Smith et al. Genome Medicine 2012, 4:45 http://genomemedicine.com/content/4/5/45 REVIEW Pharmacogene regulatory elements: from discovery to applications Robin P Smith1,2†, Ernest T Lam2,3†, Svetlana Markova1, Sook Wah Yee1* and Nadav Ahituv1,2* Pharmacogenomics and gene regulatory elements: Abstract an emerging picture Regulatory elements play an important role in the Most pharmacogenomics studies to date have focused on variability of individual responses to drug treatment. coding variants of pharmacologically important proteins. This has been established through studies on three However, well-supported examples of variants in regu la- classes of elements that regulate RNA and protein tory elements of genes involved in drug response, such as abundance: promoters, enhancers and microRNAs. drug metabolizing enzymes and transporters (see review Each of these elements, and genetic variants within by Georgitsi et al. [1]), show that variants in noncoding them, are being characterized at an exponential pace regulatory sequences are also likely to be important by next-generation sequencing (NGS) technologies. In (Table 1). Th ree classes of regulatory elements have been this review, we outline examples of how each class of studied in this context: promoters, enhancers and element aff ects drug response via regulation of drug microRNAs (miRNAs). Each of these has a direct impact targets, transporters and enzymes. We also discuss on the abundance of messenger RNA (mRNA) (in the case the impact of NGS technologies such as chromatin of promoters and enhancers) and protein (in the case of immunoprecipitation sequencing (ChIP-Seq) and RNA miRNAs). Genetic variation within each of these elements sequencing (RNA-Seq), and the ramifi cations of new has been linked to human disease as well as interindividual techniques such as high-throughput chromosome diff erences in drug response. For example, a single capture (Hi-C), chromatin interaction analysis by nucleotide polymorphism (SNP) in the promoter of paired-end tag sequencing (ChIA-PET) and massively VKORC1, the gene encoding vitamin K epoxide reductase parallel reporter assays (MPRA). NGS approaches are complex subunit 1, radically aff ects an indi vidual’s generating data faster than they can be analyzed, and response to the anticoagulant warfarin [2]. Likewise, a SNP new methods will be required to prioritize laboratory within an enhancer in the vicinity of several solute carrier results before they are ready for the clinic. However, family (SLC) drug transporters is associated with increased there is no doubt that these approaches will bring clearance of methotrexate (MTX) [3], and a SNP within a about a systems-level understanding of the interplay 3’-untranslated region (UTR) miRNA binding site between genetic variants and drug response. An prevents resistance to the chemo therapeutic cisplatin [4]. understanding of the importance of regulatory variants While a rough estimate of the number of coding genes in pharmacogenomics will facilitate the identifi cation exists [5], it is unclear how many regulatory elements of responders versus non-responders, the prevention there are in the genome. Th e diffi culty in defi ning critical of adverse eff ects and the optimization of therapies for regulatory elements is compounded by the fact that the individual patients. search space for regulatory elements is vast (98% of our Keywords ChIP-Seq, enhancers, miRNA, next- genome is noncoding) and without clear sequence cues generation sequencing, pharmacogenomics, such as open reading frames. Next-generation sequencing promoters, RNA-Seq (NGS) approaches are rapidly changing the status quo, revealing the location and function of regulatory elements on a genomic scale. Robust, high-throughput DNA sequencing platforms emerged as the Human †These authors contributed equally to this review Genome Project came to a close in 2003, as did a desire to *Correspondence: [email protected], [email protected] 1Department of Bioengineering and Therapeutic Sciences, University of California establish a reference human epigenome [6]. Key technical San Francisco, San Francisco, CA, USA advances have brought this goal closer to reality by Full list of author information is available at the end of the article enabling rapid de novo detection of DNA methylation [7,8], enhancers [9,10] and RNA transcripts [11-13] on a © 2010 BioMed Central Ltd © 2012 BioMed Central Ltd genome-wide level (Table 2). Smith et al. Genome Medicine 2012, 4:45 Page 2 of 13 http://genomemedicine.com/content/4/5/45 Table 1. Pharmacogene regulatory variants linked to drug response Gene/miRNA Drug Comments References Promoters MGMT Temozolomide Promoter methylation of the gene encoding O-6-methylguanine-DNA [78,79] methyltransferase (MGMT) is associated with treatment outcome in patients with glioblastoma multiforme and anaplastic astrocytoma treated with temozolomide UGT1A1 Irinotecan The UGT1A1*28 polymorphism (insertion of a seventh ‘TA’ repeat in the UGT1A1 [23,29-31] promoter) leads to lower UGT1A1 expression levels, causing lower clearance of the active irinotecan metabolite, SN-38. This results in severe side eff ects, such as diarrhea and neutropenia VKORC1 Anticoagulant A common promoter variant in VKORC1, -1639G>A (rs17878363), has signifi cant [22,25-27] implications for dosing algorithm of anticoagulant response. Patients with the variant G require higher dose of warfarin and have lower response to acenocoumarol Enhancers SLCO1A2 Methotrexate A screen of ECRs in the vicinity of nine pharmacologically relevant membrane [3] transporters identifi ed several enhancers, notably a region in the fi rst intron of SLCO1A2. Common SNPs within this region were linked to reduced SLCO1A2 mRNA levels (rs4148981C>T) and methotrextate clearance (rs11045981 T>G) miRNAs miR-24 Methotrexate A naturally occurring SNP near the miR-24 binding site in the 3’ UTR of human [73] DHFR (829C>T) interfered with miR-24 function and resulted in DHFR overexpression and methotrexate resistance miR-27a and miR-451 Doxorubicin P-glycoprotein, encoded by ABCB1, causes resistance in cancer cells to multiple [71] chemotherapeutics, and can be activated by miR-27a and miR-451. Antagonizing these miRNAs results in increased uptake of doxorubicin miR-27b Cyclophosphamide Overexpression of miR-27b in PANC1cells downregulated CYP3A4 and led to [69,70] cyclophosphamide resistance miR-200b/200c/429 family Cisplatin A polymorphism (rs1045385) within the 3’ UTR of TFAP2 prevents binding of this [4] family of miRNAs, leading to higher levels of its product, transcription factor AP-2α. This variant allows drugs that induce AP-2α, such as cisplatin, to more eff ectively inhibit cancer cell growth ECR, evolutionarily conserved region; miRNA, microRNA; SNP, single nucleotide polymorphism; UTR, untranslated region. In this review, we discuss the role of each class of (Table 1). Th at is not to say that other classes of regulatory element in drug response variability, and how regulatory elements are not important for pharmacogene our understanding of these mechanisms has been im- regulation; they most likely are, but have not yet been pacted by NGS approaches. We also discuss NGS tech- identifi ed. As functional validation assays improve, a nolo gies such as deoxyribonuclease I sequencing (DNase- more complete picture of regulatory mechanisms will no Seq), formaldehyde-assisted isolation of regulatory ele- doubt emerge. ments sequencing (FAIRE-Seq), high-throughput chromo- some capture (Hi-C) and chromatin interaction analysis Promoters by paired-end tag sequencing (ChIA-PET), which have Gene promoters are located at the 5’ terminus of their not yet been applied to pharmacogene regulation but will target gene, and often have two separate domains, known greatly improve our ability to interpret noncoding genetic as the core and proximal promoter regions. Th e core variation. Finally, we comment on the need for more promoter is where the transcription machinery assembles effi cient functional validation, and discuss other [16,17] and is usually 35 to 40 base pairs (bp) long. Th e challenges that need to be considered in moving NGS proximal promoter is located in the immediate vicinity data into the clinic. (-250 bp to +250 bp) of the gene’s transcription start site (TSS). It contains several transcription factor binding Pharmacogene regulatory elements sites (TFBS) and is thought to serve as a tethering Th ere are many diff erent classes of gene regulatory element for enhancers, enabling them to interact with the elements, including promoters, enhancers, miRNAs, core promoter [18]. Additional elements up to 5 kb silencers and insulators (Figure 1; for detailed reviews see upstream of the proximal promoter are often considered Maston et al. [14] and Noonan et al. [15]). In this review, to be part of the ‘promoter region’, and are designated as we will focus on the fi rst three classes, each of which has such in this review. Mammalian promoters often contain been linked to multiple pharmacogenomic phenotypes CpG islands: sequences at least 200 bp in length with Smith et al. Genome Medicine 2012, 4:45 Page 3 of 13 http://genomemedicine.com/content/4/5/45 Table 2. Current next-generation sequencing technologies that are suitable for pharmacogene regulatory element discovery Technology Description References DNA methylation MethylC-Seq Unmethylated cytosines are converted into uracil by bisulfi te treatment, and the converted DNA is sequenced. [7,8] Methylation is detected with single-base resolution