Identification of a Family of Camp Response Element-Binding Protein Coactivators by Genome-Scale Functional Analysis in Mammalian Cells

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Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Vadim Iourgenko*†, Wenjun Zhang*†, Craig Mickanin*, Ira Daly*, Can Jiang*, Jonathan M. Hexham*, Anthony P. Orth‡, Loren Miraglia‡, Jodi Meltzer*, Dan Garza*, Gung-Wei Chirn*, Elizabeth McWhinnie*, Dalia Cohen*, Joanne Skelton*, Robert Terry*, Yang Yu*, Dale Bodian*, Frank P. Buxton*, Jian Zhu*, Chuanzheng Song*, and Mark A. Labow*§

*Department of Functional Genomics, Novartis Institute for Biomedical Research, 100 Technology Square, Cambridge, MA 02139; and ‡Genomics Institute, Novartis Research Foundation, 10675 John Jay Hopkins Drive, Suite F117, San Diego, CA 92121

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved July 30, 2003 (received for review May 8, 2003) This report describes an unbiased method for systematically de- of screening data and further experiments demonstrated that the termining gene function in mammalian cells. A total of 20,704 IL-8 promoter contained an unrecognized cAMP response predicted human full-length cDNAs were tested for induction of element (CRE)-like element that was activated by a protein, the IL-8 promoter. A number of genes, including those for cyto- termed transducer of regulated cAMP response element-binding kines, receptors, adapters, kinases, and transcription factors, were protein (CREB) TORC1, which is the founding member of a identified that induced the IL-8 promoter through known regula- conserved family of CREB coactivators. Thus, screening of tory sites. Proteins that acted through a cooperative interaction arrayed cDNAs represents an unbiased approach for identifica- between an AP-1 and an unrecognized cAMP response element tion of gene function and elucidation of pathways that regulate (CRE)-like site were also identified. A protein, termed transducer complex biological processes. of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the Materials and Methods variant CRE and consensus CRE sites. TORC1 potently induced Reporter DNA Constructs. pIL-8-luciferase (Luc) was constructed known CREB1 target genes, bound CREB1, and activated expres- by insertion of the Ϫ1491 to ϩ43 region of the human IL-8 gene sion through a potent transcription activation domain. A functional into pGL3Basic (Promega). PCR was used to generate a reporter Drosophila TORC gene was also identified. Thus, TORCs represent containing the first 160 nucleotides of the IL-8 promoter for a family of highly conserved CREB coactivators that may control the introduction of AP-1, C͞EBP, and NF-␬B site mutations as potency and specificity of CRE-mediated responses. described (11). The variant CRE (CRE-like) site was mutated to 5Ј-TCGATCAA-3Ј. Promoter constructs carrying six copies of ͉ ͉ ͉ IL-8 genomics high-throughput screening transducer of regulated the IL-8 CRE-like sequence (pCREL-Luc) or five copies of a cAMP response element-binding protein member of the S-100 Caϩ2-binding protein family (CAPL) CRE-like sequence 5Ј-TGACACAA-3Ј (pCREL2-Luc) were he completion of draft mouse and human genome sequences prepared by inserting PCR-amplified sequences into pTAL-Luc Thas underscored the need for a systematic approach to (Becton Dickinson Biosciences, Clontech, Palo Alto, CA). analyzing mammalian gene function. The human and mouse pCRE-Luc (Stratagene) contains four copies of a consensus Ϸ Ϸ genomes contain 30,000 genes (1–4). However, 28% of the CRE. predicted human and mouse protein coding genes have no known functional domain or functional classification. The de- Full-Length Human cDNA Clones. A number of cDNA libraries were velopment of large collections of characterized full-length hu- constructed in pCMV-Sport6 vector (Invitrogen) or its deriva- man cDNAs, like that of the Mammalian Gene Collection Ј ͞͞ tives for 5 end sequencing (Celera Genomics, Rockville, MD). (http: mgc.nci.nih.gov) (5), and the ability to construct gene Predicted full-length cDNA clones were isolated and arrayed by specific knockdown reagents using RNA interference (short using a Q-Bot (Genetix, Boston), and were placed into 384-well interfering RNA) (6), should provide a means to systematically plates (Genetix) containing 60 ␮l of Luria Broth Base (Invitro- assess gene function by reiteratively testing these reagents in gen), 8% glycerol, and 100 ␮g͞ml ampicillin. Bacteria were cell-based assays. As a test of this approach, a collection of 20,704 grown in 96-deep-well plates containing 1 ml of Terrific Broth predicted full-length cDNAs was assembled and tested individ- (KD Medical, Columbia, MD), and were then used for produc- ually for activation of the IL-8 promoter. The IL-8 promoter was tion of plasmid DNA by using a BioRobot 8000 with a QIAprep used as a model complex biological process because its expres- Turbo96 PB protocol (Qiagen, Valencia, CA). For high- sion is affected by a variety of signaling pathways. throughput screening, the 20,704 cDNA clones were dispensed The IL-8 gene, expressed by a number of cell types, encodes in 384-well PCR plates at 4 ␮l per well at 7.5 ng͞␮linOPTI- a chemokine that induces neutrophil migration and activation. MEM (Invitrogen). IL-8 expression is associated with the development of diseases such as asthma, arthritis, and cancer. The promoter contains

binding sites for the transcription factors AP-1, NF-␬B, and CELL BIOLOGY ͞ This paper was submitted directly (Track II) to the PNAS ofﬁce. C EBP (for review see ref. 7), which act independently or Abbreviations: TNF, tumor necrosis factor; CRE, cAMP response element; CRE-like, a variant synergistically in response to extracellular stimuli. Activation by CRE; CREB, cAMP response element-binding protein; TORC, transducer of regulated CREB; the cytokines IL-1 and tumor necrosis factor ␣ (TNF-␣) likely act hTORC, human TORC, dTORC, Drosophila TORC; PKA, protein kinase A; Luc, luciferase. primarily through NF-␬B, whereas other stimuli, including met- Data deposition: The sequences reported in this paper have been deposited in the GenBank als, hypoxia, reactive oxygen species, and proteosome inhibition, database [accession nos. AY360171 (hTORC1), AY360172 (hTORC2), and AY360173 use a variety of other pathways (8–10). (hTORC3)]. The high-throughput approach used here identified many †V.I. and W.Z. contributed equally to this work. components of the AP-1, NF-␬B, and C͞EBP pathways, and a §To whom correspondence should be addressed. E-mail: [email protected]. number of uncharacterized proteins. Computational comparison © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1932773100 PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12147–12152 Downloaded by guest on October 2, 2021 Fig. 1. High-throughput screening of arrayed full-length cDNAs. (A) Percentage of cDNAs with speciﬁc InterPro annotations. (B) Fold induction of pIL-8-Luc after transfection of the 10% most active cDNAs. (C) Fold induction (shown by color) by cDNAs in the primary (IL-8࿝1) and conﬁrmatory IL-8-Luc (IL-8࿝2) assays, CRE-Luc, and serum response element-Luc reporter assays. (D) HeLa cells were treated with IL-1␤ or transfected with an empty vector (CMV), or with relA or MAP3K11 constructs (x axis), individually or in combination with a second construct as shown in the legend. The level of secreted IL-8 protein in the media 48 h posttransfection is indicated.

Cell Culture and High-Throughput Transfection. Trypsinized HeLa The results in Fig. 2A are absolute values, because different cells were resuspended in complete growth medium at 105 cells activators affected the control Luc gene differently. For ELISAs, per ml and distributed into 51 white 384-well plates (Costar) at HeLa cells were cotransfected by using 100 ng of one plasmid, 30 ␮l per well by using a Multidrop 384 (Thermo Labsystems, listed on the x axis, and 25 ng of a second plasmid, as indicated Vantaa, Finland), and were incubated overnight at 37°Cin5% in the legend, in 96-well plates (Costar). Secreted IL-8 was CO2. For screening, pIL-8-Luc reporter plasmid was added to measured 72 h posttransfection by using an IL-8 ELISA kit OptiMEM I serum-free medium (50 ng of reporter per trans- (Sigma). Cells transfected with empty vector and treated with fection). FuGENE 6 transfection reagent (Roche Diagnostics, IL-1␤ (R & D Systems) at 5 ng͞ml for 16 h were used as a positive Indianapolis) was used for high-throughput transfections at 3 ␮l control. of FuGENE 6 per ␮g of total DNA. Three microliters of OptiMEM-reporter-FuGENE 6 mix was added per well con- Gene Expression Profiling with Affymetrix DNA Microarray Chips. taining 4 ␮l of prediluted cDNAs by using a BiomekFX (Beck- HeLa cells were transfected by using Targefect F1 reagent man Coulter). After 15 min incubation at room temperature, 6 (Targeting Systems, Santee, CA), according to the manufactur- ␮l of the mix from each well was transferred to a 384-well tissue er’s instructions. Total RNA was extracted 72 h posttransfection culture plate and incubated for 48 h. Cells were treated with by using TRIzol (Invitrogen) and purified by using a Qiagen forskolin (1 ␮M final) or PMA (1 ng͞ml), respectively, for 16 h, RNeasy kit (Qiagen), according to the manufacturer’s instruc- before Luc assay for some experiments as described. tions. RNA was quantified by using Ribogreen (Molecular Probes). Isolated RNA (5–10 ␮g) was amplified and labeled by Luc and ELISA. Luc and ELISA data represent at least triplicate using a modified Eberwine protocol (12). Biotin-labeled RNA values and SD, except for those of Fig. 1 B and C. Forty-eight was hybridized to HG࿝U95A chips, according to the manufac- hours posttransfection, firefly Luc activity was measured by turer’s instructions (Affymetrix, Santa Clara, CA). The scanned using a BrightGlo Luc assay system (Promega), following the images were quantified with Affymetrix GeneChip microarray manufacturer’s protocol. Luminescence was determined with a suite 4 with a target intensity of 500. At least two chips were used LUMINOSKAN ascent luminometer (Thermo Labsystems) for each condition. with a 200- or 400-msec integration time. Results were normalized for transfection efficiency by using a cotransfected Renilla Association of TORC1 with CREB1. FLAG-tagged constructs were Luc plasmid (pRL-SV40; Promega), unless otherwise indicated. transfected into HEK293 cells in 100-mm dishes (Falcon) by Luc activities were measured by using a DualGlo Luc assay using FuGENE 6, according to the manufacturer’s protocol. system (Promega), according to the manufacturer’s protocols. Lysates were prepared 40 h after transfection in 800 ␮lof10mM

12148 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1932773100 Iourgenko et al. Downloaded by guest on October 2, 2021 hsp-Luc contains four copies of a Drosophila CRE, followed by a heat shock 70-kDa protein minimal promoter. Drosophila melanogaster Schneider cells (S2) were transfected in six-well plates (Costar) by using calcium phosphate. A total of 25 ␮gof DNA was transfected into a six-well dish containing 4 ml of cells (106 cells per ml). Luc assays were performed 48 h later. The UAS-transgenes were activated by cotransfection with the actin promoter-GAL4 plasmid provided by Norbert Perrimon (De- partment of Genetics, Harvard Medical School, Boston). Values were normalized by using a cotransfected Renilla Luc gene driven by a heat shock promoter. As a negative control S2, cells were cotransfected with CRE-hsp-Luc reporter and empty pUAST vector. Results Putative full-length cDNAs (20,704) were tested for the ability to induce the IL-8 promoter on transient transfection in human cells. The cDNA clones were selected, based on the presence of a start codon or 5Ј end of a predicted or characterized gene. The 12,905 clones matched RefSeq genes, of which 5,463 were functionally annotated (Fig. 1A). Complete sequencing of Ͼ100 clones suggested that Ϸ70% of the rescued cDNAs contained full-length cDNAs (data not shown). The cDNAs were cotransfected with a firefly Luc gene controlled by the IL-8 promoter (pIL-8-Luc). Sixty-four cDNAs induced the reporter by Ͼ5-fold (Fig. 1B). The verified active cDNAs included one to three copies of 28 unique genes. The entire collection was also screened for activation of CRE- or serum response element- driven reporters. Twenty-two unique cDNAs were chosen for further investigation, and the activities of these clones in the primary screens and a secondary IL-8-reporter assay are shown after hierarchical clustering analysis (Fig. 1C). Genes relatively specific for the IL-8 reporter (where the induction of the IL-8 reporter was Ͼ5-fold higher than other reporter constructs) included several known inducers of NF-␬B represented by relA, Fig. 2. TORC1 activates the IL-8 promoter through a CRE-like site. (A) IL-8 a subunit of NF-␬B, the TNF receptor TNFSFR1A, TNF-related promoter constructs with mutations in the C͞EBP␤ (␦C͞EBP), NF-␬B(␦NF-␬B), molecule TNFSF12, RIPK2, TRAF6, ACT1, and the kinase ␦ ␦ AP-1 ( AP-1), or CRE-like ( CRE-like) sites were transfected with MAP3K11, ANKRD3. Genes known to activate AP-1 were also identified, relA, or TORC1 as indicated. Fireﬂy Luc levels are shown as arbitrary units (AU). including the JNK-inducing kinases MAP3K11 and MAP3K12, (B) CRE-like and consensus CRE-containing reporters were transfected with junD, and c-jun (data not shown). C͞EBP␤, predicted to bind the the activators shown in the key. pTAL-Luc contains the same promoter as the ͞ ␤ CRE vectors without any response element. Luc values are shown as AUs after C EBP site, was also identified. Several cDNAs were identified normalization. (C) A dominant mutant of CREB1, KCREB, but not IkB␣, blocked whose mechanisms of action were not clear, including two TORC1 induction. The pIL-8-Luc reporter was cotransfected with vector (CMV) guanine nucleotide exchange factors for the Rho GTPase (Rho- or TORC1, as indicated in the key, and inhibitor as indicated on the x axis. (D) GEFs), p114 and ARHGEF1, C16orf15, thyrotroph embryonic pIL-8-Luc was cotransfected with either ␦59 or an empty vector (CMV), as factor 1 (TEF1), fibronectin (FN1), and nuclear receptor family indicated in the key, with various activators as shown on the x axis. (E) member NR2F2. C16orf15 encodes a proline-rich protein of ␦ pAP-1(PMA)-Luc was transfected with control vector or 59 as indicated in the unknown function (13). TEF1 is a member of the basic leucine key, and cells were treated with PMA or transfected with MAP3K11 as indi- zipper transcription factors, which acts directly through a TEF cated on the x axis. response element (14). FN1 is a matrix glycoprotein highly expressed in injured tissues reported to induce the IL-1 gene Hepes, pH 7.6͞250 mM NaCl͞5 mM EDTA͞1mMDTT͞0.1% through AP-1 (15). Nonidet P-40, and freshly dissolved protease inhibitors. Immu- The accumulation of secreted endogenous IL-8 from HeLa noprecipitation was carried out by using anti-FLAG-M2-agarose cells was measured after transfection with relA and MAP3K11 beads (Sigma). Precipitated proteins were separated on SDS͞ alone or in combination (Fig. 1D). MAP3K11 and relA induced small increases, but the combination of both induced levels of 4–20% PAGE (Invitrogen) and transferred to a nitrocellulose secreted IL-8 comparable to that observed after treatment with membrane (Invitrogen). Western blots were performed by using IL-1␤, one of the most potent inducers of IL-8 known. Thus, antibody against CREB1 (Cell Signaling Technology, Beverly, whereas the reporter gene was potently induced by proteins MA) and anti-FLAG M2 monoclonal antibodies (Sigma). activating a single pathway, activation of multiple signaling CELL BIOLOGY pathways synergistically induced expression of the endogenous TORC1-Related Proteins. Human TORC2 and 3 (hTORC2 and IL-8 gene. hTORC3) were identified by searching public and inhouse EST Surprisingly, a number of CRE-binding proteins CREB1, ࿝ databases. hTORC2 was identified from XP 117201. hTORC3 CRE-BPa, and XBP1, potently activated the IL-8 reporter, was identified with FLJ00364. Full-length cDNAs were con- suggesting that it contains an unrecognized CRE site. Further, structed in pCMV-Sport6. The sequences shown are predicted cDNAs encoding C͞EBP␤, JunD, and a clone overlapping with from clones isolated here. Drosophila TORC (dTORC) was KIAA0616 were potent inducers of both the CRE and IL-8 derived from the predicted gene CG6064. A 2.3-kb cDNA reporters. The CRE-like site was identified with the sequence encoding dTORC was used to produce pUAS-dTORC. CRE- 5Ј-TGACATAA-3Ј at Ϫ69 to Ϫ61 of the IL-8 promoter. This

Iourgenko et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12149 Downloaded by guest on October 2, 2021 sequence contains two changes from a consensus CRE, both of which were reported to be tolerated for binding to either CREB1 or CREB2 (16). The mechanism of induction by MAP3K11 and KIAA0616 was pursued, because they were the strongest activators of the IL-8 promoter found. No function is known for KIAA0616, except that sequence encoding its first 44 amino acids was recently found translocated onto the Mastermind-like gene, MAML2, in mucoepidermoid carcinoma (MEC) (17). As KIAA0616 and several related genes are shown here to specifically activate CREB-dependent gene expression, (see below) this gene was designated as TORC1. IL-8 promoters carrying mutations in the CRE-like and other regulatory sites were tested for induction by MAP3K11, TORC1, or relA (Fig. 2A). Mutation of the C͞EBP␤-binding site had no effect. The NF-␬B site mutation had little effect on induction by MAP3K11 or TORC1, but eliminated induction by relA. Mu- tation of the AP-1 site did not significantly affect response to relA, but severely reduced induction by MAP3K11, which was consistent with its known ability to activate the JNK͞SAPK pathway and AP-1. This mutation also significantly reduced, but did not eliminate, activation by TORC1. Mutation of the CRE-like site dramatically decreased induction by both TORC1 and MAP3K11. The ability of these proteins to activate a minimal promoter carrying concatamer- ized CRE-like sites (pCREL-Luc) was then examined. pCREL- Luc was strongly activated by TORC1, but not by the MAP3K11 or PMA, an AP-1 activator (Fig. 2B). TORC1, but not MAP3K11 or PMA, also potently induced a promoter driven by consensus CRE-sites (pCRE-Luc). In contrast, MAP3K11 potently induced an AP-1 reporter, which was relatively unaffected by TORC1 (data not shown). Thus, TORC1 acts through CREs, whereas MAP3K11 acts as an AP-1 inducer. The effect of a KCREB1 construct, containing an inactivating mutation in the DNA-binding domain (18), was examined to determine whether TORC1 acted through a CREB-related Fig. 3. TORC1 induces known CREB1 target genes. (A) Fold activation of protein. Coexpression of KCREB with TORC1 significantly mRNA levels was measured on Affymetrix U95a chips after TNF-␣ treatment or reduced induction of the IL-8 promoter (Fig. 2C). In contrast, transfection of HeLa cells with MAP3K11, p65, or TORC1 constructs. Values TORC1 activity was unaffected by cotransfection with I-␬B␣, represent the averages of two experiments. (B) TORC1 activates expression of ␬ a CRE-like sequence (pCREL2-Luc) found in the CAPL and PEPCK promoters. which potently inhibits NF- B. These data suggest that TORC1 Each reporter, indicated on the x axis, was cotransfected with TORC1 and acts at least partly through CREB1 or a related protein. analyzed for activation compared with vector transfected cells. (C) Cells Both the AP-1 and the CRE-like sites were required for transfected with the vector indicated on the x axis were treated with forskolin activation of the IL-8 promoter by MAP3K11 or TORC1. To (FSK), cotransfected with CRE-BPa, or both, as indicated. determine whether TORC1 participated in this interaction between these sites, a dominant interfering variant of TORC1 was tested for its ability to affect activation. A TORC1 mutant, ␦59, by MAP3K11, which induced PAI-2, a known AP-1 target gene missing the first 59 amino acids, was largely inactive for reporter (24), or relA. Thus, TORC1 specifically induced authentic induction, and reduced activation by cotransfected wild-type CREB1 target genes. The endogenous IL-8 gene was modestly TORC1 by 70% (Fig. 2D). This mutant greatly inhibited acti- induced (2.5- to 5-fold) by each activator tested, which was vation of the IL-8-Luc by MAP3K11, but had no effect on consistent with a requirement for activation of multiple pathways activation by relA. These data suggest that TORC1 may specif- for efficient induction of the endogenous IL-8 gene. Both relA ically regulate activation of the IL-8 promoter through the AP-1 and TORC1 potently activated expression of the chemokine and CRE-like sites. Activation of an AP-1 specific reporter by MIP3␣ and KIAA0467, suggesting their promoters have both PMA or MAP3K11 was also blocked by ␦59 (Fig. 2E). No effect ␬ ␬ ␣ NF- B and CRE-like sites. was seen by over expression of an I- B construct (data not CAPL, potently induced by TORC1, is not known to contain shown). These data suggest that TORC1 may interact with an a CRE site. A sequence similar to the CRE-like site was unidentified protein, which is essential for AP-1-mediated tran- identified designated CRE-like2, (5Ј-TGACACAA-3Ј)atϪ385 scription, and that TORCs may regulate a subset of AP-1- Ϫ dependent responses. and 392 of the predicted CAPL promoter. The CRE-like2 HeLa cells were transfected with TORC1, MAP3K11, or relA element, when placed upstream of a minimal promoter, allowed constructs, or treated with TNF-␣ and gene expression profiles induction by TORC1 (Fig. 3B). To determine whether the were compared by using DNA microarrays to determine whether TORC1-responsive elements acted as true CREs, their response TORC1 specifically induced expression of endogenous CRE- to forskolin or a CREB-related protein (CRE-BPa) was exam- dependent genes. TORC1 transfection induced seven genes by ined. The IL-8 promoter, CRE-like, and CRE-like2 reporters Ͼ10-fold (Fig. 3A), including several well known targets of were modestly activated by forskolin and synergistically induced CREB1, namely TSH␣ (19), phosphoenol pyruvate carboxyki- by both forskolin and CRE-BPa (Fig. 3C). Thus, to date, all nase (PEPCK) (20), crytallin ␣-B (21), amphiregulin (22), and TORC1-responsive elements represented true CREs and sup- to a lesser extent, CREM (23). This set of genes was unaffected ported the notion that TORC1 works primarily through CREB1

12150 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1932773100 Iourgenko et al. Downloaded by guest on October 2, 2021 Fig. 4. TORCs are a conserved gene family. Shown is alignment of hTORC- and dTORC-predicted proteins. Conserved amino acids are shaded. Align- ments were produced with CLUSTALW.

or other CREB-related proteins that might have higher affinity to variant CRE-sites. Sequence databases were searched for TORC1-related proteins. Whereas no significant homologies to characterized proteins were found, a number of uncharacterized proteins highly related to TORC1 were discovered. TORC1 orthologs were identified in mice and fugu, which shared 90% and 66% amino acid identity, respectively (data not shown). Two TORC1-related human genes (hTORC2 and hTORC3, which are 32% identical to TORC1) and a single dTORC (which is 20% identical to TORC1), were identified as shown (Fig. 4). The proteins display a highly conserved predicted N-terminal coil–coil domain (hTORC1 residues 8–54) and an invariant sequence matching a protein kinase A (PKA) phosphorylation consensus sequence (RKXS). hTORC2 and hTORC3 cDNAs were isolated and their ability to activate CRE-driven expression was tested. Both TORC1-related genes potently induced expression from the pCRE-Luc and pIL-8-Luc reporters similar to TORC1 (Fig. 5A). A cDNA for a potential Drosophila ortholog, dTORC, was Fig. 5. TORC proteins are CREB1 coactivators. (A) Constructs encoding the isolated and tested for activity in Drosophila S2 cells. Transfec- three human TORC genes (indicated on the x axis) were transfected with either tion of the dTORC expression plasmid potently induced a heat pIL8-Luc or pCRE-Luc, as indicated. Values shown are fold induction, as com- shock promoter when linked to Drosophila CREs in S2 cells. pared with vector-transfected cells. (B) dTORC expressed in S2 cells induced a reporter carrying Drosophila CREs. Cells were transfected with the indicated (Fig. 5B). Thus, these genes represent a functionally conserved reporter and activator as indicated on the x axis. (C) The N-terminal domain of family of proteins, which potently induce CRE-driven gene hTORC1 interacts with CREB1. HEK293 cells were transfected with the indi- expression. cated FLAG-tagged constructs encoding either amino acids 1–170 or 170–650 Several observations suggest that TORCs are CREB1 coac- of TORC1, or human histone deacetylase 1 (HDAC1) as indicated. Protein

tivators. Epitope-tagged TORCs are localized in the nucleus complexes isolated with anti-FLAG antibody were tested for the presence of CELL BIOLOGY (data not shown). However, their coding regions contain no CREB1 by Western blot (Upper). The ﬁrst lane shows CREB1 detected in obvious DNA-binding domain, and experiments failed to dem- whole-cell extracts. The same ﬁlter was also tested with anti-FLAG M2 anti- onstrate DNA binding by TORC1 (data not shown). Each body (Lower). (D) hTORCs contain potent transcriptional activation domains. protein contained a serine͞glutamine-rich domain (hTORC1 HEK293 cells were cotransfected with UAS-Luc reporter plasmid and full- length TORC constructs or GAL4BD-TORC1 (amino acids 300–650), TORC2 residues 289–559) and a negatively charged C terminus similar (amino acids 296–694), or TORC3 (amino acids 335–635) fusions. Fold induc- to transcription activation domains. Regions of TORC1 were tion is relative to transfection with the GAL4-DNA-binding domain vector, tested for association with endogenous CREB1. The N-terminal pCMV-BD. The reporter was also transfected with a plasmid encoding GAL4- 170 amino acids of TORC1, containing the highly conserved CREB fusion protein alone (GAL4-CREB), or with a PKA catalytic subunit coil–coil domain essential for TORC activity, was found to be expression construct (GAL4-CREB/PKA) as a positive control.

Iourgenko et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12151 Downloaded by guest on October 2, 2021 associated with endogenous CREB1 in human 293 cells (Fig. not effect a consensus CRE in CREB1-deficient fibroblasts. 5C). To determine whether the TORCs contained a transcrip- Alternatively, TORCs may selectively activate a subset of re- tion activation domain, various regions of the human TORCs sponsive genes by interaction with other transcription factors, were expressed as fusion proteins with the DNA-binding domain which is consistent with the observation that induction of IL-8 by of GAL4 (GAL4BD), and tested for induction of a minimal TORC1 required both the AP-1 and CRE-like sites. Elucidation promoter linked to GAL4-binding sites (UAS-GAL4). UAS- of TORC-mediated specificity should shed light on the selective GAL4 was potently induced by GAL4BD fusions containing the activation of CRE-containing genes. C-terminal portion of all three human TORCs, but not by the As discussed above, a chromosomal translocation between the full-length TORC proteins (Fig. 5D). Thus, the TORC proteins first 44 amino acids of TORC1 and the Mastermind-like 2 gene represent coactivators that bind CREB1, and, presumably, bring (MAML2) was found to create a transforming gene (17). Be- into the complex a potent activation domain. It should be noted cause the first 59 amino acids of TORC1 are shown here to be that activation by GAL4-TORCs or TORCs was greater than critical for recognition of CREs, transformation by the TORC1– that observed by either activated GAL4-CREB1 or CREB1, MAML2 fusion might result from uncontrolled activation of respectively. In other experiments, all three TORCs enhanced CREB-responsive genes. activation by a GAL4-CREB1 fusion, which was consistent with The experiments described here raise the question of the TORCs acting as CREB1 coactivators (data not shown). importance of the CRE-like site in regulating IL-8. In this ␤ ␤ Discussion regard, 2-adrenergic agonists ( 2-AR), which increase intracellular cAMP levels, were shown to induce IL-8 secretion in CREB1 is one of the best studied inducible eukaryotic tran- monocytes and bronchial epithelial cells (26, 27). This fact is scription factors. CREB1 is induced by phosphorylation by PKA, ␤ particularly important, because the use of 2-AR agonists as which allows association with the coactivator CBP. CREB1 bronchodilators can exacerbate asthma and should be used in regulates a large number of genes controlling cell growth, conjunction with anti-inflammatory steroids (28). This study survival, metabolic control, and memory. How CREB1 and suggests ␤ -AR may directly induce IL-8 transcription through related proteins achieve cell-type- or stimulus-specific induction 2 the CRE-like site. of subsets of genes is not understood. The evolutionary conser- This article demonstrates the utility of high-throughput vation of TORCs strongly suggests they will have an important screening of curated cDNAs in mammalian cells. Activities were role in regulating CREB1-dependent responses. As the TORCs assigned to several previously uncharacterized genes and added potently activate CREB-dependent gene expression in the ab- sence of extracellular stimuli, TORCs may act as a rheostat for to the characterization of several known genes. The utility of controlling the magnitude of CREB responses, and may provide reiteratively testing gene sets against many assays and building an intracellular mechanism to control CREB-responsive gene databases of function was illustrated by the prediction that the expression during development. The presence of a conserved, IL-8 gene would have a CRE sequence. potential PKA phosphorylation site in all TORCs suggests that Note. TORCs and CREB1 could be coordinately regulated. While this work was being completed, a similar approach was reported that used a smaller set cDNAs encoding potentially secreted The ability of the human TORCs to activate CRE-like ele- proteins (29). This article and the work presented here suggest that ments weakly responsive to cAMP may be explained in several high-throughput screening for function of cDNAs will find wide appli- ways. TORCs may interact with other CREB proteins such as cation in the analysis of complex biological systems. CREB2 or CREB-BPa with alternate DNA-binding specificities. It should be noted that TORCs were also identified in an We thank John B. Hogenesch and Marc Montminy for helpful independent study (25) that demonstrated that TORC1 could discussions.

1. Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., 15. Roman, J., Ritzenthaler, J. D., Fenton, M. J., Roser, S. & Schuyler, W. (2000) Devon, K., Dewar, K., Doyle, M., FitzHugh, W., et al. (2001) Nature 409, 860–921. Cytokine 12, 1581–1596. 2. Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., 16. Benbrook, D. M. & Jones, N. C. (1994) Nucleic Acids Res. 22, 1463–1469. Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., et al. (2001) Science 291, 17. Tonon, G., Modi, S., Wu, L., Kubo, A., Coxon, A. B., Komiya, T., O’Neil, K., 1304–1351. Stover, K., El Naggar, A., Griffin, J. D., et al. (2003) Nat. Genet. 33, 208–213. 3. Waterston, R. H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J. F., 18. Walton, K. M., Rehfuss, R. P., Chrivia, J. C., Lochner, J. E. & Goodman, R. H. Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., An, P., et al. (1992) Mol. Endocrinol. 6, 647–655. (2002) Nature 420, 520–562. 19. Jameson, J. L., Jaffe, R. C., Deutsch, P. J., Albanese, C. & Habener, J. F. (1988) 4. Okazaki, Y., Furuno, M., Kasukawa, T., Adachi, J., Bono, H., Kondo, S., J. Biol. Chem. 263, 9879–9886. Nikaido, I., Osato, N., Saito, R., Suzuki, H., et al. (2002) Nature 420, 563–573. 20. Liu, J. S., Park, E. A., Gurney, A. L., Roesler, W. J. & Hanson, R. W. (1991) 5. Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G., Klausner, R. D., J. Biol. Chem. 266, 19095–19102. Collins, F. S., Wagner, L., Shenmen, C. M., Schuler, G. D., Altschul, S. F., et 21. Cvekl, A., Kashanchi, F., Sax, C. M., Brady, J. N. & Piatigorsky, J. (1995) Mol. al. (2002) Proc. Natl. Acad. Sci. USA 99, 16899–16903. Cell. Biol. 15, 653–660. 6. Shi, Y. (2003) Trends Genet. 19, 9–12. 22. Bianco, C., Tortora, G., Baldassarre, G., Caputo, R., Fontanini, G., Chine, S., 7. Roebuck, K. A. (1999) J. Interferon Cytokine Res. 19, 429–438. Bianco, A. R. & Ciardiello, F. (1997) Clin. Cancer Res. 3, 439–448. 8. Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H. & Kracht, M. (2002) 23. Stehle, J. H., Foulkes, N. S., Molina, C. A., Simonneaux, V., Pevet, P. & J. Leukocyte Biol. 72, 847–855. Sassone-Corsi, P. (1993) Nature 365, 314–320. 9. Barchowsky, A., Soucy, N. V., O’Hara, K. A., Hwa, J., Noreault, T. L. & 24. Arts, J., Grimbergen, J., Bosma, P. J., Rahmsdorf, H. J. & Kooistra, T. (1996) Andrew, A. S. (2002) J. Biol. Chem. 277, 24225–24231. 10. Hipp, M. S., Urbich, C., Mayer, P., Wischhusen, J., Weller, M., Kracht, M. & Eur. J. Biochem. 241, 393–402. Spyridopoulos, I. (2002) Eur. J. Immunol. 32, 2208–2217. 25. Conkright, M., Canettieri, G., Guzman, E., Mariglia, L., Hogenesch, J. & 11. Wu, G. D., Lai, E. J., Huang, N. & Wen, X. (1997) J. Biol. Chem. 272, Montminy, M. (2003) Mol. Cell 12, 413–423. 2396–2403. 26. Kavelaars, A., van de Pol, M., Zijlstra, J. & Heijnen, C. J. (1997) J. Neuroim- 12. Eberwine, J., Kacharmina, J. E., Andrews, C., Miyashiro, K., McIntosh, T., munol. 77, 211–216. Becker, K., Barrett, T., Hinkle, D., Dent, G. & Marciano, P. (2001) J. Neurosci. 27. Linden, A. (1996) Br. J. Pharmacol. 119, 402–406. 21, 8310–8314. 28. Cockcroft, D. W., McParland, C. P., Britto, S. A., Swystun, V. A. & Rutherford, 13. Bhalla, K., Eyre, H. J., Whitmore, S. A., Sutherland, G. R. & Callen, D. F. B. C. (1993) Lancet 342, 833–837. (1999) J. Hum. Genet. 44, 383–387. 29. Chen, C., Grzegorzewski, K. J., Barash, S., Zhao, Q., Schneider, H., Wang, Q., 14. Hunger, S. P., Li, S., Fall, M. Z., Naumovski, L. & Cleary, M. L. (1996) Blood Singh, M., Pukac, L., Bell, A. C., Duan, R., et al. (2003) Nat. Biotechnol. 21, 87, 4607–4617. 294–301.

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