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Functional Retinoic Acid Response Element GORDON Vansantr AD WANDA F Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8229-8233, August 1995 Biochemistry The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element GORDON VANSANTr AD WANDA F. REYNOLDS* Sidney Kimmel Cancer Centert, 3099 Science Park Road, San Diego, CA 92121 Communicated by Roy J. Britten, California Institute of Technology, Corona del Mar, CA, May 22, 1995 ABSTRACT Alu repeats are interspersed repetitive DNA changes, most of the source gene sequence has been conserved elements specific to primates that are present in 500,000 to 1 throughout this period. An analysis of the known Alu se- million copies. We show here that an Alu sequence encodes quences indicates that mutations have been strongly sup-. functional binding sites for retinoic acid receptors, which are pressed at a number of positions, implying that Alu sequences members of the nuclear receptor family of transcription have sequence-dependent functions important for primate factors. The consensus sequences for the evolutionarily recent evolution (13). One postulated function is that these inserts Alu subclasses contain three hexamer half sites, related to the influence the expression of nearby genes (3). In support of this consensus AGGTCA, arranged as direct repeats with a spac- concept, the data presented here indicate that the consensus ing of 2 bp, which is consistent with the binding specificities sequences of evolutionarily recent Alu subfamilies contain of retinoic acid receptors. An analysis was made of the DNA binding sites for retinoic acid receptors (RARs), transcrip- binding and transactivation potential of these sites from an tional regulators that are present in most cell types and play Ala sequence that has been previously implicated in the important roles in development and cell growth (14-16). regulation ofthe keratin K18 gene. TheseAlu double half sites are shown to bind bacterially synthesized retinoic acid recep- AND tors as assayed by electrophoretic mobility shift assays. These MATERIALS METHODS sites are further shown to function as a retinoic acid response The DNA sequence preceding the keratin K18 gene, including element in transiently transfected CV-1 cells, increasing tran- the proximal Alu element, has been reported (ref. 7 and scription of a reporter gene by a factor of -35-fold. This references therein). Plasmid constructions are detailed in the transactivation requires cotransfection with vectors express- figure legends. The procedures used for CV-1 cell culture, ing retinoic acid receptors, as well as the presence of all-trans- transfection assays, and gel shift assays have also been de- retinoic acid, which is consistent with the known function of scribed (7, 17-19) and are detailed in the figure legends. retinoic acid receptors as ligand-inducible transcription fac- tors. The random insertion of potentially thousands of Alu RESULTS AND DISCUSSION repeats containing retinoic acid response elements through- out the primate genome is likely to have altered the expression Alu Sequences Contain Consensus Hormone Response El- of numerous genes, thereby contributing to evolutionary ements (HREs). This study began as an investigation into the potential. role of an upstreamAlu element in the regulation of the human keratin K18 gene. This Alu confers copy number-dependent The genomes of most higher eukaryotes contain repetitive expression to the K18 gene in transgenic mice, suggesting that DNA elements derived from genes transcribed by RNA poly- it insulates the associated gene from negative effects of merase (pol) III (1, 2). These interspersed repetitive sequences sequences surrounding random insertion sites (7). This ele- were initially proposed to represent regulatory networks, ment is also coincident with a DNase I hypersensitive site, allowing the coordinate expression of multiple, unlinked genes which correlates with K18 transcriptional activity (20). We (3). Further analysis indicated considerable interspecies vari- examined the K18-associated Alu element for possible regu- ation in these DNA sequences and in their sites of insertions, latory sites and found several potential binding sites for RARs which argued against a fundamental role in gene regulation (Fig. 1A). Since the mouse K18 homolog is retinoic acid (RA) and gave rise to the concept that interspersed repetitive inducible in embryonal carcinoma cells (23), as is the human sequences are selfish DNA with no function or selective K18 gene (data not shown), this suggested the Alu element advantage to the organism (4). In support of a regulatory might be involved in this regulation. function, recent findings indicate that certain of the primate- RARs are members of the nuclear receptor superfamily of specificAlu repeats are involved in tissue-specific regulation of ligand-activated transcription factors, which also includes re- nearby genes (5-7). ceptors for steroid hormones, thyroid hormone, glucocorti- Alu elements are functional pol III genes with internal A and coids, and vitamin D (14-16). There are three forms of RARs B box promoter elements and are probably derived from 7SL (RARa, -4, -y) and three forms of retinoid X receptors genes. The Alu sequences have been amplified and reinserted (RXRa, -3, -y). These receptors bind most typically as RAR- throughout the genome by a retroposition process involving a RXR heterodimers to two adjacent HREs consisting of vari- RNA intermediate. A few highly conserved source genes ants of the consensus sequence AGGTCA. The consensus produce the transcripts, which serve as intermediates for HRE sequence shown in Fig. 1A was deduced from a com- retroposon formation (8). During the preceding 30-60 million pilation of naturally occurring and experimentally derived years of primate evolution, a succession of source genes has recognition sequences (14, 21). Many naturally occurring given rise to extensiveAlu subfamilies whose members share a HREs deviate at one or more positions from this motif few common diagnostic base changes, indicative of mutations in the parental source gene (8-12). Except for these few base Abbreviations: pol, RNA polymerase; HRE, hormone response ele- ment; RA, retinoic acid; DR2, HREs with a spacing of 2 bp; RARE, RA response element; CAT, chloramphenicol acetyltransferase; The publication costs of this article were defrayed in part by page charge RAR, retinoic acid receptor; RXR, retinoid X receptors. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. accordance with 18 U.S.C. §1734 solely to indicate this fact. tFormerly San Diego Regional Cancer Center. 8229 Downloaded by guest on September 29, 2021 8230 Biochemistry: Vansant and Reynolds Proc. Natl. Acad. Sci. USA 92 (1995) A (shown in Fig. 1B) are made up of those residues that appear most frequently at each position for members of that class and I A box B box r are thus thought to represent the sequence at the time of -004 bi insertion and therefore the probable sequence of the parental 1 2 3 4 source genes. Class I, the oldest subfamily, is most similar to B Box promoter the 7SL sequence (11) and contains HRE 2 as well as HRE 4, AGGTGG GC GGATCA CG AGGTCA GG GATCG AGAC TCCGT but it lacks two adjacent HREs that fit the consensus with only one base deviation. The class II earlyAlu subfamily represents so 8 of Alu relative number of HRE conensus AGGTCA the majority repeats (estimated G T GG 437,000 copies, based on an estimate of 750,000 total copies; ref. 9). An excellent HRE 3 appears in this subclass, separated B cons QOTTCGANrCC from HRE 4 by 2 bp, resulting in one potential DR2 binding B box promotr site. HRE 2 and HRE 3 remain separated by 4 bp, as in the 7SL 30.1 AGGTGG ga GGaTCG cttg AG ccCA AGTTC t gggct class IAlu and the 7SL sequence. However, in the more recent c , ii 7SL la Ct T Alu classes, III and IV, a 2-bp deletion between HRE 2 and 3 94.s 1700Earl Aka AGG ctG GGaTCG AG ccCA changed the spacing from 4 to 2 bp, such that there are three class I gp ctg ng AGTTCG agacc direct repeat HREs with a 2-bp spacing (DR2), representing 437,000 Aku clss 11 AGG ctG ga GGTCG ctig AGGTCA gg AGTTCG agacc two potential dimer sites, HRE 23 and HRE 34. Certain Alu 136,000 RecentAu AGGcGG gc G cg AGGTCA gg AGaTCG agacc as dases Il-IHV elements within this recent family, such the K18-associated - Alu, also contain HRE 1 (present in 7SL30.1 but not class IAlu KIS Akl AGGTGG gc GGaTCA cg AGGTCA gg AGaTCG agacc HRE1 2 3 4 sequences), resulting in four HRE arranged as DR2, repre- senting three potential dimer sites, HRE 12, 23, and 34. Thus, FIG. 1. Alu elements contain several consensus HREs. (A) A the majority ofAlu elements (class II, relative copy number schematic representation of an Alu gene (-281 bp) indicates the 437,000; ref. 9) contain two adjacent HREs making up one relative positions of the A and B box pol III promoter elements and potential DR2 receptor binding site, whereas the more evo- the several potential HREs present in the K18-associated Alu se- lutionarily recent Alu subfamilies (classes III and IV, relative quence. The K18-associatedAlu sequence including the several HREs copy number 136,000; ref. 9) have at least three HREs, (arrows) is shown below. The fourth HRE overlaps with the B box making up two DR2 sites. promoter element (boxed). The HREs are arranged as a 2-bp spacing The Alu HREs Constitute Functional Binding Sites for (DR2). The HRE consensus sequence is derived in part from site- To determine if the HREs functional selection gel shift assays (21). (B) Acquisition of HREs during RARs. K18Alu represent evolution ofAlu elements from 7SL genes. The sequences of the HRE binding sites for RARs, gel shift assays were performed.
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