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Expression Profiling of UVB Response in Melanocytes Identifies a Set of p53-Target Guang Yang1, Guoqi Zhang1, Mark R. Pittelkow2, Marco Ramoni3 and Hensin Tsao1,4,5

Epidermal melanocytes execute specific physiological programs in response to UV radiation (UVR) at the cutaneous interface. Many melanocytic responses, including increased dendrite formation, enhanced melanogenesis/melanization, and cell cycle arrest impact the ability of melanocytes to survive and to attenuate the UVR insult. Although some of the molecules that underlie these UVR programs are known, a coherent view of UVR-induced transcriptional changes is lacking. Using primary melanocyte cultures, we assessed for UVR-mediated alterations in over 47,000 transcripts using Affymetrix U133 Plus 2.0 microarrays. From the 100 most statistically robust changes in transcript level, there were 84 genes that were suppressed 42.0-fold by UVR; among these transcripts, the identities of 48 of these genes were known. Similarly, there were 99 genes that were induced 42.0-fold by UVR; the identity of 57 of these genes were known. We then subjected these top 100 changes to the Ingenuity Pathway analysis program and identified a group of p53 targets including the cell cycle regulator CDKN1A (p21CIP), the WNT pathway regulator DKK1 (dickkopf homolog 1), the receptor tyrosine kinase EPHA2, growth factor GDF15, ferrodoxin reductase (FDXR), p53-inducible TP53I3, transcription factor ATF3, DNA repair enzyme DDB2, and the b- ADBR2. These genes were also found to be consistently elevated by UVR in six independent melanocyte lines, although there were interindividual variations in magnitude. WWOX, whose protein product interacts and regulates p53 and p73, was found to be consistently suppressed by UVR. There was also a subgroup of neurite/axonal developmental genes that were altered in response to UVR, suggesting that melanocytic and neuronal arborization may share similar mechanisms. When compared to melanomas, the basal levels of many of these p53-responsive genes were greatly dysregulated. Three genes – CDKN1A, DDB2 and ADRB2 – exhibited a trend towards loss of expression in melanomas thereby raising the possibility of a linked role in tumorigenesis. These expression data provide a global view of UVR-induced changes in melanocytes and, more importantly, generate novel hypotheses regarding melanocyte physiology. Journal of Investigative Dermatology (2006) 126, 2490–2506. doi:10.1038/sj.jid.5700470; published online 3 August 2006

INTRODUCTION much of the deleterious effects of UVR (Walker et al., 2003). Epidermal melanocytes, which comprise about 10% of The importance of an adequate melanin ‘‘shield’’ to all epidermal cells, function as the guardian against minimize UVR chronic damage has been further reinforced excessive UV radiation (UVR) damage to the skin. Although by more recent discoveries that genetic determinants of the epidermis may thicken with chronic UVR exposure, pigmentation dictate skin cancer risk (Rees, 2003). the melanocyte undergoes critical physiological changes – Constitutive pigmentation (i.e. skin color), over the course such as increased dendrite formation, melanogenesis, and of evolution, and facultative pigmentation (i.e. tanning), over melanosome translocation –that is responsible for attenuating the course of physiological response, comprise the spectrum of pigmentary responses to UVR controlled by melanocytes. In vivo, there is an inverse relationship between level of 1Wellman Center for Photomedicine, Massachusetts General Hospital, constitutive pigmentation and amount of in situ photopro- Boston, Massachusetts, USA; 2Department of Dermatology, Mayo Clinic, 3 ducts after UV irradiation; moreover, melanin content in the Rochester, Minnesota, USA; Children’s Hospital Informatics Program and Harvard Partners Center for Genetics and Genomics, Boston, Massachusetts, skin increases to a greater extent in individuals with darker USA; 4Department of Dermatology, Massachusetts General Hospital, Boston, skin (Tadokoro et al., 2003). Similar results have been found 5 Massachusetts, USA and MGH Cancer Center, Massachusetts General in culture where more heavily pigmented melanocytes Hospital, Boston, Massachusetts, USA appear to better stimulate melanogenesis in response to Correspondence: Dr Hensin Tsao, Department of Dermatology, Bartlett 622, 48 Blossom Street, Massachusetts General Hospital, Boston, Massachusetts UVR and are more resistant to the toxic effects of UVR 02114, USA. E-mail: [email protected] (Friedmann and Gilchrest, 1987; Barker et al., 1995). Abbreviations: UVR, UV radiation; NHM, normal human melanocyte Kobayashi et al. (1993) also documented in culture that Received 3 May 2005; revised 1 May 2006; accepted 16 May 2006; melanoma cells with greater melanization harbored fewer published online 3 August 2006 photoproducts after UVB exposure. Melanocytes appear to be

2490 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology G Yang et al. UVR Response Genes

less sensitive to UVR-induced cytotoxicity than keratinocytes 2003; Wang et al., 2004). The response network is quite rich in all three UV spectra (de Leeuw et al., 1994). with signaling kinases (Atr, Jnk, p38, Akt), transcription Studies of UVR responses at the molecular level has factors (Id1, N-oct3, p73, p53, cAMP response element advanced most quickly using primary melanocyte cultures binding protein, Mitf) and target effectors for cell cycle and more readily cultivated melanoma cells. UVR exposure regulation (p16INK4a, Gadd45, p21CIP), DNA repair induces a G1 arrest in melanocytes that is at least partially (Gadd45, Ddb2) and survival (Bax, Bcl-2). Interactions attributable to both p53 (Barker et al., 1995) and p16INK4a among these known and yet-to-be discovered components (Pavey et al., 1999). This cell cycle arrest is essential refine and define a patterned set of responses on the cell for effective removal of potentially mutagenic photoproducts, cycle, genomic repair, and apoptosis/survival. Furthermore, a repair mechanism termed nucleotide excision repair mutations, altered expression, or activation of many of these (Aboussekhra et al., 1995; Lehmann, 1995; Bootsma et al., molecules and pathways (e.g. p16INK4a, p53, Ddb2, Mitf, 1998). Studies of patients with defects in nucleotide excision Bcl-2, Akt) have been shown to play roles in the pathogenesis repair (i.e. xeroderma pigmentosum) suggest that this of melanoma. Along these lines, an inherited polymorphism genomic caretaker function is critical in maintaining genetic in TP53 (Arg72Pro) has been reported to dictate melanoma homeostasis within melanocytes as individuals with xero- risk (Shen et al., 2003); individual variation in the capacity of derma pigmentosum exhibit a 600–8,000-fold increase in the p53 to induce downstream targets may, in part, explain this rate of melanoma (Kraemer et al., 1994). observation. Thus, the impact of perturbations on these early Melanocyte survival after UVR exposure also reflects a UVR response modifiers may lead to more enduring and complex balance between apoptotic and antiapoptotic carcinogenic outcomes over time. programs. Upon UVB irradiation, cultured human melano- Although much of understanding of UVR responses in cytes can undergo apoptosis in a dose-dependent manner melanocytes are based on single-/pathway approaches, (Kadekaro et al., 2003). Although the apoptotic response Valery et al. (2001) undertook a 9,000-transcript human may, in part, result from increased p53 levels (Barker et al., cDNA microarray approach to study UVR in melanocytes at 1995), there is also evidence of mitochondrial participation the transcriptome level. These investigators selected a as Bax levels increase and Bcl-2 levels decrease after UVR polygenic (pooled) melanocyte population from several exposure (Im et al., 1998; Kadekaro et al., 2003). Bcl-2 itself individuals and analyzed changes at 4 hours post-irradiation. plays an essential role in melanocyte maintenance as Overall, 198 out of 9,000 genes were shown to be altered mice deficient for BCL2 undergo loss of follicular melano- (41.9-fold) with 117 clones and 81 clones suppressed and cytes (Veis et al., 1993). There are also several survival induced by UVR, respectively (Valery et al., 2001). Although inputs that antagonize these apoptotic responses. Irradia- this study represents an early attempt at a systematic tion of melanocytes with UVR leads to activation of the classification of UVR-response genes in melanocytes, the Akt pathway, which in turn, results in phosphorylation and restricted number of genes interrogated and the narrow inhibition of the proapoptotic functions of Bad (del dynamic range (induction ranged from 1.9- to 2.6-fold) limit Peso et al., 1997; Kadekaro et al., 2003). In addition, UVR the generalizability of these results. exposure induces phosphorylation of cAMP response With the availability of much higher density microarrays, element binding protein through activation of mitogen- a more thorough annotation of the human genome activated protein kinase p38 (Tada et al., 2002). One of and a stronger technical and technological platform to the targets of cAMP response element binding protein is analyze expression data, we set out to undertake a global Mitf, a transcription factor known to enhance melanocyte view of UVR-response gene expression in order to survival through increased expression of Bcl-2 (McGill generate novel hypotheses regarding melanocyte physio- et al., 2002). logy in the context of its major epidermal function-UVR Local paracrine and possibly autocrine circuits also attenuation. regulate melanocyte function in response to UVR. For In contrast to the previous microarray study (Valery et al., instance, keratinocytes, when exposed to UVR, have been 2001), we (1) utilized the Affymetrix Human Genome U133 shown to elaborate a-melanocyte stimulating hormone Plus 2.0 Chips, which interrogate approximately 47,000 (MSH) and endothelin 1 (Yohn et al., 1993; Chakraborty transcripts, (2) used a monogenic approach, and (3) selected et al., 1996), both of which can stimulate melanogenesis a 24-hour time point of analysis in order to enrich for more (Abdel-Malek et al., 1995; Tada et al., 1998) and drive downstream effectors post-immediate early-gene responses. melanocytes to overcome the UV-induced G1 arrest (Im Using the Ingenuitys Pathway Analysis program, we et al., 1998; Tada et al., 1998). In addition to melanocyte identified a group of p53 targets that appears to be selectively proliferation, endothelin 1 of keratinocyte origin also induced by UVR. In an independent panel of primary promotes melanocyte dendricity in response to UV irradia- melanocytes, these genes were all found to be consistently tion (Hara et al., 1995). increased by UVR, although the levels of induction varied Many molecules have been shown to be induced by by individual. As p53 loss, either through direct mutagenesis UVR specifically in melanocytes (Hu et al., 1997; Bulavin or ARF inactivation (Yang et al., 2005), plays a central role et al., 1999; Huang et al., 1999; Tibbetts et al., 1999; Lefort in melanoma pathogenesis, we also examined the levels of et al., 2001; Chouinard et al., 2002; McGill et al., 2002; these genes in melanoma cell lines relative to primary Tada et al., 2002; Kadekaro et al., 2003; Zhang and Rosdahl, melanocytes.

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5 (such as line normal human melanocyte (NHM) No. 5) that were generally more responsive than other lines. 4 However, all of the lines showed 42-fold induction for most, if not all of the genes. Likewise, all of the genes 3 exhibited UVR-induction in at least one cell line. WWOX -value) 2 appeared on the Ingenuity search because the protein ( P

10 product interacts with p53 (Chang, 2002; Chang et al.,

Log 1 2003) but is not necessarily a transcriptional target. This gene − is consistently reduced by UVR exposure in melanocytes, a 0 phenomenon that has been reported only in cancer cell lines (Chang et al., 2005). −6 −4 −2 0246 Other expression changes Log2 (fold UV stimulation) Using designations and the published Figure 1. Volcano plot of transcriptional response to UV radiation. The literature, we were able to assign some functional attributes X-axis represents the relative fold change by UVR transformed by log2, that is, to some of the annotated suppressed and induced genes unchanged by UVR ¼ 0. The Y-axis is the significance of the change by T-test (Table 4). These have been classified by their involvement, P-value transformed by log10. The two lines represent 2-fold induction and suppression by UVR – a cutoff used in our analysis. including neuronal/axonal guidance and development, apoptosis, protein/vesicular transport, DNA/RNA modifica- tion, cell cycle, metabolism, redox regulation/steroid metabolism and angiogenesis. We have also grouped all RESULTS the known transcription factors. It is clear that some genes Microarray results participate in multiple processes and are thus classified into An overall representation of the results is presented as a several groups. ‘‘volcano’’ plot in Figure 1. To prioritize these changes, we Bcl-2 is known to be induced by UVR (Kadekaro et al., used GeneCluster 2.0 to rank the top 100 most-suppressed 2003). We thus interrogated our microarray for entire BH (Figure 2) and top 100 most-induced (Figure 3) genes by their family of including prosurvival members Bcl-2, statistical significance (from highest (top) to lowest (bottom)). Bcl-XL, A1, Bcl-w, and Mcl-1 and proapoptotic members Subsequent annotation of these transcripts revealed 84 genes Bax, Bad, Bok, Bid, Bim, Bik, Hrk, Noxa, and Puma (Cory that were suppressed 42.0-fold by UVR; among these and Adams, 2002). Overall, there was a general trend for transcripts, the identities of 48 of these genes are known these genes to be elevated in UVR-exposed cells, although and listed in the Table 1 (sorted by fold-suppressed). most of these changes were not statistically significant Similarly, there were 99 genes that were induced 42.0-fold (Figure 5; Supplementary Table S1). by UVR; the identity of 57 of these genes are known and Similarly, we examined genes known to be involved in enumerated in Table 2 (sorted by fold-induced). pigmentation either through biochemistry or genetics by mutations affecting the pigmentary phenotype. These genes, Ingenuity pathway analysis including melanocortin-1-receptor (MC1R), tyrosinase (TYR), In order to derive a more coherent view of the microarray tyrosinase-related protein 1 (TYRP1), dopachrome tautomer- changes, we then subjected the top 100 most-induced and ase (DCT), pink-eye dilution homolog (OCA2), lysosomal s most-suppressed genes to the Ingenuity Pathway Analysis trafficking regulator (LYST), solute carrier family 24, member software package. The largest cluster of transcriptional 5(SLC24A5), myosin 5a (MYO5A), Rab27a (RAB27A), activity was found to be related to p53 (Table 3), whose melanophilin (MLPH), Hermansky-Pudlak 1, 3, 4,5 (HPS1, activity is known to be stimulated by UVR by post- HPS3, HPS4, HPS5), c-kit (KIT), Pax3 (PAX3), Mitf (MITF), transcriptional mechanisms. The genes IER3 (also known as B (EDNRB), endothelin 3 (EDN3), and IEX-1, DIF-2, PRG1, IEX-1L), a Myc target, and NR4A1 (also SRY (sex determining region Y)-box 10 (SOX10), did not known as TR3, N10, NAK-1, NGFIB, NUR77), an Egr-1 exhibit any consistent or significant patterns of response to target, were also identified. We thus focused our validation UVR (Supplementary Table S2). efforts on this list of genes culled from the Ingenuity database (Table 3 – designated UVR clusters, or UVRc). Relative expression in melanoma cell lines Many of the UVRc genes have already been implicated Individual variation in UVRc response in human oncogenesis including ATF3 (Pelzer et al., 2006), We then examined possible variations in UVR response CDKN1A (p21CIP) (Gartel and Radhakrishnan, 2005), among the UVRc genes by singly irradiating (35 and 50 mJ/ GDF15 (Cheung et al., 2004), EPHA2 (Kinch and Carles- cm2) a panel of six primary melanocyte cultures and assessing Kinch, 2003), WWOX (Iliopoulos et al., 2006), DDB2 (Yoon for gene expression differences through quantitative PCR. As et al., 2005), FDXR (Hwang et al., 2001), NR4A1 (Li et al., shown in Figure 4, there are indeed individual variations in 2006), and DKK1 (Gonzalez-Sancho et al., 2005). This raises response to UVR. In general, the UVR induction was more the possibility that the UVRc genes may also participate robust with 50 than 35 mJ/cm2. There were some cell lines in melanoma tumorigenesis. To this end, we evaluated

2492 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

Control UV Gb:AW274503 B1-parathyroid harmone-responsive B1 gene LILRA2-leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 2 Gb:AI800713 AURKB-aurora kinase B NLGN-neuroligin 1 Gb:BF968243 Gb:AK024621.1 WWOX-WW domain containing oxidoreductase TPK1-thiamin pyrophosphokinase 1 Gb:AK000115.1 Gb:AL353746 ANK3-ankyrin 3, node of Ranvier (ankyrin G) HDAC6-histone deacetylase 6 PPM1L-protein phosphatase 1 (formerly 2C)-like Gb:AW291402 ROBO2-roundabout, axon guidance receptor, homolog 2 (Drosophila) Gb:AB028966.1 CNTN4-contactin 4 DPYD-dihydropyrimidine dehydrogenase CNGB1-cyclic nucleotide gated channel beta 1 GLS-glutaminase Gb:Au146864 KCNK3-potassium channel, subfamily K, member 3 ZNF291-zinc finger protein 291 RABGAP1L-RAB GTPase activating protein 1-like POU3F1-POU domain, class 3, transcription factor 1 DCDC2-doublecortin domain containing 2 Gb:AW16772 MSRA-methionine sulfoxide reductase A ALS2CR19-amyotrophic lateral sclerosis 2 (juvenile) region, candidate 19 Gb:AI435595 Gb:AF267860.1 WWOX-WW domain containing oxidoreductase EGLN3- egl nine homolog 3 (C. elegans) Gb:AL136607.1 SMYD3- SET and MYND domain containing 3 C1GALT1-core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta Gb:AK024568.1 SHOX2-short stature homeobox 2 SEC15L2-SEC15-like 2 (S. cerevisiae) SCDF2- sec1 family domain containing 2 LAMA2-laminin, alpha 2 (merosin, congenital muscular dystrophy PRKG1-protein kinase, cGMP-dependent, type I GMDS-GDP-mannose 4,6-dehydratase GDP-mannose 4,6-dehydratase Gb:H16409 GPHN-gephyrin HMGN2-high-mobility group nucleosomal binding domain 2 Gb:NM_023037.1 ZFP91-zinc finger protein 91 homolog Gb:BC038746.1 Gb:AL355688.1 SLC22A3-solute carrier family 22 (extraneuronal monoamine transporter), member 3 Gb:AL389983.1 ZAN-zonadhesin LRBA-LPS-responsive vesicle trafficking, beach and anchor containing DIAPH2-diaphanous homolog 2 (Drosophila) Gb:AK026748.1 ITGA2B-integrin, alpha 2b CSRP2BP-CSRP2 binding protein BCAP29-B-cell receptor-associated protein 29 Gb:BF109461 Gb:AU154991 CILP2-cartilage intermediate layer protein 2 Gb:NM_014152.1 MRPL49- mitochondrial ribosomal protein L49 Gb:AK025009.1 AKAP28-A-kinase anchoring protein 28 Gb:AK097655.1 Gb:AA058834 Gb:AK098098.1 Gb:BE393431 Gb:AI217900 SYNE2-spectrin repeat containing, nuclear envelope 2 COH1-Cohen syndrome 1 TFCP2L1- transcription factor CP2-like 1 STIM1- stromal interaction molecule 1 JMJD2B- Jumonji domain containing 2B KIF27- kinesin family member 27 Gb:U95737 RNASE-ribonuclease, RNase A family, 7 NAP5-Nck-associated protein 5 LRCH3- leucine rich repeats and calponin homology (CH) containing 3 ENPEP-glutamyl aminopeptidase (aminopeptidase A) Gb:NM_018607.3 Gb:AJ300461.1 Gb:N31985 Gb:AL050097.1 Gb:U55983 TBC1D5- TBC1 domain family, member 5 WFIKKNRP-WFIKKN-related protein Gb:AB051486.1 Gb:AI979276 CNTN3-contactin 3 (plasmacytoma assciated) MGMT-O-6-methylguanine-DNA methyltransferase CAMK1D-calcium/calmodulin-dependent protein kinase ID Gb:AV657587 Gb:BC033368.1 Gb:BF939798 Gb:AL162007.1

Figure 2. Top 100 genes suppressed by UVR ranked by P-value. This is a heat map showing the most significant (top) to least significant (bottom) predictors. The P-values were derived from the corrected T-statistic as outlined in Golub et al. (1999). the relative basal levels of these genes in four primary melanoma cells lines (Figure 6: UACC903, A375, WM164, melanocyte lines (Figure 6: NHM-3 and 5 (lanes 1, 2) from SK-Mel-28, WM35, and MM-LH in lanes 5, 6, 7, 8, 9, and 10, the University of Vermont, NHM-5 (lane 3) from Clonetics, respectively). In all samples, we normalized the levels of and NHM-6 (lane 4) from Mayo Clinic) vis-a`-vis a panel of six these genes to endogenous b-glucuronidase, which remained

www.jidonline.org 2493 G Yang et al. UVR Response Genes

Control UV NM_025089.1 SEC31L1- sec31-like 1 CYP3A7- cytochrome P450, family 3, subfamily A, polypeptide 7 Gb:AF131783.1 Gb:BC041900.1 EPHA2 Gb:BF508774 RAB25 FBX032- F-box protein 32 CENTA1-centaurin, alpha 1 PITPNB- phosphatidylinositol transfer protein Gb:AW295105 ZNF-175-zinc finger protein 175 Gb:AI393479 Gb:BF590917 Gb:BF515755 RPS6KA6- ribosomal protein S6 kinase A6 Gb:BC015135.1 FDXR- ferrodoxin reductase TCEA3- transcription elongation factor A (S11), 3 NFIX- nuclear factor I/X (CCAAT-binding transcription factor) IGSF4-immunoglobulin superfamily, member 4 MGC23401 Gb:AI190575 DKK1- dickkopf homolog 1 (Xenopus laevis) Gb:AU157313 STX7- syntaxin 7 Gb:AC004381 PLGL- plasminogen-like GDF-15- growth differentiation factor 15 CDA08 GPLD1- glycosylphosphatidylinositol specific phospholipase D1 Gb:AI821779 AGGF1- angiogenic factor with G patch and FHA domains 1 ONECUT1-one cut domain, family member 1 Gb:AW451432 Gb:AW340987 Gb:AF070587.1 Gb:AW072559 Gb:BC038725.1 Gb:AW444673 AKR1C4-aIdo-keto reductase family 1, member C4 MCOLN1 MYO1F- myosin 1F PAPLN- papillin FLRT2- fibronectin like domain-containing leucine-rich transmembrane protein 2 CDKN1A- cyclin dependent kinase inhibitor 1A ARHGEF12- Rho guanine nucleotide exchange factor (GEF) 12 CSPG2- chondroitin sulfate proteoglycan 2 (versican) F12- Factor XII SNCAIP- synphilin 1 Gb:AA625583 ANAPC11-APC11 anaphase promoting complex subunit 11 homolog (yeast) ATF3- activating transcription factor 3 Gb:AL008627 BRUNOL4-bruno-like 4, RNA binding protein (Drosophila) IER3- immediate early response 3 Gb:NM_024751.1 ATP6VOD2- ATPase, H+ transporting, lysosomal 38kDa, V0 subunit disoform 2 WFDC6- WAP four-disulfide core domain 6 Gb:AI808830 pyruvate dehydrogenase kinase 4 FLJ12242 Gb:AK025072.1 DDB2- damage specific DNA binding protein 2 GPR115- G protein-coupled receptor 115 Gb:BF678298 LIPH- lipase, member H ARHGAP23- Rho GTPase activating protein 23 Gb:AV649275 Gb:AK094730.1 Gb:AF309700.1 CYP3A5- cytochrome P450, family 3, subfamily A, polypeptide 5 Gb:R02328 UNC B-unc-5 homolog B (C. elegans) CDH26- cadherin like 26 Gb:AW469714 Gb:AL021937 BAT1- HLA-B associated transcript 1 RAB40B Gb:AI818951 TP53I3- quinone oxidoreductase NR4A1- nuclear receptor subfamily 4, group A, member 1 PLEKHA6- pleckstrin homology domain containing, family A member 6 NSUN4- NOL1/NOP2/Sun domain family, member 4 Gb:AW269970 PBEF- pre-B-cell colony enhancing factor 1 Gb:AA815055 Gb:AW300612 KRTAP4.5- keratin associated protein 4.5 ADRB2- adrenegic, beta-2, receptor AF5Q31- ALL1 fused gene from 5q31 RBPMS- RNA binding protein with multiple splicing NYD-SP29- testis development protein NYD-SP29 GPR55- G protein-coupled receptor 55 STS-1- CbI-interacting protein Sts-1 Gb:AI080505 TRIM55- tripartite motif-containing 55 Gb:N30008 Gb:ALI990151

Figure 3. Top 100 genes induced by UVR ranked by P-value. This is a heat map showing the most significant (top) to least significant (bottom) predictors. The P-values were derived from the corrected T-statistic as outlined in Golub et al. (1999).

relatively constant through the various experiments (mean expression and loss of expression, respectively. Although 7 CT ¼ 25.12 0.85 after 360 independent assays). the number of samples examined were small, in aggregate, We assumed, on genetic grounds, that canonical onco- CDKN1A (P ¼ 0.10, two-tailed t-test), b-2 adrenergic receptor genic and tumor suppressor genes would exhibit over- (ADRB2; P ¼ 0.093, two-tailed t-test), and DNA damage

2494 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

Table 1. Top 100 T-statistic ranked annotated genes (UVR-suppressed) Fold suppression P-value Gene Description

7.32 0.0003 B1 Parathyroid hormone-responsive B1 gene 6.52 0.0064 CNTN4 Contactin 4 6.33 0.0084 GLS gGlutaminase 5.59 0.0079 CNGB1 Cyclic nucleotide gated channel beta 1 5.32 0.0292 LRCH3 Leucine-rich repeats and calponin homology (CH) domain containing 3 4.97 0.0273 JMJD2B Jumonji domain containing 2B 4.77 0.005 HDAC6 Histone deacetylase 6 4.77 0.0021 AURKB Aurora kinase B 4.69 0.0288 NAP5 Nck-associated protein 5 4.35 0.027 SYNE2 Spectrin repeat containing, nuclear envelope 2 4.25 0.0273 TFCP2L1 Transcription factor CP2-like 1 4.18 0.0149 PRKG1 Protein kinase, cGMP-dependent, type I 4.01 0.0092 KCNK3 Potassium channel, subfamily K, member 3 3.97 0.0095 DCDC2 Doublecortin domain containing 2 3.94 0.0056 ROBO2 Roundabout, axon guidance receptor, homolog 2 (Drosophila) 3.76 0.0296 ENPEP Glutamyl aminopeptidase (aminopeptidase A) 3.74 0.0277 KIF27 Kinesin family member 27 3.71 0.0342 CAMK1D Calcium/calmodulin-dependent protein kinase ID 3.56 0.0008 LILRA2 Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 2 3.28 0.0162 GMDS GDP-mannose 4,6-dehydratase 3.27 0.0034 WWOX WW domain containing oxidoreductase 3.16 0.0219 CSRP2BP CSRP2 binding protein 3.06 0.0093 RABGAP1L RAB GTPase activating protein 1-like 2.99 0.0076 DPYD Dihydropyrimidine dehydrogenase 2.89 0.0273 STIM1 Stromal interaction molecule 1 2.86 0.0337 CNTN3 Contactin 3 (plasmacytoma associated) 2.76 0.0116 C1GALT1 Core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 2.65 0.0198 SLC22A3 Solute carrier family 22 (extraneuronal monoamine transporter), member 3 2.65 0.0237 MRPL49 Mitochondrial ribosomal protein L49 2.62 0.0031 NLGN1 Neuroligin 1 2.62 0.005 ANK3 Ankyrin 3 2.54 0.0219 ITGA2B Integrin, alpha 2b 2.47 0.0112 EGLN3 egl nine homolog 3 (C. elegans) 2.46 0.0111 WWOX WW domain containing oxidoreductase 2.45 0.0102 ALS2CR19 Amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 19 2.42 0.0175 HMGN2 High-mobility group nucleosomal binding domain 2 2.41 0.0208 DIAPH2 Diaphanous homolog 2 (Drosophila) 2.35 0.0094 POU3F1 POU domain, class 3, transcription factor 1 2.34 0.0228 CILP2 Cartilage intermediate layer protein 2 2.34 0.0099 MSRA Methionine sulfoxide reductase A 2.32 0.0114 SMYD3 SET and MYND domain containing 3 2.24 0.0141 LAMA2 Laminin, alpha 2 (merosin, congenital muscular dystrophy 2.18 0.0054 PPM1L Protein phosphatase 1 (formerly 2C)-like 2.16 0.0243 AKAP28 A kinase (PRKA) anchor protein 14 2.15 0.0136 SEC15L2 SEC15-like 2 (S. cerevisiae) 2.11 0.0288 RNASE7 Ribonuclease, RNase A family, 7 2.10 0.0337 MGMT O-6-methylguanine-DNA methyltransferase 2.06 0.0035 TPK1 Thiamin pyrophosphokinase 1

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Table 2. Top 100 T-statistic ranked annotated genes (UVR-Induced) Fold induction P-value Gene Description

8.01 0.0007 FBXO32 F-box protein 32 6.52 0.0003 CYP3A7 Cytochrome P450, family 3, subfamily A, polypeptide 7 5.54 0.0057 TRIM55 Tripartite motif-containing 55 5.54 0.0002 SEC31L1 SEC31-like 1 5.43 0.0046 CYP3A5 Cytochrome P450, family 3, subfamily A, polypeptide 5 4.66 0.0055 RBPMS RNA binding protein with multiple splicing 4.55 0.0007 RAB25 RAB25, member RAS oncogene family 4.51 0.0016 IGSF4 Immunoglobulin superfamily, member 4 4.46 0.0029 MYO1F Myosin 1F 4.43 0.0039 GPR115 G protein-coupled receptor 115 4.39 0.0024 ONECUT1 Onecut domain, family member 1 4.3 0.0051 NSUN4 NOL1/NOP2/Sun domain family, member 4 4.15 0.0038 ATP6VOD2 ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d isoform 2 4.12 0.0013 ZNF-175 Zinc-finger protein 175 4.07 0.005 NR4A1 Nuclear receptor subfamily 4, group A, member 1 4.06 0.0009 PITPNB Phosphatidylinositol transfer protein 4.03 0.0005 EPHA2 EPH receptor A2 3.97 0.0021 PLGL Plasminogen-like 3.93 0.0009 CENTA1 CENTA1-centaurin, alpha 1 3.83 0.0049 BAT1 HLA-B associated transcript 1 3.81 0.0043 ARHGAP23 Rho GTPase activating protein 23 3.58 0.0037 BRUNOL4 Bruno-like 4, RNA binding protein (Drosophila) 3.55 0.0028 AKR1C4 Aldo-keto reductase family 1, member C4 3.46 0.0054 AF5Q31 ALL1 fused gene from 5q31 3.27 0.0047 UNC5B unc-5 homolog B (C. elegans) 3.16 0.0036 ANAPC11 APC11 anaphase promoting complex subunit 11 homolog (yeast) 3.1 0.0038 WFDC6 WAP four-disulfide core domain 6 3.03 0.0016 NFIX Nuclear factor I/X (CCAAT-binding transcription factor) 3.01 0.002 STX7 Syntaxin 7 3.01 0.0033 ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 3.01 0.0034 F12 Factor XII (Hagemann factor) 2.99 0.0014 RPS6KA6 Ribosomal protein S6 kinase A6 2.97 0.0022 GPLD1 Glycosylphosphatidylinositol specific phospholipase D1 2.8 0.0054 ADRB2 Adrenergic, beta-2-, receptor, surface 2.78 0.0017 DKK1 Dickkopf homolog 1 (Xenopus laevis) 2.77 0.0041 LIPH Lipase, member H 2.72 0.0053 KRTAP4.5 Keratin-associated protein 4, 5 2.6 0.0032 FLRT2 Fibronectin-like domain-containing leucine rich transmembrane protein 2 2.58 0.0035 SNCAIP Synuclein, alpha interacting protein (synphilin) 2.57 0.0024 AGGF1 Angiogenic factor with G patch and FHA domains 1 2.5 0.0051 PLEKHA6 Pleckstrin homology domain containing, family A member 6 2.49 0.0056 GPR55 G protein-coupled receptor 55 2.47 0.0015 FDXR Ferredoxin reductase 2.44 0.0016 TCEA3 Transcription elongation factor A (SII), 3 Table 2 continued on following page

2496 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

Table 2. continued Fold induction P-value Gene Description

2.43 0.005 TP53I3 Tumor protein p53 inducible protein 3 2.39 0.0057 STS-1 Cbl-interacting protein Sts-1 2.26 0.0022 GDF-15 Growth differentiation factor 15 2.26 0.0022 CDA08 2.23 0.0039 DDB2 Damage-specific DNA binding protein 2, 48 kDa 2.23 0.0036 ATF3 Activating transcription factor 3 2.22 0.0031 PAPLN Papilin, proteoglycan-like sulfated glycoprotein 2.11 0.0056 NYD-SP29 Testis development protein NYD-SP29 2.1 0.0032 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, CIP1) 2.09 0.0047 CDH26 Cadherin-like 26 2.08 0.0034 CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 2.04 0.0052 PBEF Pre-B-cell colony enhancing factor 1 2.04 0.0038 IER3 Immediate-early response 3 EPH, erythropoeitin-producing hepatocyte receptor; NOP2, nucleolar protein gene 2; RAS, rat sarcoma viral oncogene homologue; UVR, UV radiation.

Table 3. Genes mapped to specific pathways by the Across all genes examined, the mean coefficients of variation Ingenuity Pathway Software (UVR clusters) for the melanocyte lines and melanoma cell lines were 44.4 ¼ Transcripts Fold change and 104.3, respectively (P 0.00073). This suggests that the genes may be under tighter regulation in primary cells but p53 transcriptional node they become dysregulated, either through genetic or epige- CDKN1A (p21CIP) +2.1 netic means in melanoma cells. FDXR +2.47 TP53I3 +2.43 DISCUSSION DKK1 +2.78 In this study, we set out to catalog transcriptome-wide GDF15 +2.26 changes in melanocytes so as to clarify relevant physiological ATF3 +2.23 programs mobilized by these cells in response to UVR EPHA2 +4.03 exposure. We adopted several strategic modifications com- DDB2 +2.23 pared with the previously published lower density microarray ADRB2 +2.8 studies (Jean et al., 2001; Valery et al., 2001). First, microarrays obviously exhibit great variability in design and WWOX (interacts with p53) À3.27 density. We wanted a microarray with the greatest level of Myc transcriptional node empirical testing. Thus, we selected the Affymetrix platform IER3 +2.04 coupled with the Affymetrix U133 Plus 2.0 gene chip for our EGR-1 transcriptional node analysis. This represents almost 5- to 60-fold increase in NR4A1 +4.07 interrogated species compared to previous UVR analyses EGFR, epidermal growth factor receptor; EPH, erythropoeitin-producing (Jean et al., 2001; Li et al., 2001; Valery et al., 2001; Sesto hepatocyte receptor; UVR, UV radiation. et al., 2002). Second, we used a later timepoint of analysis (24 hours) as early stress responses may elicit differential effects downstream in different cell types; thus, we biased our analysis to detect more secondary effectors that may play melanocyte-specific functional and physiological roles. binding protein 2 (DDB2; P ¼ 0.049, two-tailed t-test) Third, rather than pooling melanocytes from different showed a tendency towards expression loss in melanomas individuals (i.e. polygenic analysis), we utilized a primary when compared to melanocytes. Dickkopf homolog 1 melanocytes from a single individual (i.e. monogenic (DKK1) exhibited a trend towards expression loss except in analysis); we then followed the global assessment with a single melanoma cell line (SK-Mel-28). EPHA2 was strongly validating quantitative PCR on a subset of bioinformatically elevated in some melanoma cell lines whereas all of the filtered genes using an expanded panel of six independent melanocytes showed relatively low levels consonant with primary melanocyte lines. This approach has its own prior reports (Seftor et al., 2002). advantages and disadvantages. Monogenic approaches Overall, the variation observed among melanocyte lines may yield a more robust signal-to-noise ratio as other also seemed more blunted than among melanoma cell lines. genetic influences are minimized, for example variations in

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14 1 12 0.8 10 8 0.6 6

0.4 WWOX CDKN1A 4 0.2 2 0 0 123456 123456

8 9 7.5 6 6 4 4.5 DDB2 TP53/3 3 2 1.5 0 0 123456 123456 24 9 20 7.5 16 6 12 4.5 DKK1 8 3 ADRB2 4 1.5 0 0 123456 123456

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4 10 8 3 6 ATF3

2 NR4A1 4 1 2 0 0 1 2345 6 123456

18 10 15 8 12 6 9 IER3

EPHA2 4 6 3 2 0 0 123456 123456

Figure 4. Induction of UVR cluster genes in primary melanocytes. The genes of interest are labeled outside the individual graphs. The uncolored, dark grey, and light grey boxes represent transcriptional activities clustered around p53, Myc, and Egr-1, respectively. The gray and black bars represent relative levels of mRNA transcript upon exposure to 35 and 50 mJ/cm2 of UVB, respectively. The X-axis designates six independent primary melanocyte lines: Lanes 1–6 correspond to primary lines 1–6 (NHM1–6) – two unrelated primary melanocyte lines from the Mayo Clinic (NHM-1, NHM-6) that were different than the microarray line, two unrelated primary melanocyte lines from Dr Marcus Bosenberg (University of Vermont, Burlington, VT; NHM-3, NHM-4), one primary melanocyte line from Clonetics (Cambrex Bio Science, Walkersville, Inc., MD; NHM-5), and one primary melanocyte line from Cascade Biologics (Portland, OR; NHM-2). The Y-axis specifies the fold induction relative to unirradiated control. All samples were normalized to endogenous b-glucuronidase (GusB).

2498 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

Table 4. Functional classes of differentially expressed genes Fold change Putative function Genes (UVR/control) Molecular structure Location

Neuron/axon development IGSF4 +4.51 Surface receptor Membrane EPHA2 +4.03 Surface receptor Membrane UNC5B +3.27 Surface receptor Membrane ATF3 +2.23 Transcription factor Nucleus POU3F1 À2.35 Transcription factor Nucleus NLGN1 À2.62 Carboxylic ester hydrolase activity Membrane ANK3 À2.62 Surface receptor Golgi CNTN3 À2.86 Surface receptor Membrane STIM1 À2.89 Matrix protein Membrane ROBO2 À3.94 Surface receptor Membrane CNTN4 À6.52 Surface receptor Membrane Apoptosis NR4A1 +4.07 Steroid receptor Nucleus UNC5b +3.27 Surface receptor Membrane FDXR +2.47 Ferrodoxin reductase Mitochondrion TP53I3 +2.43 Quinone oxidoreductase GDF15 +2.26 Growth factor Extracellular PBEF +2.04 Growth factor Extracellular IER3 +2.04 Surface receptor Membrane BCAP29 À1.69 Surface receptor Endoplasmic reticulum LAMA2 À2.24 Basement membrane protein Basement membrane WWOX À3.27 Oxidoreductase Mitochondrion Protein/vesicular transport RAB25 +4.55 GTP binding Cytoplasm STX7 +3.01 SNARE complex Endosome RAB40B +1.95 GTP binding Cytoplasm SCFD2 +1.69 COH1 À1.67 Metallopeptidase activity Membrane LRBA À1.68 Regulates EGFR internalization Membrane BCAP29 À1.69 Surface receptor Endoplasmic reticulum SEC15L2 À2.15 Vesicle fusion with plasma membrane Exocyst DNA/RNA modification BAT1 +3.83 ATP-dependent RNA helicase Nucleus BRUNOL4 +3.58 Bell response element RNA binding Nucleus TCEA3 +2.44 Transcription factor Nucleus DDB2 +2.23 Nucleotide excision repair Nucleus MGMT À2.1 DNA methyltransferase Nucleus HMGN2 À2.42 Chromatin restructuring Nucleus Cell cycle NSUN4 +4.3 NOL1 domain protein Nucleus ANAPC11 +3.16 Anaphase promoting complex Nucleus CDKN1A +2.1 Protein kinase inhibitor Nucleus AURKB À4.77 Protein kinase Nucleus Metabolism PITPNB +4.06 Phospholipid transfer Cytoplasm LIPH +2.77 Lipase Extracellular DPYD À2.99 Dihydroorotate dehydrogenase Intracellular GMDS À3.28 GDP-mannose 4,6-dehydratase activity Intracellular Redox regulation; steroid metabolism CYP3A7 +6.52 Cytochrome p450, subfamily 3, peptide 7 Endoplasmic reticulum CYP3A5 +5.43 Cytochrome p450, subfamily 3, peptide 5 Endoplasmic reticulum AKR1C4 +3.55 Aldoketo reductase Cytoplasm FDXR +2.47 Ferrodoxin reductase Mitochondrion TP53I3 +2.43 Quinone oxidoreductase

Table 4 continued on following page

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Table 4. continued Fold change Putative function Genes (UVR/control) Molecular structure Location

MSRA À2.34 Methionine sulfoxide reductase Mitochondrion/cytoplasm WWOX À3.27 Oxidoreductase Mitochondrion Angiogenesis EPHA2 +4.03 Surface receptor Membrane AGGF1 +2.57 Secreted angiogenic factor Extracellular ENPEP À3.76 Glutamyl aminopeptidase Membrane Transcription factors ONECUT1 +4.39 Nucleus ZNF-175 +4.12 Nucleus NR4A1 +4.07 Nucleus AF5Q31 +3.46 Nucleus NFIX +3.03 Nucleus TCEA3 +2.44 Nucleus ATF3 +2.23 Nucleus SHOX2 À1.75 Nucleus ZNF291 À1.78 Nucleus POU3F1 À2.35 Nucleus EGFR, epidermal growth factor receptor; EPH, erythropoeitin-producing hepatocyte receptor; NOL1, nucleolar protein 1; SNARE, soluble N-ethylmale- imide-sensitive factor attachment protein receptor; UVR, UV radiation.

pigmentary status, promoter variants that regulate expression, 2 Prosurvival and varying age of cells. Because these factors likely impact Proapoptosis UVR response to some extent, important signals may be lost 1.6 in pooled samples as subsets of melanocytes may respond 1.2 differentially and the overall aggregate transcript profile may -value)

not detect significant changes. However, monogenic assess- ( P ments may fail to identify some transcripts because, as we 10 0.8 Log

have demonstrated, individuals are likely to be differentially − sensitive to UVR. 0.4 In our analysis, we focused on the top 100 statistically 0 most significant gene expression changes modulated by UVR. −2 −1 012 Using the Ingenuitys Pathway database, we focused largely Log2 (fold change) on a cluster of transcriptional activity around p53, a known UVR-responsive transcription factor in melanocytes (Kade- Figure 5. Relative UVR-induced expression changes of BH family members. karo et al., 2003). The actual levels of p53 mRNA did not The relative levels of induction by UVR of proapoptotic (Bax, Bad, Bok, appear to be significantly altered in our microarray analysis Bid, Bim, Bik, Hrk, Noxa, and Puma) and prosurvival (Bcl-2, Bcl-XL, A1, Bcl-w, and Mcl-1) members of the BH family are shown in red and blue, (data not shown). This is consistent with the known UVR- respectively. There is a general trend towards induction of all genes although mediated post-translational stabilization of p53 protein rather individually, these genes did not reach significance at P ¼ 0.01. The actual than an effect on p53 transcript levels. Although p53 protein values are enumerated in Table S1. levels are known to be increased in response to UVR, the subset of p53 targets in melanocytes that lead to downstream effector functions are largely unknown. In this study, we have of dickkopf homolog 1, DKK1, a regulator of Wnt signaling, identified and confirmed a subset of genes regulated by p53 directly implicates this signaling stream in the pigment cell’s and responsive to UVR in melanocytes. Some previously response to UVR. Although DKK1 has been shown to be undisclosed p53 targets are worthy of mention. The elevated in response to genotoxic stress (Grotewold and b-adrenergic receptor, Adrb2, has recently been shown to Ruther, 2002), the loss of DKK1 in many melanoma cell lines be expressed in melanocytes and to play a role in raises the possibility of a more direct role for Dkk1 in catecholaminergic control of pigmentation (Gillbro et al., proliferative control. Regulation of intracellular redox states 2004). Thus, elevation in ADRB2 levels may be a novel UVR- appears to be a critical as oxidative stress is caused by response to increase intracellular cAMP levels and melano- UVR (reviewed in Halliday, 2005). Transcriptional changes genesis. Although the Wnt pathway is known to be critical for occurred in two p53-regulated genes – FDXR and TP53I3 – pigment cell development (Dunn et al., 2005), the induction and several other genes, such as aldo-keto reductase family 1,

2500 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

14 1.5 12 1.2 10 8 0.9 6

0.6 WWOX CDKN1A 4 0.3 2 0 0 12345678910 12345678910

2.0 18 1.6 15

1.2 12 9

TP53/3 0.8 6 DDB2 0.4 3 0 0 12345678910 12345678910

2.4 3 2.0 2.5 1.6 2 1.2 1.5 DKK1 0.8 1 ADRB2 0.4 1.5 0 0 12345678910 12345678910

320 5

240 4 3 160 GDF15

2 FDXR 80 1

0 0 12345678910 12345678910

8 2

6 1.5

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0 0 12345678910 1 2 345678910

6 30 5 25 4 20 3 15 IER3 EPHA2 2 10 1 5 0 0 12345678910 1 2 3 4 5 6 7 8 910

Figure 6. Relative levels of UVR cluster genes in unirradiated primary melanocytes and melanoma cell lines. The genes of interest are again labeled outside the individual graphs. The uncolored, dark grey, and light grey boxes represent transcriptional activities clustered around p53, Myc, and Egr-1, respectively, as in Figure 4. The gray bars represent levels of transcript normalized to endogenous b-glucuronidase (GUSB). The X-axis designates four independent primary melanocyte lines (NHM-3 and 5 (lanes 1, 2) from University of Vermont, NHM-5 (lane 3) from Clonetics, and NHM-6 (lane 4) from Mayo)) and six melanoma cell lines (UACC903, A375, WM164, SK-Mel-28, WM35, MM-LH in lanes 5–10, respectively) and the Y-axis specifies the relative transcript levels. As an example, for p21CIP, there was 12 times the amount of p21CIP transcript as GusB in NHM-3 (lane 1) but only 4 times the amount of p21CIP compared to GusB in melanoma cell line A375 (lane 6). As in Northern blotting, assuming GusB levels to be similar between the two lines, p21CIP can then be interpreted to be expressed at a higher level in NHM-3 relative to A375.

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member C4 (AKR1C4) and methionine sulfoxide reductase A arborization. As axonal outgrowth and neural patterning (MSRA), not known to be targets of p53. during neural development occurs through spatially restricted One gene that is consistently downregulated by UVR in expression of the same molecules, it is possible that melanocytes is WWOX. This gene has been reported to melanocytes coordinate critical developmental genes from interact with p53 and consistently exhibits downregulation by its neuroectodermal lineage in order to regulate dendrite UVR is WWOX. There is extant evidence that Wwox can outgrowth in a targeted orientation. bind both p53 (Chang, 2002) and p73 (Aqeilan et al., 2004b), The redistribution of melanosomes down dendrites with although the full functional consequences of this interaction subsequent transfer to keratinocytes is also a known UVR is still unknown. The WWOX gene is located at the response (Gibbs et al., 2000; Virador et al., 2002; Boissy, chromosome 16q23 common fragile site and reported to be 2003). The expression of several vesicular transport proteins underexpressed in some cancers (Aqeilan et al., 2004a). From were altered including two RAB genes (RAB25 and RAB40B) our data, WWOX expression is not significantly diminished and syntaxin-7, a SNARE complex protein. Both Rab in melanomas as compared to melanocytes. and syntaxin proteins have been previously reported to be Other known p53 targets, such as TYR (Khlgatian et al., associated with melanosome trafficking (Virador et al., 2002; 2002) or TYRP1 (Nylander et al., 2000) were not found to Boissy, 2003). be elevated. This may reflect differences in constitutive In summary, we used a transcriptome-wide approach to pigmentation or possibly the kinetics of induction. Alterna- identify potential genes that participate in the melanocytic tively, enforced expression of p53 in experimental assays response to UVR. Many of these changes can be assigned to may lead to transcriptional induction of these genes, but, functional groups that parallel known melanocyte physio- in vivo, UVR may in fact regulate these genes in more logy. The identification of select p53 targets confirms a complex circuitries. known role for p53 in the UVR response; more importantly On the one hand, many prosurvival genes, including IER3 however, the specific identity of these genes generates new and survival members of the BH family, are induced along avenues for investigation in the UVR biology of melanocytes with DNA repair enzymes such as DDB2. On the other hand, and possibly melanoma tumorigenesis. proapoptotic genes, such as TP53I3, ferrodoxin reductase (FDXR), and apoptotic members of the BH family are also MATERIALS AND METHODS elevated. The simultaneous expression of these ostensibly Source of cells antagonistic processes may in fact result because adjunctive For the microarray analysis, we obtained NHMs from a single functions of these genes may not be known and thus neonate (Mayo Clinic, Rochester, MN; designated NHM-Mayo) suppositions regarding function are only partially informative. through an Institutional Review Board-approved protocol at the Alternatively, in any culture system, some cells may be Mayo Clinic. Since the discarded tissue was de-identified, informed undergoing apoptosis whereas other cells may be in a consent was not obtained. The work was conducted according to the survival/repair mode. In performing a monogenic analysis, Declaration of Helsinki Principles. For validation, six additional lines we have attempted to minimize genetic heterogeneity, such were obtained (NHM1–6): two other unrelated primary neonatal as degree of pigmentation, but other factors, such as position melanocyte lines from the Mayo Clinic (NHM-1, NHM-6), two in the cell cycle, may also play a role in determining unrelated neonatal foreskin melanocyte lines from the University of vulnerability to UVR. Vermont, (NHM-3, NHM-4), one primary neonatal melanocyte As the full function of most human genes is unknown, it is line from Clonetics (Cambrex Bio Science, Walkersville, Inc., not yet possible to construct a coherent model capturing all MD; NHM-5) and one primary neonatal melanocyte line from the relevant physiological activities. However, several func- Cascade Biologics (Portland, OR; NHM-2). Melanocytes were all tional themes emerge. For instance, a number of genes that lightly pigmented. regulate neuronal development, including axonal and synap- Cells were grown in Medium 154 supplemented with tic guidance (Table 4), appear to be UVR responsive. These Human Melanocyte Growth Supplement (Cascade Biologics, include contactin-3 (CNTN3, also known as BIG1), con- Portland, OR) and penicillin/streptomycin. Melanocytes were tactin-4 (CNTN4, also known as BIG2) (Yoshihara et al., expanded to approximately 20 million cells and 18 hours 1994, 1995), Robo2/Slit (Miyasaka et al., 2005), SynCAM (or before the irradiation, the cells were re-plated at 2.5 million IGSF4) (Watabe et al., 2003), EphA2 (Brittis et al., 2002), cells per 150-cm dish. The cultures were subconfluent and Unc5b (Lu et al., 2004), neuroglin-1 (or NLGN1) (Scheiffele replicating at the time of irradiation. On the morning of UV et al., 2000), ankyrin-G (also known as ANK3) (Ango et al., irradiation, the cells were washed with phosphate-buffered 2004), and Stim1 (Hansen et al., 2004). Two transcription saline and checked for attachment and viability. For validation factors – Atf3 (Pearson et al., 2003) and Pou3F1 (Jaegle et al., purposes, additional NHM were purchased from Cascade Biologics 1996) – that have also been shown to participate in axonal (Portland, OR) and cultured in the same media with the Human outgrowth and neural development were also differentially Melanocyte Growth Supplement. expressed. It has long been observed that melanocytes will In order to examine the relative levels of gene expression among form dendrites in response to UVB irradiation often in the primary melanocyte lines and melanoma cell lines, we used direction of light exposure (Iyengar, 1994; Yamashita et al., unstimulated primary melanocyte lines NHM-3, NHM-4, NHM-5, 2005). Thus, UVR may induce trophic factors, such as NHM-6 and melanoma cell lines UACC903, A375, WM164, SK- endothelin-1 (Hara et al., 1995), that mediate the surface Mel-28, WM35 and MM-LH (Yang et al., 2005).

2502 Journal of Investigative Dermatology (2006), Volume 126 G Yang et al. UVR Response Genes

UV irradiation management for the entire GeneChip System. Percent present calls We subjected the NHMs to UVB/UVA exposure using a bank of (ranges between 30 and 55%), background (o100), and spike-in pre-heated 12 fluorescent FS36T12-UVB-HO tubes (Ultralite bulbs, control measurements were made using the GeneChip Operating Ultralite Enterprises, Inc., Lawrenceville, GA) filtered with Kodacel Software algorithms and represented the fraction of detected (Kodak, Rochester, NY) to remove all radiation below 290 nm. The transcripts out of the total number of transcripts present on the temperature within the irradiation chamber was kept constant with a array. Image scanning and data pre-processing to obtain trans- rotating fan. Unirradiated controls were kept from light exposure but cript measurements were performed using Affymetrix MAS subjected to all other manipulations. The fluence was 0.56 mW/cm2 5.0 following the protocol recommended by the manufacturer measured with a Portable High Accuracy UV-Visible Spectro- (http://www.affymetrix.com). radiometer (Optronic Laboratories, Inc., Orlando, FL). The spectral distribution of our light source is diagrammed in Supplementary Analysis Figure S1. In determining a dose, we wanted to document a clear We then used the GeneCluster 2.0. (Golub et al., 1999; Tamayo cellular response to the UVR without obvious evidence of toxicity. et al., 1999) software to assess differences in expression pattern. A There was some evidence of growth arrest between 10 and 30 mJ/ class prediction method was used to select genes most correlated cm2 at 24 hours but no evidence of cell death (data not shown). We with UVR exposure. Although these features or ‘‘marker genes’’ are thus selected a dose of 25 mJ/cm2, which is in line with other studies themselves biologically interesting, they were used as iterative inputs (Lefort et al., 2001; Pedeux et al., 2002; Sesto et al., 2002; Zhang into a classification algorithm that uses existing ‘‘labeled’’ samples to and Rosdahl, 2003). Immediately after irradiation, cells were washed build a model to predict the labels for future samples. Genes with phosphate-buffered saline, re-fed with complete media and correlated with unirradiated and irradiated sets were then directly returned to 371C. identified and selected by using a ‘‘distance’’ metric. The corrected T-statistic (Golub et al., 1999), as implemented in GeneCluster 2.0 Microarray and currently in GenePattern (www.broad.mit.edu), was used to Total RNA from quadruplicate samples of UV-irradiated (exposed generate a ranking of ‘‘most suppressed’’ and ‘‘most induced’’ genes in pairs to minimize local fluctuations in fluence) and quadruplicate by UVR; when needed, the T-statistic was converted to P-values samples of control unirradiated NHMs (from Mayo Clinic) using 6 degrees of freedom. were isolated using the Qiagen RNAeasy kit (Valencia, CA) and The top 100 ranked UVR-suppressed and top 100 ranked UVR- then submitted to the Harvard/Partners Center for Genetics induced genes were then subjected to the Ingenuitys Pathway and Genomics Microarray facility for analysis with the Affymetrix Analysis (http://www.ingenuity.com/) for identification of interacting Human Genome U133 Plus 2.0. Chips. This Microarray contains genes and transcription factor nodes. 1,300,000 unique oligonucleotides covering over 47,000 transcripts, which in turn represent approximately 39,000 of the best-character- ized human genes. RNA purity was determined initially by 260/ Quantitative PCR 280 ¼ 1.85–2.01 and finally by scanning with an Agilent 2100 Selected genes exhibiting differential expression in response to Bioanalyzer using the RNA 6000 Nano LabChips. Total RNA UVR were validated using quantitative PCR. Primers to assess was subjected to subsequent labeling and hybridization only if the the expression of select genes were available as Taqmans Gene 18S/28S ratio was approximately 2.0 as determined by the Expression Assays (Applied Biosystems, Foster City, CA). All Bioanalyzer. target genes were labeled with FAM as the fluorescent reporter The steps of cDNA synthesis were carried out using standard while the endogenous control, GUSB, was labeled with VIC as the Affymetrix kits, which provided all necessary reagents in one kit fluorescent reporter. for synthesis of double-stranded cDNA containing the T7 promoter For validation, we subjected six additional unrelated primary sequence from 1 to 15 mg of total RNA or 0.2–2 mg of mRNA. melanocyte lines to UVR irradiation at two doses – 35 and 50 mJ/ Cleanup of cDNA was also carried out using the Affymetrix cm2. Twenty-four hours after the UV irradiation, all cells were cDNA cleanup kits. In vitro transcription and labeling of cRNA washed with phosphate-buffered saline and total RNA was extracted were performed using Affymetrix reagents designed for in vitro from irradiated and non-irradiated NHMs using the Qiagen RNAeasy transcription amplification and biotin-labeling to prepare targets kit (Valencia, CA). Five micrograms of total RNA were then reverse- for GeneChips brand arrays. Hybridization was carried out transcribed using SuperScriptTM First-strand Synthesis (Invitrogen according to the Affymetrix GeneChips Manual. Twenty micro- Life Technologies, Carlsbad, CA). For quantitative PCR, 3 ml of the grams of labeled, fragmented in vitro transcription material was the reverse-transcribed product (diluted 1:8) was then used in a 50 ml nominal amount used on the GeneChips arrays. A model 320 reaction along with 0.25 mlofTaq,5ml of a 1:10 dilution of 10 Â hybridization oven was used to incubate the arrays at a constant PCR reaction, 4 ml 2.5 mM dNTP (all from TaKara, Fisher Scientific, temperature of 451C overnight. Preparation of microarrays for Hampton, NH) and 1:20 dilution of the Taqmans Gene Expression scanning was carried out with Affymetrix wash protocols on a Assay primers. Real-time PCR was performed using the MX4000 Model 450 Fluidics station under the control of Affymetrix GeneChip (Stratagene, La Jolla, CA) cycling 2 minutes at 501C, 10 minutes at Operating Software. 951C, 1 minute at 60–621C for 40 cycles. Serial dilutions for each Scanning was carried out on an Affymetrix Model 3000 scanner primer set was used to determine efficiency and quantitation of with an autoloader. We used the Affymetrix GeneChip Operating target gene product relative to endogenous control was obtained

Software operating system to control the Model 3000 scanner using the Pfaffl equation (Pfaffl, 2001) with Etarget and Eref being data acquisition functions, first-level data analysis and desktop data the efficiencies (E ¼ 2.0 at 100% efficiency) of the target and

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endogenous control, respectively: Chang NS, Doherty J, Ensign A (2003) JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1- DCTtargetðnonUVÀUVÞ mediated apoptosis. J Biol Chem 278:9195–202 ðEtargetÞ ðE ÞDCTrefðnonUVÀUVÞ Chang NS, Doherty J, Ensign A, Schultz L, Hsu LJ, Hong Q (2005) WOX1 is ref essential for tumor necrosis factor-, UV light-, staurosporine-, and where Eref and Etarget are the efficiencies of the endogenous control p53-mediated cell death, and its tyrosine 33-phosphorylated form and target gene, respectively, and C is the threshold cycle. binds and stabilizes serine 46-phosphorylated p53. J Biol Chem 280: T 43100–8 Cheung PK, Woolcock B, Adomat H, Sutcliffe M, Bainbridge TC, Jones EC CONFLICT OF INTEREST et al. (2004) Protein profiling of microdissected prostate tissue links The authors state no conflict of interest. growth differentiation factor 15 to prostate carcinogenesis. 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