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.