG). In previous studies, we demonstrated that Ipc1 regulates the expression of the antiphagocytic protein 1 (App1), a novel fungal factor involved in pathogenicity of Cryptococcus neoformans. Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in the C. neofor-mans GAL7:IPC1 strain, in which the Ipc1 expression can be modulated, and found that the luciferase activity was indeed regulated when Ipc1 was modulated. Next, using the luciferase reporter assay in both C. neoformans wild-type and GAL7:IPC1 strains, we investigated the role of DAG and sphingolipids in the activation of the APP1 promoter and found that treatment with 1,2-dioctanoylglycerol does increase APP1 transcription, whereas treatment with phytosphingosine or ceramides does not. Two putative consensus sequences were found in the APP1 promoter for ATF and AP-2 transcription factors. Mutagenesis analysis of these sequences revealed that they play a key role in the regulation of APP1 transcription: ATF is an activator, whereas AP-2 in a negative regulator. Finally, we identified a putative Atf2 transcription factor, which is required for APP1 transcription and under the control of Ipc1-DAG pathway. These studies provide novel regulatory mechanisms of the sphingolipid pathway involved in the regulation of gene transcription of C. neoformans. Inositol-phosphorylceramide synthase 1 (Ipc1) is a fungal-specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG). In previous studies, we demonstrated that Ipc1 regulates the expression of the antiphagocytic protein 1 (App1), a novel fungal factor involved in pathogenicity of Cryptococcus neoformans. Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in the C. neofor-mans GAL7:IPC1 strain, in which the Ipc1 expression can be modulated, and found that the luciferase activity was indeed regulated when Ipc1 was modulated. Next, using the luciferase reporter assay in both C. neoformans wild-type and GAL7:IPC1 strains, we investigated the role of DAG and sphingolipids in the activation of the APP1 promoter and found that treatment with 1,2-dioctanoylglycerol does increase APP1 transcription, whereas treatment with phytosphingosine or ceramides does not. Two putative consensus sequences were found in the APP1 promoter for ATF and AP-2 transcription factors. Mutagenesis analysis of these sequences revealed that they play a key role in the regulation of APP1 transcription: ATF is an activator, whereas AP-2 in a negative regulator. Finally, we identified a putative Atf2 transcription factor, which is required for APP1 transcription and under the control of Ipc1-DAG pathway. These studies provide novel regulatory mechanisms of the sphingolipid pathway involved in the regulation of gene transcription of C. neoformans. Inositol-phosphorylceramide synthase 1 (Ipc1) 3The abbreviations used are:Ipc1inositol-phosphorylceramide synthase 1DAGdiacylglycerolATFactivating transcription factorLUCluciferaseNAT1nourseothricin acetyltransferase 1HYGhygromycin BACTactinUTRuntranslated regionAPP1antiphagocytic protein 1SDWsterile-distilled waterPKAprotein kinase APKCprotein kinase C is a fungal-specific enzyme of the sphingolipid pathway (Fig. 1) that regulates the level of phytoceramide and diacylglycerol (DAG), two well established bioactive molecules in mammalian cells, which regulate key cellular functions such as cell growth and viability (1Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (130) Google Scholar, 2Chung N. Obeid L.M. Methods Enzymol. 2000; 311: 319-331Crossref PubMed Scopus (11) Google Scholar, 3Luberto C. Hannun Y.A. Lipids. 1999; 34: S5-S11Crossref PubMed Google Scholar, 4Bell R. Burns D. Okazaki T. Hannun Y. Adv. Exp. Med. Biol. 1992; 318: 275-284Crossref PubMed Scopus (10) Google Scholar, 5Pinto W.J. Srinivasan B. Shepherd S. Schmidt A. Dickson R.C. Lester R.L. J. Bacteriol. 1992; 174: 2565-2574Crossref PubMed Google Scholar, 6Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (442) Google Scholar). On the other hand, the role of these lipids in signaling in yeast cells is poorly understood. inositol-phosphorylceramide synthase 1 diacylglycerol activating transcription factor luciferase nourseothricin acetyltransferase 1 hygromycin B actin untranslated region antiphagocytic protein 1 sterile-distilled water protein kinase A protein kinase C Although the presence of sphingolipid enzymes has been demonstrated in Saccharomyces cerevisiae (7Heidler S.A. Radding J.A. Antimicrob. Agents Chemother. 1995; 39: 2765-2769Crossref PubMed Scopus (91) Google Scholar), and in pathogenic fungi, such as Aspergillus fumigatus (8Kuroda M. Hashida-Okado T. Yasumoto R. Gomi K. Kato I. Takesako K. Mol. Gen. Genet. 1999; 261: 290-296Crossref PubMed Scopus (46) Google Scholar), Candida albicans, and Cryptococcus neoformans (9Heidler S.A. Radding J.A. Biochim. Biophys. Acta. 2000; 1500: 147-152Crossref PubMed Scopus (54) Google Scholar), studies of sphingolipid-mediated signaling transduction in pathogenic fungi are in their infancy. Studies in S. cerevisiae showed that Ipc1 modulates the level of phytoceramide and DAG (10Cerbon J. Falcon A. Hernandez-Luna C. Segura-Cobos D. Biochem. J. 2005; 388: 169-176Crossref PubMed Scopus (26) Google Scholar) but whether these lipids regulate signaling in this microorganism has yet to be elucidated. Whereas in mammalian cells DAG is a well known activator of protein kinase C (PKC), DAG does not activate the fungal homolog Pkc1 in S. cerevisiae (11Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar, 12Antonsson B. Montessuit S. Friedli L. Payton M.A. Paravicini G. J. Biol. Chem. 1994; 269: 16821-16828Abstract Full Text PDF PubMed Google Scholar) or in C. albicans (13Paravicini G. Mendoza A. Antonsson B. Cooper M. Losberger C. Payton M.A. Yeast. 1996; 12: 741-756Crossref PubMed Scopus (70) Google Scholar). Thus, if DAG regulates signaling in S. cerevisiae or C. albicans this regulation would be exerted through proteins other than Pkc1. On the other hand, C. neoformans Pkc1 contains a putative DAG-binding domain, or C1 domain, which is highly homologous to the C1 domain of DAG-dependent mammalian PKCs (14Heung L.J. Luberto C. Plowden A. Hannun Y.A. Del Poeta M. J. Biol. Chem. 2004; 279: 21144-21153Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In recent studies, we showed that Ipc1 activates Pkc1 in C. neoformans through a DAG-dependent mechanism (14Heung L.J. Luberto C. Plowden A. Hannun Y.A. Del Poeta M. J. Biol. Chem. 2004; 279: 21144-21153Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This activation is mediated by the C1 domain of Pkc1 and regulates the localization and function of laccase (15Heung L.J. Kaiser A.E. Luberto C. Del Poeta M. J. Biol. Chem. 2005; 280: 28547-28555Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), an enzyme that catalyzes melanin, which is required for the pathogenicity of C. neoformans. Additional studies revealed that Ipc1 also plays a role in the regulation of phagocytosis of C. neoformans, through the modulation of a novel fungal factor called antiphagocytic protein 1 (App1), which inhibits the attachment and ingestion of fungal cells by macrophages (16Luberto C. Martinez-Marino B. Taraskiewicz D. Bolanos B. Chitano P. Toffaletti D.L. Cox G.M. Perfect J.R. Hannun Y.A. Balish E. Del Poeta M. J. Clin. Investig. 2003; 112: 1080-1094Crossref PubMed Scopus (115) Google Scholar). Ipc1 controls App1 at the mRNA level, suggesting a transcriptional or post-transcriptional activation. Therefore, we sought to examine the possibility that Ipc1 and the lipids that it modulates, such as phytoceramide or/and DAG, may regulate the transcriptional activation of fungal factor(s), such as App1. To study the mechanism of the transcriptional regulation of the APP1 gene by Ipc1, the APP1 promoter was fused to the firefly luciferase gene in both C. neoformans wild-type and the GAL7:IPC1 strain, in which Ipc1 can be regulated by growing yeast cells in glucose or galactose. We find that the luciferase activity is modulated by the expression of Ipc1. Treatment with DAG activates luciferase in a dose- and time-dependent manner only when the luciferase gene is under the control of the APP1 promoter, whereas treatment with ceramide/phytoceramide or sphingosine/phytosphingosine does not affect APP1 transcription. We identified two consensus sequences in the APP1 promoter for AP-2 and ATF cis-acting elements. Deletion of the ATF consensus sequence in the APP1 promoter abolishes luciferase activity, whereas mutation of ATF abrogates the DAG-dependent activation. Deletion or mutation of AP-2 causes a significant increase of APP1 transcription, suggesting that this consensus sequence acts as a negative regulator. Finally, we identified and deleted the putative C. neoformans ATF2 gene by homologous recombination and found that loss of Atf2 abrogates luciferase activation driven by the APP1 promoter and regulated by Ipc1 or DAG. Thus, these studies suggest that APP1 transcription is under the control of the Ipc1-DAG pathway through the Atf2 transcription factor and two consensus sequences (AP-2 and ATF) present in the APP1 promoter. Strains, Growth Media, and Reagents—C. neoformans var. grubii serotype A strain H99 and derivative mutants used in this study are illustrated in TABLE ONE. The strains were routinely grown in yeast extract/peptone/dextrose (YPD) medium. Yeast extract peptone (YP) supplemented with 20 g/liter glucose or 20 g/liter galactose was used to down- or up-regulate the expression of IPC1 gene, respectively. Nourseothricin (Werner BioAgents, Germany) at a concentration of 100 μg/ml was added to YPD plates for selection of the IPC1/APP1:LUC, GAL7:IPC1/APP1:LUC, IPC1/ACT:LUC, GAL7:IPC1/5′UTR:LUC, and IPC1/366:LUC strains, as indicated. Hygromycin B (Calbiochem, San Diego, CA) at a concentration of 200 units/ml was added to YPD plates for selection of IPC1/APP1:LUC/Δatf2 and GAL7:IPC1/APP1:LUC/Δatf2 strains. C. neoformans strains carrying episomal plasmids (numbers 8–13, TABLE ONE), were routinely grown onto YPD medium containing 200 units/ml of hygromycin B.TABLE ONEList of C. neoformans strains used in this studyNo.StrainsRef.1IPC1 (wild-type)20Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. Hannun Y.A. Del Poeta M. Genes Dev. 2001; 15: 201-212Crossref PubMed Scopus (123) Google Scholar2GAL7:IPC120Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. Hannun Y.A. Del Poeta M. Genes Dev. 2001; 15: 201-212Crossref PubMed Scopus (123) Google Scholar3IPC1/APP1:LUCThis study4GAL7:IPC1/APP1:LUCThis study5IPC1/ACT:LUCThis study6GAL7:IPC1/5′UTR:LUCThis study7IPC1/366:LUCThis study8GAL7:IPC1/5′UTR:LUC + pTel/366:LUC (Tel/366:LUC)This study9GAL7:IPC1/5′UTR:LUC + pTel/Δap2:LUC (Tel/Δap2:LUC)This study10GAL7:IPC1/5′UTR:LUC + pTel/Δatf:LUC (Tel/Δatf:LUC)This study11GAL7:IPC1/5′UTR:LUC + pTel/Δatf+Δap2:LUC (Tel/Δatf+Δap2:LUC)This study12GAL7:IPC1/5′UTR:LUC + pTel/map2:LUC (Tel/map2:LUC)This study13GAL7:IPC1/5′UTR:LUC + pTel/matf:LUC (Tel/matf:LUC)This study14IPC1/APP1:LUC/Δatf2This study15GAL7:IPC1/APP1:LUC/Δatf2This study Open table in a new tab Nuclear Run-on Assay—The nuclear run-on assay was performed according to Hirayoshi and Lis (17Hirayoshi K. Lis J.T. Methods Enzymol. 1999; 304: 351-362Crossref PubMed Scopus (52) Google Scholar). Briefly, C. neoformans IPC1 (WT) and GAL7IIPC1 strains were grown on YP-glucose medium in a shaker incubator for 24 h at 30 °C. Cells were washed 3 times in sterile-distilled water (SDW) and then incubated in YNB broth containing 2% glucose or 2% galactose for 24 h at 30 °C. Cells were collected at 2800 × g, washed 3 times in 0.5 m NaCl, 50 mm EDTA, suspended in 9.5 ml of SDW containing 0.5 ml of β-mercaptoethanol, and incubated at 37 °C for 1 h. The cell pellet was then collected at 2800 × g, suspended in 4 ml of spheroplastic solution (1 m sorbitol, 0.1 m Na citrate, pH 5.8, 0.01 m EDTA), and placed on ice for 10 min. Then, 1 ml of spheroplastic solution containing 10 mg of lysing enzyme (Sigma number L-1412) was added, and cells were incubated at 37 °C for 1 h. Next, 2.5 × 107 cells were harvested at 3000 × g for 6 min at room temperature and washed with TMN buffer (10 mm Tris-HCl, pH 7.4, 100 nm NaCl, 5 mm MgCl2). Cells were suspended and kept on ice for 15 min in Sarkosyl solution (0.95 ml of SDW, 0.05 ml of 10% (w/w) N-lauroylsarcosine (Sigma L-9150)). After centrifugation at 2500 × g for 1 min at 4 °C, the cells were suspended in 100 μl of reaction buffer (50 mm Tris-HCl, pH 7.9, 100 mm KCl, 5 mm MgCl2, 1 mm MnCl2, 2 mm dithiothreitol, 0.5 mm ATP, GTP, CTP, 100 μCi of [α-32P]UTP, and 1 units of RNasin). The reaction was incubated at 25 °C for 8 min and terminated by the addition of 1 μl of 1 mg/ml α-amanitin and 20 μl of 50 mg/ml DNase I. The mixture was incubated for 10 min at 30 °C and then an equal volume of stop buffer (∼120 μl) containing 20 mm Tris-HCl, pH 7.4, 10 mm EDTA, 2% SDS, and 200 μg/μl proteinase K was added and the mixture incubated at 42 °C for 30 min. The labeled mRNA was extracted using phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma number P-3803) and precipitated with ethanol. The labeled mRNA was suspended in 100 μl of diethyl pyrocarbonate water and added to the hybridization chamber containing nytran membranes in which actin, IPC1, and APP1 cDNA were previously immobilized using a slot blot apparatus. Before adding the labeled mRNA, the nytran membranes were pre-hybridized for 3 h at 58 °C in hybridization solution (10% dextran sulfate, 1% SDS, 50% formamide, 6× standard saline citrate (SSC)). Once the labeled mRNA was added, the nytran membranes were hybridized at 58 °C for 16 h. After hybridization, the membranes were washed 3 times at 58 °C for 10 min each in 2× SSC and 1% SDS, 3 times at 68 °C for 10 min each in 2× SSC and 1% SDS, and 3 times at 68 °C for 10 min each in 0.1× SSC and 1% SDS. Membranes were then air-dried and exposed to phospho-screen at room temperature for 6 days. Each band was quantified by a phosphoimager STORM 840. Generation of C. neoformans Strains Carrying the Luciferase Gene under the Control of Wild-type or Mutated Forms of the APP1 Promoter—Plasmid pSK/APP1/LUC/NAT1/3′UTR was generated as follows: the NAT1 gene under the control of the C. neoformans actin (ACT) promoter was amplified from the pNAT1 vector (kindly provided by Dr. John Perfect, Duke University Medical Center, Durham, NC) using primers XB-NAT-F (5′-CTAATCTAGAGCGAGGATGTGAGCTGGAGAGCGG-3′) and XB-NAT-R (5′-CGCGTCTAGAGAAGAGATGTAGAAACTAGCTTCC-3′), which contain XbaI sites (bold and underlined). The resulting fragment was cloned into pCR2.1-TOPO vector (Invitrogen), generating pCR-NAT1. The LUC gene was amplified using primers Luc3′,5′-GATCTTTCCGCCCTTCTT-3′, and Luc5′,5′-GCATGCCAGAGATCCTAT-3′, and plasmid pGL3 basic vector (Promega) as a template. The resulting fragment was digested with HindIII and BamHI and cloned into the HindIII- and BamHI-digested pCR-NAT1 vector. The resulting plasmid (pCR-NAT1/LUC) was digested with HindIII and EcoRV, yielding a fragment containing LUC:NAT1 that was subcloned into HindIII- and EcoRV-digested pSK vector (Invitrogen). The resulting plasmid, pSK/LUC/NAT1, was digested with EcoRV and SacI to insert the EcoRI-blunted and SacI-restricted 3′-UTR region of the APP1 gene from the pΔapp1 plasmid (3Luberto C. Hannun Y.A. Lipids. 1999; 34: S5-S11Crossref PubMed Google Scholar), generating pSK/LUC/NAT1/3′UTR. Next, a 800-bp fragment corresponding to the APP1 promoter was obtained by digesting that pΔapp1 plasmid with XhoI and EcoRI and subcloned into XhoI and SalI-blunted restricted pSKLUC/NAT1/3′UTR plasmid creating plasmid pSK/APP1/LUC/NAT1/3′/UTR, which was biolistically transformed into C. neoformans WT and GAL7:IPC1 to generate IPC1/APP1:LUC and GAL7:IPC1/APP1:LUC strains. This 800-bp APP1 sequence was chosen because it was highly predicted to be the promoter of APP1: 1) it represents the 5′-UTR sequence immediately upstream of the APP1 mRNA transcribed region (GenBank™ accession number AY965856); 2) it contains the TATA box (–72 bp from ATG) and two putative consensus sequences for transcription factors AP-2 (–236 bp from ATG) and ATF (–139 bp from ATG); and 3) it was highly predicted to be a promoter region when blasted into the BioInformatics & Molecular Analysis Section (BIMAS) at the National Institutes of Health (bimas.dcrt.nih.gov/molbio/proscan/index.html). Plasmid pSK/5′UTR/LUC/NAT1/3′UTR was generated as follows: a 876-bp 5′-UTR fragment corresponding to the upstream untranslated region of the APP1 promoter was amplified from genomic DNA using primers APP15-XhoI, 5′-CATCTCGAGTGAGTACTGGATCTG-3′, and APP13-HindIII, 5′-GAAAAGCTTTCATTGCTTAACGGTATTG-3′, which contain XhoI and HindIII sites, respectively (bold and underlined). The resulting fragment was subcloned into pCR2.1 TOPO vector, generating the pCR/5′UTR plasmid. This plasmid was digested with XhoI and HindIII and the resulting 876-bp fragment was subcloned into the XhoI- and HindIII-restricted pSK/LUC/NAT1/3′UTR plasmid. The resulting vector pSK/5′UTR/LUC/NAT1/3′UTR was transformed into the GAL7:IPC1 strain to generate the negative control GAL7:IPC1/5′UTR:LUC strain. Plasmid pSK/5′UTR/ACT/LUC/NAT1/3′UTR was generated as follows: 5′UTR of the APP1 locus was amplified from genomic DNA using primers APP15-XhoI, 5′-CATCTCGAGTGAGTACTGGATCTG-3′, and APP13-ClaI, 5′-GAAATCGATTCATTGCTTAACGGTATTG-3′, which contain XhoI and ClaI sites, respectively (bold and underlined). C. neoformans actin promoter (ACT) was amplified using primers Act5, 5′-CAAATCGATGCTGCGAGGATGTGA-3′, and Act3, 5′-GTTAAGCTTTTGGCGGAGTTTACTAAT-3′, which contain ClaI and HindIII sites, respectively (bold and underlined). These fragments were digested with the corresponding enzymes and cloned into the XhoI- and HindIII-restricted pSK/LUC/NAT1/3′UTR plasmid. The resulting plasmid, pSK/5′UTR/ACT/LUC/NAT1/3′UTR, was transformed into the C. neoformans WT H99 strain to generate the positive control IPC1/ACT:LUC strain. Plasmid pSK/5′UTR/366/LUC/NAT1/3′UTR was generated as follows: pΔapp1/ADE plasmid (3Luberto C. Hannun Y.A. Lipids. 1999; 34: S5-S11Crossref PubMed Google Scholar) was digested with HindIII and EcoRI, yielding a 366-bp fragment corresponding to the end of the APP1 promoter. The 366-bp fragment was blunted and subcloned into HindIII-restricted and -blunted pSK/5′UTR/LUC/NAT1/3′UTR vector. The resulting construct, pSK5′UTR/366/LUC/NAT1/3′UTR, was transformed into the C. neoformans WT H99 strain to generate the IPC1/366:LUC strain. The above plasmids were sequenced before the biolistic transformation to confirm appropriate insertion of the corresponding fragments. Stable resistant transformants were selected for further analysis after five passages onto non-selectable YPD agar medium, and genomic DNA was extracted and subjected to Southern analysis with appropriate probes to identify a double crossover event at the APP1 locus without ectopic integrations (data not shown). Plasmid pTel/366:LUC was generated as follows: the 366:LUC fragment was amplified from the pSK/APP1/LUC/NAT1/3′UTR plasmid using primers 366A, 5′-CACGATATCCAGTAAACTGTAGTTTACTGGAAC-3′, and 366B, 5′-CACGATATCTTTACCACATTTGTAGAGGTTTTAC-3′, which contain EcoRV sites (bold and underlined). The resulting fragment was subcloned in pCR2.1 TOPO vector generating plasmid pCR-366:LUC, which was digested with EcoRV. The resulting 2,366-bp fragment was subcloned into the KpnI-restricted and -blunted pTel/ACT:HYG plasmid (kindly provided by Dr. John Perfect, Duke University Medical Center, Durham, NC), generating the pTel/366:LUC plasmid. Three deletions were generated by site-directed mutagenesis (Invitrogen) in the 366-bp fragment. The ATF consensus sequence (TGACGTCA) was deleted in the pCR-366:LUC plasmid using primers ATFdel1, 5′-TACTATGTAGTCACCTGTCAAAAGTCGTACT-3′, and ATFdel2, 5′-TGACAGGTGACTACATAGTAATCGCTGTAAG-3′, and the resulting Δatf:LUC fragment was cloned into pTel/ACT:HYG generating pTel/Δatf:LUC plasmid. The AP-2 consensus sequence (CCCCGCGGC) was deleted in the pCR-366:LUC plasmid using primers AP2del1, 5′-CCCCCACTATGGGGACATGTTCGCCTTGTCC-3′, and AP2del2, 5′-ACATGTCCCCATAGTGGGGGTCGCCGATTTT-3′, and the resulting Δap2:LUC fragment was cloned into pTel/ACT:HYG generating the pTel/Δap2:LUC plasmid. The ATF consensus sequence was also deleted in the pTel/Δap2:LUC plasmid, resulting in the pTel/Δatf+Δap2:LUC plasmid. Additionally, the ATF consensus sequence TGACGTCA was mutated into TGAAATCA by PCR site-directed mutagenesis using primers ATFmut1, 5′-TGTAGTCACCTGTCATGAAATCAAAAG TCGT-3′, and ATFmut2, 5′-TCATGACAGGTGACTACATAGTAATCGCT-3′, and the pCR-366:LUC plasmid as a template. The resulting fragment was subcloned into the pTel/ACT:HYG plasmid, generating pTel/matf:LUC plasmid. The AP-2 consensus sequence CCCCGCGGC was mutated into CCCCGCAAC by PCR site-directed mutagenesis using primers AP2mut1, 5′-ATGGGGACATGTCCCCGCAACTCGCCTTGTC-3′, and AP2mut2, 5′-GCGGGGACATGTCCCCATAGTGGGGGTCG-3′, and pCR-366:LUC plasmid as a template. The resulting fragment was subcloned into the pTel/ACT:HYG plasmid, generating pTel/map2:LUC plasmid. The above plasmids were sequenced prior to biolistic transformation to make sure that the desired deletions and mutations have occurred. These plasmids were transformed into the GAL7:IPC1/5′UTR:LUC strain according to Toffaletti et al. (18Toffaletti D.L. Rude T.H. Johnston S.A. Durack D.T. Perfect J.R. J. Bacteriol. 1993; 175: 1405-1411Crossref PubMed Google Scholar). The transformants were patched onto hygromycin (HYG) plates, then three times onto YPD, and finally onto HYG plates. Transformants that did not grow on the final HYG plates were processed from the YPD plates for genomic DNA extraction according to Casadevall and Perfect (19Casadevall A. Perfect J.R. Press A.S.F.M. Cryptococcus neoformans. ASM Press, Washington, D. C.1998: 381-405Google Scholar). Southern analysis of undigested DNA was performed to confirm episomal integration (data not shown). From the above transformation reactions, transformant numbers 20, 8, 24, 6, 7, and 24 were chosen and designated C. neoformans Tel/366:LUC, Tel/Δatf:LUC, Tel/Δap2:LUC, Tel/Δatf+Δap2:LUC, Tel/matf:LUC, and Tel/map2:LUC strains, respectively. Ipc1 and Luciferase Enzymatic Activities—Ipc1 activity was performed as described previously (20Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. Hannun Y.A. Del Poeta M. Genes Dev. 2001; 15: 201-212Crossref PubMed Scopus (123) Google Scholar). Luciferase activity was performed according to the Promega protocol described in the Luciferase Reporter Gene Assay. Proteins were extracted according to Luberto et al. (20Luberto C. Toffaletti D.L. Wills E.A. Tucker S.C. Casadevall A. Perfect J.R. Hannun Y.A. Del Poeta M. Genes Dev. 2001; 15: 201-212Crossref PubMed Scopus (123) Google Scholar). Then, 20 μl of cell lysate was added to 100 μl of luciferase assay reagent (Promega) and the production of luciferase was immediately measured by a Reporter Microplates Luminometer (Turner Designs). Results were normalized per 1 μg of proteins. Lipid Treatments—The C. neoformans strains were grown on the appropriate medium in a shaking incubator for 24 h at 30 °C. Cell pellets were washed 2 times with SDW, resuspended in fresh medium, and incubated in the appropriate medium in a shaking incubator for 16 h at 30 °C. Next, cell pellets were washed twice with SDW, resuspended in YP medium, and counted. Next, 5 × 106 cells/ml were inoculated in 40 ml of appropriate medium containing 0, 5, 10, and 20 μm 1,2-dioctanoylglycerol (DiC8) for 2 h and 30 min at 30 °C in a shaking incubator. Proteins were then extracted, quantified, and luciferase activity was measured. Identification of Putative C. neoformans ATF2 Gene, Cloning, and Disruption—To identify the potential transcription factor(s) responsible for APP1 activation, we blasted the human AP-2 gene family, AP-2α (NP_003211), AP-2β (NP_003212), AP-2 β-like (NP_758438), AP-2Δ (CAI21171), AP-2γ (NP_003213), and AP-2ϵ (CAI23520) and human ATF gene family, ATF1 (P18846), ATF2 (NM_001880), ATF3 (P18847), ATF4 (P18848), ATF5 (Q9Y2D1), ATF6 (P18850), and ATF7 (P17544) into the C. neoformans H99 Duke University Genome Data base (cneo.genetics.duke.edu/blast.html). The search identified one sequence with an E value of 1e-11 corresponding to chr2-piece9 for the human Atf2 or Atf7 transcription factors, whereas we could not find any significant homology with any other ATF or AP-2 transcription factors. Thus, we focused our attention of the sequence identified in chr2-piece9, which was named putative C. neoformans ATF2. The sequence was retrieved, translated, and the amino acid sequence was aligned with human ATF2 and ATF7 genes. A putative open reading frame containing the basic region and the leucine zipper characteristic of the bZIP domain was identified. Thus, the 5′-UTR fragment corresponding to the upstream region of the ATF2 gene was amplified using primers Atf51, 5′-CAATCTAGATTTCATCACTTCTCCCCTCTCCGC-3′, and Atf52, 5′-CAAGGATCCTGAGTGATGAAAGAGGTGGTAAAG-3′, which contain XbaI and BamHI sites, respectively (bold and underlined), and C. neoformans H99 genomic DNA as a template. Next, the 3′-UTR fragment corresponding to the downstream region of the ATF2 gene was amplified using primers Atf31, 5′-CAACTCGAGTTGGTCATGGTGTGATCATTCTTC-3′, and Atf32, 5′-CAAGGTACCAAGAGAAGGGAGATTAGATCG-3′, which contain XhoI and KpnI sites, respectively (bold and underlined), and the C. neoformans H99 genomic DNA as a template. Finally, the ACT:HYG was amplified from the pTel/ACT:HYG plasmid using primers Hyg/Act1, 5′-CAAGGATCC-TGCGAGGATGTGAGCTGGAGAGCG-3′, and Hyg/Act2, 5′-CAACCTCGAGGTCGACGGTATCGATAAGCTTTA-3′, which contain BamHI and XhoI sites, respectively (bold and underlined). The 5′UTR, ACT: HYG, and 3′-UTR fragments were digested with XbaI + BamHI, BamHI + XhoI, and XhoI + KpnI, respectively, and cloned into XbaI-and KpnI-restricted pSK vector, yielding the pSK/5′UTR/HYG/3′UTR plasmid, which was biolistically transformed into the C. neoformans WT H99 strain and GAL7:IPC1 strain producing IPC1/APP1:LUC/Δatf2 and GAL7:IPC1/APP1:LUC/Δatf2 strains. Statistical Analysis—Statistical analysis was performed using Student's t test. Ipc1 Regulates Transcription of APP1—In previous studies, we found that APP1 mRNA levels are regulated by Ipc1 modulation (16Luberto C. Martinez-Marino B. Taraskiewicz D. Bolanos B. Chitano P. Toffaletti D.L. Cox G.M. Perfect J.R. Hannun Y.A. Balish E. Del Poeta M. J. Clin. Investig. 2003; 112: 1080-1094Crossref PubMed Scopus (115) Google Scholar). Thus, we investigated whether Ipc1 regulates APP1 gene expression at the transcriptional level. A nuclear run-on assay was used to measure the level of APP1 mRNA upon Ipc1 modulation. Fig. 2 shows that when Ipc1 is up-regulated (GAL7:IPC1 strain grown in galactose) the transcription of IPC1 and APP1 mRNA increases compared with the WT strain. These results suggest that up-regulation of Ipc1 increases APP1 transcription. The nuclear run-on assay was also performed in conditions in which Ipc1 was down-regulated (glucose). We found that, using this method, neither IPC1 nor APP1 mRNA transcripts decrease in the GAL7:IPC1 strain grown on glucose compared with the WT strain (data not shown), suggesting that the sensitivity of the method is not sufficient. To investigate how the APP1 transcription is regulated by Ipc1, a ∼800-bp fragment upstream of the ATG of the APP1 gene was fused to the luciferase reporter gene of the firefly Photinus pyralis in both WT and GAL7:IPC1 strains, producing IPC1/APP1:LUC and GAL7:IPC1/APP1:LUC strains. The IPC1/APP1:LUC and GAL7:IPC1/APP1:LUC strains were grown in glucose and galactose, and luciferase activity was measured. Up-regulation of Ipc1 determined a significant increase of luciferase activity in the GAL7:IPC1/APP1:LUC strain, confirming previous results with the nuclear run-on assay, in which up-regulation of Ipc1 increases APP1 transcription. Importantly, there was no significant difference in the luciferase activity between glucose and galactose cultures of the IPC1/APP1:LUC strain, suggesting that the different carbon source does not affect APP1 transcription (Fig. 3A). Interestingly, under conditions in which Ipc1 is down-regulated (GAL7:IPC1/APP1:LUC grown on glucose), luciferase activity does not decrease below the wild-type level. These results suggest that when Ipc1 is down-regulated, potential compensatory mechanism(s) may exist leading to an increase of DAG level by pathways other than that regulated by Ipc1. This hypothesis is supported by evidence that a decrease of DAG under conditions of Ipc1 down-regulation is transitory and occurring in the very early log-phase of growth (14Heung L.J. Luberto C. Plowden A. Hannun Y.A. Del Poeta M. J. Biol. Chem. 2004; 279: 21144-21153Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). It is also possible that because of the leakiness of the GAL7 promoter, the effect of GAL7 down-regulation cannot be measured using these assays at a given time point. On the other hand, in previous studies we showed that APP1 mRNA levels analyzed by reverse transcriptase-PCR are significantly decreased when Ipc1 is down-regulated (16Luberto C. Mar" @default.
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