Current Pharmacogenomics and Personalized Medicine, 2012, 10, 33-42 33 Original Research Article Host Genomics Plasticity in Response to Ambient Temperature Change: Transcriptional Regulation Induced by Cold Temperature Perception in the Human BEAS-2B Cell Line Seyeon Park1,*, Sohyun Chun1,2 and Danuh Kim1,2 1Department of Applied Chemistry, Dongduk Women’s University, Seoul, Korea; 2Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Abstract: Pharmacogenomics has long attempted to identify genome-drug interactions and environmental exposures as guideposts for personalized medicine. However, non-drug related environmental factors, too, can interact with the host genome and potentially cause confounding in explaining drug-genome interactions. One such environmental factor that has been bracketed out in the past is the ambient temperature change, and ways in which it can influence genomic plasticity. Indeed, recognition of temperature is an important element of microsensory perception that allows cells to evaluate both their external environment and internal physiological milieu. In this paper, we report the changes in global gene expression three hours after a 30-min cold temperature (10°C) treatment in the human bronchial epithelial cell line BEAS-2B using DNA microarrays. We found 11,276 candidate genes (6,297 with increased, 4,979 with decreased expression) that were differentially expressed after low-temperature treatment of BEAS-2B compared to the untreated control cells (p<0.001). Additionally, up- and down-regulated transcription factor genes were further verified using real- time polymerase chain reaction. We found expression changes in response to cold temperature in transcription factor genes such as ZXDA, ZNF44, ZDHHC13, ZNF423, ZFYVE20, ZNF45, ZC3HAV, ZCCHC5, RUNX1T1, DMRT1, STAT4, EFCAB1 and HSFX1 that can alter the temperature-adaptive responsiveness of the bronchial epithelial cells. In summary, we herein show that a moderately cold temperature induces a genome-wide response in the human bronchial epithelial cell line BEAS-2B, and further discuss its relevance for pharmacogenomics and upstream drug discovery. For example, these observations provide a new crucial putative link between ambient temperature and genomic plasticity that together inform personalized medicine such that future pharmacogenomics biomarker discovery research can better control and account for ever-present dynamic environmental exposures such as ambient temperature. We conclude with the implications of these data in relation to rational drug design for neuropathic and other chronic pain syndromes that are in part moderated by host-ambient temperature interactions. Keywords: Ambient temperature, BEAS-2B cell line, chronic pain syndrome, environmental confounding and biomarker discovery, genome-temperature interaction, genomics plasticity, personalized medicine, transcription factor gene. 1. INTRODUCTION specific genotypes [1]. However, it is unknown how these patterns of environment-induced expression plasticity differ Personalized medicine and pharmacogenomics more between genetically divergent individuals of a biological specifically, have attempted to identify the genome-drug species, especially such as the higher vertebrates. Further- interactions. However, non-drug related environmental more, in contrast to the exposures to hot temperatures, factors, too, can interact with the host genome and potentially populations under natural conditions can often be exposed to cause confounding in explaining drug-genome interactions. longer periods of less extreme or moderate cold temperature One such environmental factor that has been bracketed out in changes. Phenotypic plasticity to temperature plays an past pharmacogenomics research is the ambient temperature important role in the evolution of life in a variable climate change and ways in which it can influence genomic [5] and is widespread among species [6-8]. plasticity and gene regulation. The mRNA levels respond rapidly to variable ambient conditions such as temperature Indeed, the ‘‘genotype-by-environment’’ interaction is change [1]. For instance, this has been shown for yeast [2], the prerequisite for adaptive evolution in a fluctuating bacteria [3], and C. elegans [4] after exposure to heat shock. environment [9]. In fact, it has been shown that more than The genomic plasticity response to such ambient temperature half of the regulatory connections in a gene expression changes can differ depending on the host genetic make-up or network are unique for specific conditions such as cell cycle, spore formation DNA damage and stress response [10]. New insights into genomic plasticity response to ambient *Address correspondence to this author at the Department of Applied temperature in higher vertebrates are more than essential for Chemistry, Dongduk Women’s University, 23-1 Wolgok-dong, Sungbuk- discovery of biomarkers that stand the test of dynamic ku, Seoul 136-714, Korea; Tel 82-2-940-4514; Fax 82-2-940-4193; changes in environmental exposures. E-mail: [email protected]; [email protected] 1875-6913/12 $58.00+.00 © 2012 Bentham Science Publishers 34 Current Pharmacogenomics and Personalized Medicine, 2012, Vol. 10, No. 1 Park et al. In this study, our hypothesis builds on, and extends upon, anti-TRPA1 antibody (diluted in 1% BSA, 1:100) (Abcam, prior observations on gene-ambient temperature adaptive Cambridge, UK) overnight at 48°C. After the primary responses, and thermosensation specifically. In brief, antibody treatment, the cells were incubated with FITC- thermosensitive afferents express ion channels of the conjugated anti-rabbit IgG (diluted in 1% BSA, 1:500) transient receptor potential (TRP) family that respond at (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room distinct temperature thresholds, thus establishing a molecular temperature. Next, cells were counterstained using 4,6- basis for thermosensation. Transient receptor potential diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, melastatin 8 (TRPM8) and transient receptor potential A1 MO). The cover-slips were mounted on glass slides, and (TRPA1) are nonselective cation channels expressed on a fluorescence was detected using a Nikon fluorescence subset of peripheral afferent fibers. TRPA1 is activated by microscope (Nikon, Tokyo, Japan). Images were taken with the pungent ingredients in mustard and cinnamon, but it also a Nikon Eclipse TE2000-U camera (Nikon). has been postulated to mediate perception of noxious cold temperatures [11,12]. In particular, the molecular and 2.3. RNA Isolation and Microarray Analysis biochemical pathways inside cells that regulate cold-induced To investigate the alterations associated with low- responses are largely unknown. In general, cold exposure temperature adaptation after sensing at the molecular level, induces adaptive thermogenesis, which elevates energy differential genome analysis of BEAS-2B cells was expenditure in mammals via mitochondrial uncoupling [13]. performed by microarray analysis 3 h after treatment at 10°C The respiratory epithelium is constantly exposed to the for 30 min. Total RNA was isolated by following protocols external environment, and prolonged inhalation of cold air is of the RNeasy Mini Kit (Qiagen, Hilden, Germany) detrimental to human airways [14]. Physiologically, mild including DNAse digestion. The RNA was checked for cold exposure is known to elevate energy expenditure in integrity and purity on the Agilent 2100 Bioanalyzer mammals, including humans [13]. This process is known as (Agilent Technologies, Böblingen, Germany). Total RNA adaptive thermogenesis. In small animals, adaptive thermo- from each control and low-temperature treatment was used genesis is mainly achieved by mitochondrial uncoupling in for genome-wide gene expression profiling. Hybridization brown adipose tissue and regulated via the sympathetic on Agilent’s human whole genome 4 44K microarrays nervous system [13]. Transcriptional activation after thermo- was performed according to standard procedures supplied sensitive receptor binding could be coupled with changes in by the manufacturer (Agilent Technologies). In brief, RNA intracellular responses of representative determinants that was amplified and labeled using 50 ng of total RNA lead to adaptive thermogenesis and compensatory events due and Agilent’s Low RNA Input Linear Amplification Kit to cooling. Although environmental temperature inevitably PLUS to generate biotin-labeled complementary RNA. The influences an organism, the mechanism of systemic fragmented complementary RNA was hybridized to the gene temperature perception remains largely unknown [15]. chips for 16 h at 45°C. Arrays were washed and stained The present genome-wide gene expression study, using under standard conditions, and scanned on an Agilent DNA the human bronchial epithelial cell line BEAS-2B as a model microarray scanner. Feature Extraction Software was used to for proximal sensor, and mediator of compensatory events in control washing and scanning; to generate DAT, CEL, and the cooling of respiratory tract cells, aimed to identify a EXP files; and to process the raw data for signal calculation broad range of molecular correlates of adaptive responses to and pairwise chip comparison [16]. cold temperature. We herein show that a moderate cold temperature induces a genome-wide response and discuss its 2.4. Real-Time Polymerase Chain Reaction (PCR) biological relevance in relation to neuropathic