Widespread Alu repeat-driven expansion of consensus DR2 retinoic acid response elements during primate evolution
© Laperriere et al; licensee BioMed Central Ltd. 2007
Received: 14 September 2006
Accepted: 19 January 2007
Published: 19 January 2007
Nuclear receptors are hormone-regulated transcription factors whose signaling controls numerous aspects of development and physiology. Many receptors recognize DNA hormone response elements formed by direct repeats of RGKTCA motifs separated by 1 to 5 bp (DR1-DR5). Although many known such response elements are conserved in the mouse and human genomes, it is unclear to which extent transcriptional regulation by nuclear receptors has evolved specifically in primates.
We have mapped the positions of all consensus DR-type hormone response elements in the human genome, and found that DR2 motifs, recognized by retinoic acid receptors (RARs), are heavily overrepresented (108,582 elements). 90% of these are present in Alu repeats, which also contain lesser numbers of other consensus DRs, including 50% of consensus DR4 motifs. Few DR2s are in potentially mobile AluY elements and the vast majority are also present in chimp and macaque. 95.5% of Alu-DR2s are distributed throughout subclasses of AluS repeats, and arose largely through deamination of a methylated CpG dinucleotide in a non-consensus motif present in AluS sequences. We find that Alu-DR2 motifs are located adjacent to numerous known retinoic acid target genes, and show by chromatin immunoprecipitation assays in squamous carcinoma cells that several of these elements recruit RARs in vivo. These findings are supported by ChIP-on-chip data from retinoic acid-treated HL60 cells revealing RAR binding to several Alu-DR2 motifs.
These data provide strong support for the notion that Alu-mediated expansion of DR elements contributed to the evolution of gene regulation by RARs and other nuclear receptors in primates and humans.
Alu repeats are SINEs (Short INterspersed Elements) whose original insertion in genomic sequences appears to have occurred shortly after the dawn of the primate lineage. While most SINEs arose from tRNA genes, Alu elements are derived from the 7SL RNA gene, which encodes a component of the protein signal recognition complex. The structure of modern Alu sequences arose from a duplication of primordial elements, which were approximately 200 bp in length and composed of an RNA polymerase III promoter at one extremity and a polyA tail at the other. Dimeric Alu elements expanded extensively throughout primate genomes as a result of a parasitic relationship with the transposition machinery encoded by L1 retrotransposons, which are LINEs (Long INterspersed Elements) [1, 2], and today occupy ~10% of the human genome.
Several lines of evidence indicate that a variety of events associated with Alu transposition, recombination and expansion have contributed to genome evolution and to altering gene expression by several mechanisms. Because of their high CpG content and the tendency of CpG dinucleotides to mutate, and because of their prevalence, Alu repeats contain a substantial percentage of the single nucleotide polymorphisms in the human genome . Their polyA tails act to seed microsatellite formation and expansion . Moreover, Alu transposition has been proposed to underlie the expansion of segmental duplications that comprise approximately 5% of the human genome . Conservative estimates suggest that 0.3–0.5% of human genetic disorders arise from mobile element insertion or from element-driven recombination events [5, 6]. There is also evidence that Alu repeats inserted in an antisense orientation to gene transcripts can introduce alternative splicing sites [7, 8], and that insertion of Alu sequences may alter epigenetic regulation of gene expression .
In addition to the above, increasing evidence suggests that Alu repeats are a source of elements that regulate transcriptional initiation by RNA polymerase II .
We have been interested in genome-wide mapping of hormone response elements in the promoter-proximal regions of human genes [11, 12] to study their evolutionary conservation in the vicinity of genes, and their accessibility to cognate members of the nuclear receptor family. Nuclear receptors are the primary targets of a range of lipophilic signaling molecules such as steroid and thyroid hormones, vitamin D, retinoids, specific prostaglandins and cholesterol metabolites that regulate many aspects of physiology and metabolism, as well as embryonic development . They directly regulate gene transcription by ligand-dependent recruitment of so-called coregulatory proteins that carry out the histone modifications, chromatin remodeling and binding of RNA polymerase II and ancillary factors necessary for initiation of transcription [13, 14].
Nuclear receptors are composed of a series of domains, with the DNA binding and ligand binding domains being the most highly conserved. Most nuclear receptors function as homo- or heterodimers, and many receptor DNA binding domains recognize variants of consensus RGKTCA motifs arranged as either direct or inverted repeats. Many non-steroid receptors recognize direct repeats in the form of heterodimers with members of the retinoid × receptor (RXR) family. For example, retinoic acid receptors (RARs) heterodimerized with RXRs specifically recognize retinoic acid response elements (RAREs) as RGKTCA direct repeats separated by 1, 2 or 5 bp (DR1, DR2 or DR5 motifs), whereas heterodimerized receptors for vitamin D (VDR) and thyroid hormone (TR) recognize DR3 and DR4 elements, respectively.
Evidence is accumulating that Alu repeats constitute a significant source of hormone response elements . Notably, a motif, AGGTCAnnAGTTCG, found within most subclasses of AluS sequences, corresponds to a non-consensus DR2 element recognized by RARs, and has been shown to function as a RARE . Signaling through RARs is of particular interest because retinoids control many aspects of embryonic development in a wide variety of organisms [13, 18, 19].
Here, we used a genome-wide screen to map DR-type hormone response elements in the human genome, and found that consensus DR2 elements are massively overrepresented relative to DR1 and DR5 RAREs and DRs recognized by other nuclear receptors, due to the presence of consensus DR2 motifs in >100,000 Alu repeats. The vast majority of these correspond to AluS sequences, which drove the Alu element expansion in primate that started 35–40 Myr ago. We also show that these "Alu-DR2" elements are present in retinoic acid regulated genes and bind RARs in vivo. Taken together, our data provide strong support for the notion that Alu-mediated expansion of DR elements contributed to the evolution of gene regulation by RARs and other nuclear receptors in primates and humans.
Mapping DR-type hormone response elements in the human and mouse genomes
Consensus RGKTCA direct repeats found in the human and mouse genomes.
outside transposable elements
Over 100,000 consensus DR elements are present in Alu repeats
RGKTCA direct repeats found in Alu elements.
# in Alu
% in DR1
% in DR2
% in DR3
% in DR4
% in DR5
The 102,359 DR2 elements, which are RAR target sequences, are by far the most frequent consensus DR motif present in Alu repeats (93.4%, Fig. 1), followed by DR4 elements, recognized specifically by TRs (5.7% or 6198 occurrences, Fig. 1). It is noteworthy that the 6198 Alu-DR4 elements represent ~50% of the total consensus DR4 elements in the genome (Fig. 1). Moreover, unlike in the DR2 consensus, RGGTCA motifs are found predominantly in the 3' repeat of the Alu DR4 sequences (Table 2, DR4 column). This position is bound by the TR when RXR-TR heterodimers bind to DR4 motifs. Since the TR has weak affinity for RGTTCA but recognizes RGGTCA motifs , these DR4 elements represent optimal TR binding sites.
The vast majority of Alu-DR elements are present in AluS sequences
Consensus Alu-DR2 elements arose predominantly via deamination of a methylated CpG dinucleotide
The distribution and frequency of DR2 elements in Alu sequences can be explained by the fact that the consensus DR2 RARE differs from the consensus sequence of several AluS subfamilies by a single G to A substitution in the 3'-most nucleotide of the DR2 repeat, whereas generation of an RARE consensus from Alu J or Y sequences would require multiple substitutions (Fig. 3B). Similarly, a single A to G or T substitution in the third position of the upstream GGATCA motif in AluS subfamilies Sx, Sq and Sp would generate a consensus DR4 element. Note however that although two different single mutations can lead to formation of a consensus DR4 versus one single replacement leading to a consensus DR2, the rate of conversion to a consensus DR2 is far superior to that to a DR4 element in all Alu subclasses.
It is noteworthy that generation of the DR2 RARE consensus could occur through deamination of a methylated C residue in the CpG dinucleotide of the AluS consensus, converting it to a T on the complementary strand of the direct repeat. A comparison of the frequencies of all four possible CpN dinucleotides in AGGTCAnnAGTTCN motifs present in AluS sequences indicates that CpA occurs far more frequently than CpT or CpC (Fig. 3C). Similarly, the conversion to T of the C residue of the CpG on the sense strand occurred far more frequently than other substitutions (Fig. 3C). Collectively, these data support the notion that the CpA dinucleotide in the DR2 element arose predominantly via a deamination-driven mechanism rather than by random base substitutions. The data are also consistent with other observations of high rates of CpG methylation  and of CpG dinucleotide decay generally in Alu repeats [22, 23]. Further, the rate of conversion to consensus DR2s was similar in different subclasses of AluS elements differing by only one nucleotide (about 15–20% except for the AluSg1, a minor subfamily of AluSg , for which the rate of conversion is about 60%, Fig. 3B), suggesting generation of DR2s via the same methylation/deamination mechanism throughout evolution of the family. In addition, it is worth noting that the conversion to a perfect DR2 from the consensus AluS sequence, while introducing a change in the sequence of the box B of the RNA polIII promoter, occurs without altering its consensus (GWTCRANNC), suggesting that Alu elements containing perfect DR2 may still be mobile . In a recent paper Price et al. defined over 200 AluS subfamilies , seven of which contain consensus DR2 elements. One of these, AluSg_14 shares the presence of the consensus RARE DR2 with three subfamilies [see Additional file 4]. These AluS subfamilies are likely examples of DR2 elements amplified by transposition instead of spontaneous mutation of a CpG.
Distribution of Alu-DR2 elements relative to the 5'ends of human genes
Association of RARs with Alu-DR2 elements in vivo
It is highly likely that most of the genes with proximal Alu-DR2 elements are not implicated in RAR-dependent gene regulation in a given cell type, with access to the sites being potentially limited by chromatin structure or binding of other proteins. For example, chromatin immunoprecipitation studies followed by microarray analysis (ChIP-on-chip) of genes regulated by related estrogen receptors in estrogen-sensitive breast carcinoma cells have suggested that only a minority of potential high affinity estrogen response elements of a given chromosome are accessible to receptors . Moreover, as mentioned above, Alu repeat sequences are highly susceptible to methylation , which correlates with a closed chromatin structure and transcriptional silencing. However, 363 Alu-DR2 were found near 193 known retinoic acid target genes [see Additional file 2], suggesting that some Alu-DR2 elements can drive retinoid-regulated gene expression. We therefore tested the accessibility of several of these Alu-DR2 sequences to RARs by chromatin immunoprecipitation (ChIP) assay. We chose multiple elements present in the vicinity of 5 genes known to be RA-responsive in cells of squamous epithelial origin, RAI1, GPRC5A (RAI3), SMYD5 (RAI15), RARRES1 and RARRES3, and assessed RAR binding to these elements in SCC25 cells, a relatively well-differentiated human head and neck squamous cell carcinoma (HNSCC) line . SCC25 cells express RARs and are retinoid-responsive , and are arrested by RA in the G0/G1 phase of the cell cycle . The genes RAI1 and GPRC5A were originally identified as RA-regulated genes in HNSCC . Similarly, SMYD5 and RARRES 1 and 3 (retinoic acid receptor responder genes 1 and 3) were found to be induced by RA in human keratinocytes . RARRES3 expression is lost in many tumors, and its product inhibits cell proliferation when transiently overexpressed .
Alu-DR2 elements within ENCODE ChIP-on-chip RARα binding regions.
RARα binding region
DR2 position in region
Nuclear receptor signaling, particularly that of retinoic acid receptors, has been widely studied in animal models, most notably the mouse. Our findings reveal striking differences in hormone response element frequency and distribution between rodent, and primate and human genomes. Results of the genome-wide mapping of high affinity hormone response elements for members of the nuclear receptor family of transcription factors presented herein indicate that consensus DR2 retinoic acid response elements are vastly overrepresented relative to other DR response elements in the human genome, and are enriched approximately 4-fold in the human genome relative to the mouse. This enrichment arose as a result of the presence of DR2 motifs in Alu repeats, which are primate-specific. Movement of Alu repeats and other classes of transposable elements has been recognized as a potential mechanism for introduction of RNA polymerase II transcriptional regulatory elements for a variety of transcription factors into promoter regions of genes [10, 35, 36], thus altering the signal transduction pathways regulating specific genes. For example, a recent study  used DNA-binding profiles of representatives of different classes of transcription factors from the TRANSFAC database  to identify transcription factor binding sites in transposable elements located in promoter regions. This study did not screen for RAREs, for which matrices are not provided in the TRANSFAC database.
While the presence of imperfect RAREs in Alu elements and their capacity to function as RAREs in reporter assays has been reported previously , the main findings from our study are the unexpected large number of consensus DR2 RAREs, and the demonstration that several of these sites are functional RAR binding sites in vivo. It is striking that the genome-wide number of imperfect DR2s corresponding to the AGGTCAnnAGTTCG consensus, which matches the consensus sequences of most AluS subclasses, is not much higher (102,657) than that of the consensus DR2s (93,278), underlining the unexpectedly high number of perfect DR2 RAREs in the human genome (Fig. 3C). The generation of consensus DR2 elements by deamination of the CpG dinucleotide within the 3' AGTTCG motif is supported by the comparably high number of AGGTCAnnAGTTTG sequences (80,444), which diverge from a perfect DR2 more than the motif found in consensus AluS sequences but represent the other possible site generated by methylation/deamination at the CpG dinucleotide in the B box of Alu repeats.
Demonstration that Alu DR2 RAREs are functional binding sites in their normal chromatin context is crucial given the high frequency of methylation of Alu sequences in the human genome , and the association between DNA methylation and silencing of transcription in general, and of Alu repeats specifically . For instance, expression and retinoic acid responsiveness of one of the genes studied above, RARRES1, is frequently inhibited in malignancies by hypermethylation , and the extent of methylation is inversely correlated with the state of differentiation of malignant cells .
The capacity of Alu-DR2 motifs to function as RAREs is supported by several lines of evidence. We identified 363 motifs within 10 kb of the 5' ends of 193 known retinoic acid-regulated genes [see Additional file 2]. It is also noteworthy that 8 Alu-DR2 within 10 kb of 9 genes overlap with DNase I-hypersensitive sites from primary human CD4+ T cells  [see Additional file 2]. We confirmed the capacity of several Alu-DR2 elements to bind RARs in vivo in retinoic acid-sensitive SCC25 cells by chromatin immunoprecipitation analysis of receptor binding to sequences located proximal to five genes known to be retinoic acid-responsive in squamous carcinoma cells. These studies indicate that access of RARs to the DR2 motifs under study was not impeded by their presence in an Alu repeats in these cells. Moreover, our analyses of ChIP-on-chip studies from the ENCODE consortium showed that several Alu-DR2 elements in human HL60 cells are sites of RA-dependent recruitment of RARs (Table 3), further supporting the function of Alu-DR2 elements as RAREs.
We found that DR2 consensus elements were present in substantial numbers in multiple AluS subfamilies, indicating that they arose throughout the course of AluS expansion. The perfect RARE DR2 motif corresponds to the consensus sequence in seven divergent AluS subfamilies defined by Price et al. , in addition to being found sporadically throughout numerous other AluS subfamily sequences (Additional file 4 and unpublished results). It should be noted that the downstream half-site of the DR2 element lies within box B of the Alu RNA polymerase III promoter, but that the conversion of RGTTCG to RGTTCA to generate the consensus DR2 motif does not necessarily impair its competence for retrotransposition, consistent with transcriptional activity of Alu RNA polymerase III promoters containing RGTTCAANNC box B motifs [25, 42].
Consistent with their presence predominantly in AluS sequences, which represent relatively ancient Alu elements, we found that the vast majority of human Alu-DR2 motifs are present in the chimp and macaque genomes. A few Alu-DR2 elements were apparently unique to the human genome, although in some cases they corresponded to regions that were not sequenced in the simian genomes. While none of these uniquely human Alu-DR2s are adjacent to known retinoid-regulated genes in humans, it will be of interest in the future to characterize the potential of RA to regulate their expression in appropriate cell model systems. It is also noteworthy that one of these genes, CYP2A6, was shown to be regulated by the nuclear receptor hepatic nuclear factor 4 (HNF4) , which, like RARs, can recognize DR2 motifs .
Our previous work identified a consensus DR3 element recognized by the VDR in vitro and in vivo in the proximal promoter region of the cathelicidin antimicrobial peptide gene , which was found to be strongly regulated by vitamin D in a variety of human cell types. However, neither the regulation nor the response element was conserved in mouse, and studies by others  showed the DR3 to be conserved in primates, but not other species, and present in an Alu repeat. This represents a clear example of an Alu-driven divergence in nuclear receptor gene regulation between primates/humans and other species. Results presented here demonstrate that Alu repeat expansion similarly has led to the generation of functional binding sites for retinoic acid receptors. Further studies of DR2-containing Alu repeats will be necessary to determine precisely to which extent these elements have contributed to the primate-specific regulation of target genes by retinoic acid.
We find that consensus DR2 motifs are heavily overrepresented in the human genome relative to other DR response elements due to their presence in a subset of Alu motifs, in particular in AluS sequences. Although DR4s are found far less frequently in Alu repeats, those present account for 50% of consensus DR4 motifs in the human genome. Consensus Alu-DR2 elements arose predominantly through deamination of a methylated CpG dinucleotide present in AluS elements rather than through random base substitutions. While Alu elements can be transcriptionally repressed by methylation-driven mechanisms, our screen identified Alu-DR2 elements in promoter proximal regions of numerous retinoid regulated genes. Importantly, we found that Alu-DR2s are accessible to RARs in vivo in two RA-responsive human cell lines, indicating that the expansion of DR2 motifs within Alu elements during the course of primate evolution contributed to altering regulation of gene expression by retinoic acid.
Genome-wide identification of DR1-DR5 within Alu sequences
The human (hg17, May 2004) and mouse (mm6, March 2005) reference sequence chromosomes from the UCSC Genome Browser database [47, 48] were scanned for RGKTCA motifs separated by 1 to 5 bp (DR1-DR5) using a previously described program perl program [11, 12]. The RefSeq Genes track , downloaded September 21 2005, was used to identify the DR2 located -10 to +10 kb of gene 5'-ends. A custom SQL program used the chromosomal location of Alu sequences from the RepeatMasker track  to identify included DR1-DR5. The same program used the chromosomal location of regions bound by RARα from the ENCODE Affy Sites track to identify DR2 elements within these regions. Another SQL program was used to identify the DR2 in AluY that are not included within Human/Chimp (hg17 vs panTro1) and Human/Rhesus (hg17 vs rheMac2) pairwise BLASTZ alignment . The χ2 test to compare the distribution of Alu-DR2 to Alu repeat was carried out with the chisq.test function of the R statistical computing environment . The cladogram of AluSg subfamilies was generated with PHYLIP 3.6  using the consensus of AluS families from Repbase  and AluS subfamilies with RARE DR2 in their consensus from Price et al . All programs were run on the bioinformatics cluster of The Quebec Bioinformatics Network (BioneQ) and are available upon request.
Approximately 2 × 107 SCC25 cells were treated for 1 h with RA 10-6M or vehicle. Then cells were rinsed with PBS and incubated for 10 min in PBS containing 1% formaldehyde at 37°C. Cells were washed with ice-cold PBS and the cell pellets were resuspended in 300 μl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1) and 1× protease inhibitor cocktail (Roche Diagnostics, Laval, Que, Canada). Sonication was performed by pulsing three times for 15 s to reduce DNA to fragments of ~300–500 bp in length. Centrifugation was then performed to obtain cleared cell lysates. Lysates were diluted 1:10 in ChIP dilution buffer (1% Triton X-100, 1 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl pH 8.1). A portion of the lysate was taken as INPUT control sample. Pre-clearing was done by incubating the cell lysates with salmon sperm DNA (1 μg/ml), BSA (1 μg/ml) and protein A-agarose slurry (Santa Cruz Biotechnology, Santa Cruz CA) for 1 h. Cell lysates were then incubated by overnight rotation at 4°C with 4 μg of either normal rabbit IgG or primary antibody, followed by rotation-incubation with protein A-agarose for 2 h. The beads were rinsed three times sequentially with TSE I buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 150 mM NaCl; 20 mM Tris-HCl, pH 8.1), TSE II buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 500 mM NaCl; 20 mM Tris-HCl, pH 8.1), LiCl Wash Buffer (0.25 M LiCl; 1% NP-40; 1% deoxychlolate (Na salt); 1 mM EDTA; 10 mM Tris-HCl, pH 8.1) and 2 times TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0). The beads were eluted twice with 250 μl elution buffer (1% SDS; 0.1 M NaHCO3). To reverse the cross-linking, the eluates were incubated in a 65°C incubator overnight and then the samples were purified with PCR purification kit (Qiagen, Mississauga, Ont, Canada). PCR primers used for ChIP assays are described in Additional file 3.
List of abbreviations
hepatic nuclear factor 4
long interspersed elements
retinoic acid receptor
retinoic acid response element
retinoid × receptor
Short INterspersed Elements
thyroid hormone receptor
vitamin D receptor.
This work was supported by an operating grant from the Canadian Institutes of Health Research to JHW and SM (MOP-74571) and by a grant from the Natural Sciences and Engineering Research Council of Canada to SM. JHW and SM are Chercheurs-boursier of the Fonds de Recherche en Santé du Québec. DL was supported by a scholarship from a CIHR Strategic Training Program Grant in Bioinformatics.
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