Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach Carmela Beger*†, Leigh N. Pierce‡, Martin Kru¨ ger*§, Eric G. Marcusson‡, Joan M. Robbins‡, Piri Welcsh¶, Peter J. Welch‡ʈ, Karl Welte**, Mary-Claire King¶, Jack R. Barber‡, and Flossie Wong-Staal*†† *Department of Medicine, University of California San Diego, La Jolla, CA 92093-0665; ‡Immusol Inc., San Diego, California 92121; ¶Departments of Medicine and Genetics, University of Washington, Seattle, WA 98195-7720; and **Department of Pediatric Hematology and Oncology, Hannover Medical School, D-30625 Hannover, Germany Edited by Carlo M. Croce, Thomas Jefferson University, Philadelphia, PA, and approved October 30, 2000 (received for review July 23, 2000) Expression of the breast and ovarian cancer susceptibility gene reproducibly confer phenotypic changes is used to identify the BRCA1 is down-regulated in sporadic breast and ovarian cancer corresponding target gene(s). We have recently used this ap- cases. Therefore, the identification of genes involved in the regu- proach to identify cellular genes involved in Hepatitis C virus lation of BRCA1 expression might lead to new insights into the translation mediated by its internal ribosome entry site (18). In pathogenesis and treatment of these tumors. In the present study, the present application, ribozyme-library-transduced cells were an ‘‘inverse genomics’’ approach based on a randomized ribozyme selected for altered BRCA1 promoter-driven reporter gene gene library was applied to identify cellular genes regulating expression. By using this approach, we identified the dominant BRCA1 expression. A ribozyme gene library with randomized negative transcriptional regulator Id4 as a crucial gene involved target recognition sequences was introduced into human ovarian in BRCA1 regulation, and provide further insights into the cancer-derived cells stably expressing a selectable marker [en- biological properties of Id4 protein in the context of BRCA1 hanced green fluorescence protein (EGFP)] under the control of the expression. BRCA1 promoter. Cells in which BRCA1 expression was up- regulated by particular ribozymes were selected through their Materials and Methods concomitant increase in EGFP expression. The cellular target gene Construction of the Ribozyme Library Vector. The Moloney retro- of one ribozyme was identified to be the dominant negative viral genome based vector pLHPM-RzLib was constructed to transcriptional regulator Id4. Modulation of Id4 expression re- contain a ribozyme (Rz) expression cassette with eight random sulted in inversely regulated expression of BRCA1. In addition, nucleotides in helix 1 and four random nucleotides in helix 2 increase in Id4 expression was associated with the ability of cells driven by the tRNAval promoter as described (18). To demon- to exhibit anchorage-independent growth, demonstrating the bi- strate the randomness of the Rz library insert, 50 individual ological relevance of this gene. Our data suggest that Id4 is a crucial bacterial colonies were analyzed by standard sequencing tech- gene regulating BRCA1 expression and might therefore be impor- niques using primer NL6H6 (5Ј-CTGACTCCATCGAGC- tant for the BRCA1 regulatory pathway involved in the pathogen- CAGTGTAGAG-3Ј). Additionally, in vitro cleavages of several esis of sporadic breast and ovarian cancer. short RNA transcripts by a comparable ribozyme library con- firmed the randomness (data not shown). An unrelated control RCA1 has been linked to the genetic susceptibility of a ribozyme, directed against the negative strand of the 5ЈUTR of Bmajority of familial breast and ovarian cancers (1, 2). Its the Hepatitis C Virus (‘‘CNR3’’) was cloned into pLHPM and dominant nature as a tumor suppressor gene has been supported used as a control ribozyme. by the finding of frequent loss of heterozygosity (LOH) of the wild-type allele in breast tumors from mutation carriers (1, 3, 4). Reporter Vector Construction. To construct a BRCA1 promoter- Although mutational loss of function of BRCA1 does not seem driven enhanced green fluorescence protein (EGFP) reporter to contribute to sporadic cancers (5–7), BRCA1 mRNA levels are vector, a 2.9-kb fragment was amplified from a pGL2 vector reduced or undetectable in sporadic breast and ovarian carci- containing a 3.8-kb genomic BRCA1 PstI-fragment (19) using nomas and breast cancer cell lines (8–11). CpG methylation of primers BA-5Pst (5Ј-ATCTTTCTGCAGCTGCTGGCCCGG- the promoter may contribute to this decreased expression (12, 3Ј) and BA-3Age (5Ј-GTGTAAACCGGTAACGCGAAGAG- 13), but is unlikely to be the sole mechanism (14). Thus, the CAGATA-3Ј) and cloned into pEGFP-1 (CLONTECH). Vector regulatory pathway leading to the reduction of BRCA1 expres- pCMV-EGFP, with a CMV promoter driving EGFP gene ex- sion in sporadic breast cancer remains unclear. The identifica- tion of candidate genes in this pathway can potentially lead to the development of new therapeutic options. This paper was submitted directly (Track II) to the PNAS office. Here, we report a ribozyme-library-based ‘‘inverse genomics’’ Abbreviations: EGFP, enhanced green fluorescence protein; FACS, fluorescence activated approach to identify cellular regulators of BRCA1 expression. cell sorting; RT-PCR, reverse transcription–PCR; HLH, helix–loop–helix; Rz, ribozyme; VRz, Ribozymes are catalytic RNA molecules that bind to defined ‘‘validation’’ ribozyme. RNA targets based on sequence complementarity, and enzy- †Present address: Department of Pediatric Hematology and Oncology, Hannover Medical matically cleave these RNA targets. Typically, hairpin ribozymes School, D-30625 Hannover, Germany. cleave upstream of a GUC triplet in the target sequence, thereby §Present address: Department of Gastroenterology and Hepatology, Hannover Medical School, D-30625 Hannover, Germany. inhibiting the expression of the corresponding gene (15–17). A ʈ library of hairpin ribozymes with randomized substrate binding Present address: Invitrogen Corp., 1600 Faraday Avenue, Carlsbad, CA 92008. sequences could potentially cleave any RNA substrate contain- ††To whom reprint requests should be addressed. Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0665. E-mail: ing a GUC. Expression of the ribozyme library in cells and [email protected]. subsequent selection for a given phenotype enables the identi- The publication costs of this article were defrayed in part by page charge payment. This fication of single ribozymes responsible for this particular phe- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. notype. The target recognition sequence of ribozymes that §1734 solely to indicate this fact. 130–135 ͉ PNAS ͉ January 2, 2001 ͉ vol. 98 ͉ no. 1 Downloaded by guest on September 27, 2021 pression, was used as a control vector. The hygromycin reporter Construction of Id4 Expression Vectors. The Id4 cDNA sequence vector was constructed by inserting the hygro͞HSV-Tk fusion was amplified by using primers ID4-3 (5Ј-TCCGAAGGGAGT- gene from pCMV-HyTk into pcDNA3.1(Ϫ) (Invitrogen), gen- GACTAGGACACCC-3Ј) and ID4-7 (5Ј-TTCTGCTCTTC- erating the control vector pc-HyTk (18). The BRCA1-driven CCCCTCCCTCTCTA-3Ј). The amplified product was cloned HyTk plasmid pBR-HyTK was obtained by replacing the CMV with the retroviral expression vector pLPCX (CLONTECH) in promoter with a 2.9-kb genomic BRCA1 fragment (BglII–Sap I) sense- or antisense-orientation. containing the promoter region. Construction of Target Validation Ribozymes. Hairpin ribozyme Construction and Characterization of Reporter Cell Clones. PA-1 cells cleavage sites were identified within the sequence of the candi- were transfected with pBR-EGFP or pCMV-EGFP by using a date target gene according to target recognition sequence standard calcium phosphate transfection method. SK-BR-3 cells requirements (xxxxNGTCxxxxxxxx; x representing bases comple- were transfected with pBR-EGFP, pCMV-EGFP, pc-HyTk, and mentary to helices 1 & 2): GCGCNGTCCAGGTGTG [‘‘vali- pBR-HyTk; T47-D cells were transfected with pBR-EGFP and dation’’ ribozyme (VRz)1], GACCNGTCCAGCCGCG pCMV-EGFP by using Lipofectamine Plus (GIBCO͞BRL) ac- (VRz2). Ribozymes were constructed by annealing of overlap- cording to the manufacturer’s instructions. Following selection ping ribozyme specific oligonucleotides (20). Double-stranded DNA was cloned into pLHPM and retroviral particles were with G418 (GIBCO͞BRL), single colonies were functionally produced. analyzed by flow cytometry analyzing EGFP expression (EGFP reporter cells), or characterization for their resistance against RNA Analysis. Total RNA was extracted from cultures grown to hygromycin B (hygromycin reporter cells). The integrity of the ͞ 80% confluency and Northern blot analysis was performed as BRCA1 promoter EGFP cassette in SK-BR-3 cells transfected described elsewhere (18). For quantitative real-time reverse with pBR-EGFP was confirmed by PCR amplification. Clones transcription–PCR (RT-PCR) analysis, the TaqMan technology from SK-BR-3 cells transfected with pBR-HyTk were tested for ͞ (7700 Sequence Detector, Perkin–Elmer) was applied according the integrity of the BRCA1 promoter HyTk cassette by genomic to the manufacturer’s instructions for one-tube RT-PCR by southern blot. using the standard curve method. Primers and probes (IDT) were chosen with the help of PRIMER EXPRESS Software (Perkin– Viral Vector Production. Retroviral particles were produced by Elmer)—sequences