To test the power of primate shadowing to identify functional elements in large genomic intervals, we sequenced the primate orthologs of eight human loci containing LXRα and eight of its target genes: SREBF1, CYP7A1, LDLR, ABCG5, ABCG8, APOE cluster, APOCIII cluster, and HMGCR. The sequenced species comprised six anthropoid primates (baboon, colobus, dusky titi, marmoset, owl monkey and squirrel monkey) and one prosimian (lemur). The targeted genomic segments included all exons, introns and flanking intergenic regions of the above mentioned genes, encompassing 558 Kb of human genomic DNA.

We identified sequences evolutionarily conserved among seven anthropoid primates (the six targeted anthropoids plus human) or among all eight primates (anthropoid plus lemur) using Gumby, an algorithm that detects sequence blocks evolving significantly more slowly than the local neutral rate 3910. Most of the conserved regions overlapped exons (see, for example, Figure 1). The true-positive rate, defined as the fraction of conserved regions overlapping exons or known regulatory regions, was 80% in the 7-primate comparison (Figure 2a) and 81% using the 8-primate set (data not shown). The human-dog comparison, which approximately matches the combined branch length of the primate comparison, had a similar true-positive rate of 84% (Figure 2c). The more distant human-mouse comparison displayed a marginally higher true-positive rate of 90% (Figure 2b). This is consistent with the theoretical prediction that statistical power increases with the total branch length of the species set 11. It should be noted, though, that regulatory sequence annotation of the eight loci we analyzed is probably highly incomplete. Therefore, these true-positive rates are lower bounds; some or all of the 'false positives' could eventually be reassigned as true positives upon expansion of the set of sequences annotated as cis-regulatory. Thus, due to incomplete annotation of functional elements, it is not clear if the difference between the primate and human-mouse true positive rates reflects a significant difference in reliability between the two sets of predictions. On average, 64% of the exons in the 8 loci overlapped conserved regions in both the 7-primate and 8-primate comparisons. Similar sensitivity was obtained in human-mouse (65%) and human-dog (71%) comparisons. Thus, primate sequence comparison was approximately equivalent to pairwise human-mouse or human dog analysis in identifying exons.

To identify cis-regulatory sequences not detectable in comparisons between human and distant mammals, we searched the 8 gene loci for non-coding sequences highly conserved among primates (p value ≤ 0.005) but not detectable in human-mouse or human-dog comparisons (p value > 0.1). Gumby analysis of human and six other anthropoid primates identified six anthropoid-primate-conserved non-coding regions (Additional data file 4). These sequences were either undetectable (p value > 0.1) or less significantly conserved (p value > 0.005) when the prosimian lemur was included in the primate set (data not shown).

To independently confirm the (anthropoid) primate-specific nature of sequence conservation in these six regions, we compared their nucleotide substitution rate to that of non-exonic sequences in the same locus. Evolutionarily conserved sequences are defined by a constraint factor (ratio of the substitution rate of test sequences to the non-exonic average) smaller than 1. We found that the six primate-conserved sequences had constraint factors well below 1 among anthropoid primates, as expected, and much closer to 1 in the human-mouse and human-dog comparisons (Figure 3). Finally, none of the 6 sequences overlapped significantly conserved segments identified by the phastCons program 12 in a 17-species alignment of the human genome to (mostly) non-primate mammals and more distant vertebrates 13, which further confirms the primate-specificity of their evolutionary conservation.

To explore the potential regulatory function of these primate-conserved elements, we examined their ability to drive reporter gene expression in both a transient transfection assay in human HepG2 cells and an in vivo mouse liver gene transfer assay. Since it is possible that the computational prediction only captures part of the entire regulatory module, each human element plus 200 to 400 base-pairs (bp) of flanking sequence on either side was cloned upstream of the human promoter of the gene closest to each element and fused to a luciferase reporter gene (see Materials and methods). Therefore, the included flanking sequences may also contribute to the observed regulatory activity. Two elements showed enhancer activity, increasing the expression of a luciferase reporter gene 1.6- to 5-fold in both the human liver cell line HepG2 and in vivo in mouse liver, while a third element appeared to be a silencer, suppressing luciferase expression by 50% (Figure 4, Table 1 and data not shown). While a third element, LDLR_PS2, showed modest enhancer activity in HepG2 cells (approximately 1.6-fold increase over promoter alone), its activity in 293T cells was much stronger (approximately 5-fold increase over promoter alone), presumably due to availability in these cells of appropriate transcription factors, such as SREBFs, that are capable of activating LDLR 14. In an independent assay of transcription potential, both enhancer elements were shown to be DNase I hypersensitive sites in HepG2 cells (Figure 4, Table 1 and data not shown), suggesting that the corresponding DNA elements are involved in transcriptional regulation of the endogenous genes. To confirm that the primate orthologs of these identified human regulatory elements are also functional, we cloned the aligned LDLR PS4 sequences from baboon, dusky titi, marmoset and lemur into the luciferase reporter vector and tested their ability to drive reporter gene expression in HepG2 cells. All orthologous non-human primate sequences showed enhancer activity (Additional data file 3).

Since these functional human regulatory elements exhibited primate-specific sequence conservation, we explored whether their functional role is unique to primates. Although human-mouse comparison failed to identify these sequences as constrained (Gumby p value > 0.1; Figures 1 and 3b), we were able to identify the aligned counterparts to the three primate-conserved functional sequences in mouse using the global alignment program MLAGAN 15. To explore the regulatory function, if any, of these mouse orthologs, we cloned the aligned sequences into the luciferase reporter vector described above and compared their activity to that of the human sequence. Despite the lack of statistically significant conservation between the rodent and human sequence, all three mouse orthologs exhibited regulatory activity in the same direction to that observed for the human elements (data not shown). Thus, the silencer and the two enhancers identified through primate-specific sequence conservation are ancestral mammalian regulatory elements, rather than newly evolved functional regions specific to primates.

The primate set we used to identify known functional elements and the three divergent mammalian regulatory sequences comprises human, baboon, colobus, marmoset, squirrel monkey and owl monkey. However, it is unlikely that all the corresponding genome sequences will be available in the near future. Of the set of most informative primate genomes for comparative analysis 6, the human and rhesus genome sequences are already publicly available, and the marmoset genome is currently being sequenced. We tested whether comparisons among these three species were sufficient to detect functional sequences in the eight lipid gene loci. Human-rhesus-marmoset comparison identified 55% of the 160 exons (versus 7 primates: 64%) with a true-positive rate of 72% (79% for 7 primates) (Figure 2a,d), suggesting that a significant fraction of exons can be detected using a limited number of primates 16. We subsequently assessed the ability of human-rhesus-marmoset comparison to detect the three newly identified regulatory sequences. As was observed in the comparison of 7 anthropoid primates, both LDLR enhancers were highly conserved (p value <0.005) in the 3-way primate analysis and ranked among the three most conserved non-coding sequences in the 75 kb genomic region (data not shown). The smaller set of 3 primates was also sufficient to detect the silencer in the SREBF1 locus (non-coding rank 1), though not as strongly (p value = 0.044; Figure 1c). The lower statistical significance of the SREBF1 silencer in the three-primate analysis relative to the seven-primate analysis is due to the lower combined branch length of the former. These results suggest that availability of the marmoset genome sequence will facilitate genome-wide analysis of primate-specific conservation, and uncover regulatory sequences that are undetectable in distant, non-primate comparisons.

Primate bacterial artificial chromosome (BAC) clones containing targeted loci were purchased from Children's Hospital Oakland Research Institute in Oakland, California 20. Draft sequences of baboon, colobus, dusky titi, marmoset, owl monkey, squirrel monkey, and lemur BACs were determined by sequencing ends of 3 kb subclones to 8- to 10-fold coverage using BigDye terminators (Applied Biosystems, Foster City, CA, USA) and assembling reads into contigs with the Phred-Phrap-Consed suite as described previously 21. All BAC sequences were submitted to GenBank (see Additional data file 6 for accession numbers). Human, mouse, dog and rhesus sequences were downloaded from the UCSC Genome Bioinformatics worldwide web site 13. Based on the human March 2006 assembly hg18, the coordinates for the analyzed human loci are: SREBF1, chr17:17653939-17690020; CYP7A1, chr8: 59525742-59605413; LDLR, chr19:11054146-11127904; ABCG5/ABCG8, chr2:43835998-43966668; APOE cluster, chr19:50080832-50149764; APOCIII cluster, chr11:116137283-116217351; HMGCR, chr5:74646522-74714122; LXRα, chr11:47231580-47253367. Exon annotations of these regions were obtained from the UCSC Genome Bioinformatics website 13. The promoter sequence of a gene is defined as the 1 kb region upstream of the transcription start site. Four enhancers in the APOE locus were previously described 2223. These four APOE enhancers, together with 15 promoters in the 8 genomic loci, comprise the set of known regulatory regions.

All sequence alignments were carried out using MLAGAN 15. Aligned sequences were scanned for statistically significant (p value ≤ 0.1) evolutionarily conserved regions using Gumby 3910. We defined primate-specific conserved elements as those human sequences that were highly conserved (Gumby p value ≤ 0.005) among anthropoid primates, but not conserved in more distant mammalian comparisons. Mammalian sequence conservation was defined as: p value ≤ 0.1 in human-mouse or human-dog comparison; or 70% human-mouse sequence identity over at least 100 bp 24; or significant conservation in an alignment of 17 vertebrate genomes 12.

Evolutionarily conserved regions identified by Gumby were visualized using RankVISTA 25. Conservation scores in the RankVISTA plots were calculated as the negative logarithm of the Gumby p value.

The constraint factor of a conserved non-coding sequence (CNS) was defined as the nucleotide substitution rate (summed over all branches of the phylogenetic tree) within the element divided by the local background substitution rate at intronic and intergenic positions in the locus (neutral rate). We estimated substitution rates along each lineage by maximum likelihood using fastDNAml 26.

The human promoter was cloned in the proper orientation upstream of the luciferase cDNA in the pGL3Basic construct (Promega, Madison, WI, USA). The primate-specific elements from human or mouse were PCR cloned into polylinker sites upstream of the promoter of the closest gene (see Additional data file 5 for primer sequences). Each human element includes the primate-conserved sequence plus approximately 200 to 400 bp of flanking sequence. Thus, approximately 1,000 bp of total sequence are tested in each reporter construct.

Cells were grown at 37°C and 5% CO₂ in the minimum essential medium (ATCC, Manassas, VA, USA) (HepG2), or Dulbecco's modified Eagle's medium (ATCC) (293T cells), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), L-glutamine and penicillin-streptomycin. The cells were grown in 12-well plates (6 × 10⁴ cells/well for HepG2, 4 × 10⁴ cells/well for 293T) and transfected using Fugene (Roche Molecular Biochemicals, Indianapolis, IN, USA) following the manufacturer's protocol. Briefly, 500 ng (for HepG2) or 100 ng (for 293T) of each assayed plasmid and 50 ng (for HepG2) or 10 ng (for 293T) pCMVβ (BD Biosciences, San Jose, CA, USA) were mixed with 1.5 μl Fugene and added to each well. Following 42 to 48 hours of incubation, cells were harvested and lysed. Activity of luciferase and β-galactosidase was measured using the Luciferase Assay System (Promega) and galacto-Light Plus (Applied Biosystems), respectively. Luciferase activity for each sample was normalized to the β-galactosidase assay control. Transfections were carried out in duplicates. All experiments are representative of at least three independent transfections.

Tail vein injection was performed as described by Herweijer and Wolff 27 following the TransIT^® In Vivo Gene Delivery System Protocol (Mirus Corporation, Madison, WI, USA). Six to nine FVB male mice (Charles River Laboratory, Wilmington, MA, USA) at age 7 to 8 weeks were used for each reporter gene construct; 10 μg of each reporter construct, along with 2 μg of pCMVβ (BD Biosciences) to correct for delivery efficiency, were injected into each mouse. The entire content of the syringe was delivered in 3 to 5 seconds. Animals were sacrificed 24 hours later, livers extracted, measured to correct for size, homogenized, and centrifuged for 15 minutes at 4°C, 14,000 rpm. Activity of luciferase and β-galactosidase was measured as described above. All p values are from the two-sample Wilcoxon rank-sum (Mann-Whitney) test using STATA (STATA Corporation, College Station, TX, USA). All experimental results are representative of two independent plasmid DNA transfer assays.

DNase I-hypersensitive site mapping was performed as described previously 28. Briefly, cell pellets were resuspended in DNase I digestion buffer containing 0.5% IGEPAL (tert-Octylphenoxy poly(oxyethylene)ethanol) at 2 × 10⁷ cells/ml buffer. Aliquots (100 μl) of the resuspended cells were mixed with equal volumes of DNase I buffer containing varying concentrations of DNase I.The DNase I digestion reaction was incubated at 23°C for 5 minutes before being stopped with the addition of 8 μl of 0.5 M EDTA and 2 μl of 100 mg/ml RNase A.Following 5 minutes of RNase A treatment, genomic DNA was isolated using the QiaQuickPCR Kit (Qiagen, Valencia, CA, USA). Then, 10 μg of each of the DNA samples was digested with an appropriate restriction enzyme and resolved in a 1.2% agarose gel by electrophoresis. The DNA from the gel was then transferred onto a nylon membrane. Southern blotting was carried out with a radiolabeled DNA probe generated by PCR amplification (see Additional data file 5 for primer sequences), and corresponds to regions of approximately 1,000 bp from Gumby predicted elements. Following hybridization and washing, the blot was exposed to Biomax film (Kodak Co., Rochester, NY, USA) with intensifying screens at -80°C for 48 hours.