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Upregulation of UGT2B4 Expression by 3’-Phosphoadenosine 5’-Phosphosulfate Synthase

Knockdown: Implications for Coordinated Control of Bile Acid Conjugation

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Kathleen G. Barrett, Hailin Fang, Daniela Cukovic, Alan A. Dombkowski, Thomas A. Kocarek, dmd.aspetjournals.org and Melissa Runge-Morris

Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B., at ASPET Journals on October 2, 2021 H.F., T.A.K, M.R.-M.) and Department of Pediatrics, Wayne State University, Detroit, Michigan

(D.C. and A.A.D.).

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Running Title: UGT2B4 upregulation by PAPS deficiency

Address correspondence to: Dr. Melissa Runge-Morris, Institute of Environmental Health

Sciences, 259 Mack Avenue, Room 5148, Wayne State University, Detroit, MI 48201, USA. Tel:

(313) 577-5598; FAX: (313) 577-0082; E-mail: [email protected]

Number of text pages: 39 Downloaded from Number of tables: 0 (4 Supplemental)

Number of figures: 11 (4 Supplemental)

Number of references: 56 dmd.aspetjournals.org Number of words in Abstract: 244

Number of words in Introduction: 449

Number of words in Discussion: 1474 at ASPET Journals on October 2, 2021

ABBREVIATIONS: ANOVA, analysis of variance; BA, bile acid; CDCA, chenodeoxycholic acid;

Ct, cycle threshold; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s Modified Eagle Medium; EU, ethynyl uridine; FBS, fetal bovine serum; FXR, farnesoid X ; FXRE, FXR response element; GW4064, 3-(2,6-dichlorophenyl)-4-(3’-carboxy-2-chlorostilben-4-yl)oxymethyl-5- isopropylisoxazole; HBSS, Hanks’ Balanced Salt Solution; HD, homeodomain; HDCA, ; HRP, horseradish peroxidase; LS, linker scanning; LCA, lithocholic acid;

Luc, firefly luciferase; NT, nontargeting; PAPS, 3’-phosphoadenosine-5’-phosphosulfate;

PAPSS, 3’-phosphoadenosine-5’-phosphosulfate synthase; PXR, ; qRT-

PCR, quantitative reverse transcription-polymerase chain reaction; shRNA, short hairpin RNA; siRNA, small interfering RNA; SNP, single nucleotide polymorphism; SULT, cytosolic sulfotransferase; SULT2A, hydroxysteroid sulfotransferase; Sult2a1, mouse cytoplasmic sulfotransferase 2a1; SULT2A1, cytoplasmic sulfotransferase 2A1; UGT, UDP- 2

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; UGT2B4, human UDP-glucuronosyltransferase 2B4; 3’-UTR, 3’- untranslated region; WT, wild-type. Downloaded from dmd.aspetjournals.org at ASPET Journals on October 2, 2021

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Abstract

During cholestasis, the bile acid-conjugating , cytosolic sulfotransferase 2A1

(SULT2A1) and UDP-glucuronosyltransferase 2B4 (UGT2B4), work in concert to prevent the accumulation of toxic bile acids. To understand the impact of sulfotransferase deficiency on human hepatic expression, we knocked down 3’-phosphoadenosine 5’-phosphosulfate synthases (PAPSS) 1 and 2, which catalyze synthesis of the obligate sulfotransferase , in HepG2 cells. PAPSS knockdown caused no change in SULT2A1 expression; however Downloaded from

UGT2B4 expression increased markedly (~41-fold increase in UGT2B4 mRNA content).

Knockdown of SULT2A1 in HepG2 cells also increased UGT2B4 expression. To investigate the dmd.aspetjournals.org underlying mechanism, we transfected PAPSS-deficient HepG2 cells with a luciferase reporter plasmid containing ~2 Kb of the UGT2B4 5’-flanking region, which included a response element for the bile acid-sensing , (FXR). FXR activation or at ASPET Journals on October 2, 2021 overexpression increased UGT2B4 promoter activity; however, knocking down FXR or mutating or deleting the FXRE did not significantly decrease UGT2B4 promoter activity. Further evaluation of the UGT2B4 5’-flanking region indicated the presence of distal regulatory elements between nucleotides -10090 and -10037 that negatively and positively regulated UGT2B4 transcription. Pulse-chase analysis showed that increased UGT2B4 expression in PAPSS- deficient cells was attributable to both increased mRNA synthesis and stability. Transfection analysis demonstrated that the UGT2B4 3’-untranslated region decreased luciferase reporter expression less in PAPSS-deficient cells than in control cells. These data indicate that knocking down PAPSS increases UGT2B4 transcription and mRNA stability as a compensatory response to the loss of SULT2A1 activity, presumably to maintain bile acid-conjugating activity.

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Introduction

Cytosolic sulfotransferases (SULTs) and UDP- (UGTs) are biotransformation enzymes that catalyze the conjugation of a variety of xenobiotics and endogenous compounds to sulfonate or glucuronate moieties. SULTs and UGTs recognize similar substrates and are regulated by nuclear signaling pathways involved in normal metabolic processes suggesting some coordinated action. For example, SULT2A1 (hydroxysteroid sulfotransferase) and UGT2B4 metabolize steroids and bile acids (BAs) and play important Downloaded from roles in bile acid homeostasis. Due to the cytotoxic properties of BAs, bile homeostasis is a tightly regulated process. During pathophysiological conditions where bile flow is obstructed dmd.aspetjournals.org (i.e., cholestasis), urinary elimination of BAs increases to circumvent the accumulation of intracellular BAs and damage, and more urinary BAs are in the sulfonated and glucuronidated forms (van Berge Henegouwen et al., 1976; Pillot et al., 1993; Takikawa et al., at ASPET Journals on October 2, 2021 1986). Under these conditions enhanced metabolism by SULT2A1 and UGT2B4 acts as a defensive mechanism preventing BA cytotoxicity.

Recently we reported the upregulation of hepatic Sult2a1 in hyposulfatemic NaS1 null mice (Barrett et al., 2013). These mice are unable to reabsorb sulfate and display physiological alterations that include elevated serum bile acid levels and altered hepatic lipid metabolism

(Dawson et al., 2003; Dawson et al., 2006). We hypothesized that Sult2a1 upregulation in NaS1 null mice was attributable to reduced hepatic sulfotransferase activity, resulting in increased levels of bile acids able to activate the bile acid-sensing farnesoid X receptor (FXR) in an attempt to restore bile acid homeostasis (Barrett et al., 2013). To investigate this regulatory mechanism, a human liver cell model with diminished sulfonation capacity was created by knocking down 3’-phosphoadenosine 5’-phosphosulfate synthases (PAPSS) 1 and 2 in HepG2 cells (shPAPSS1/2 cells) (Barrett et al., 2013). The suppression of PAPSS1 and 2 would decrease the levels of obligate cofactor and sulfate donor 3’-phosphoadenosine 5’-

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phosphosulfate (PAPS) and reduce cellular sulfotransferase activity (Klaassen and Boles,

1997). Higher mouse Sult2a1 promoter activity was observed when reporter constructs containing an intact inverted repeat of AGGTCA with 0 intervening nt motif were transiently transfected into the shPAPSS1/2 cells compared to control cells. However, a species difference was noted in that endogenous SULT2A1 was not upregulated in PAPSS1/2 double knockdown

HepG2 cells, whereas the amount of UGT2B4 mRNA was significantly increased. UGT2B4 is the predominant UGT in human liver that conjugates bile acids and is a known target of FXR

(Pillot et al., 1993; Barbier et al., 2003; Izukawa et al., 2009; Ohno and Nakajin, 2009; Court et Downloaded from al., 2012). The upregulation of UGT2B4 may be a human-specific compensatory response to the loss of bile acid sulfonation to prevent liver damage in the event of sulfate depletion. In this dmd.aspetjournals.org study we investigate the mechanism(s) responsible for the PAPSS1/2 knockdown-mediated upregulation of human UGT2B4. at ASPET Journals on October 2, 2021

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Materials and Methods

Cell Culture. HepG2 cells were engineered for stable knockdown of PAPSS1 and

PAPSS2 (shPAPSS1/2 cells) or SULT2A1 (shSULT2A1 cells) as described previously (Barrett et al., 2013). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with non-essential amino acid mix, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) (all purchased from Life Technologies, Grand Island, NY). Cells were Downloaded from maintained under a humidified atmosphere of 95% air, 5% CO2 at 37˚C.

Microarray Analysis. Total RNA was prepared from four independent batches of cultured HepG2 (shNT and shPAPSS1/2) cells using the RNeasy Mini Kit (Qiagen, Valencia, dmd.aspetjournals.org CA) and evaluated for quality using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara,

CA). The RNA samples were labeled with Alexa 647 or Alexa 555, using the Agilent Low Input

Fluorescent Linear Amplification Kit. Microarray analysis was performed using Human v.2. GE at ASPET Journals on October 2, 2021 4x44K Microarrays (Agilent Technologies), using a basic two-color hybridization design to determine relative levels in shPAPSS1/2 vs. shNT samples. Dye swaps were performed to account for dye bias effects such that for the four arrays, in two of the arrays the shNT samples were labeled with Alexa 647 and the shPAPSS1/2 samples were labeled with

Alexa 555, while in the other two arrays the dye orientation was reversed. Microarrays were scanned using the Agilent dual laser DNA microarray scanner, model G2565AA, and image analysis was performed using Agilent Feature Extraction software. Outlier features having aberrant image characteristics were flagged and excluded from subsequent analysis.

Fluorescent intensity values were adjusted using local background subtraction. For each probe on the array, a log2 ratio was calculated, representing the relative abundance of transcript in shPAPSS1/2 cells relative to shNT cells. Statistical analysis of microarray data was performed using GeneSpring, version 7.3. The data from the 4 replicate arrays were analyzed using t-tests

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(against zero) and the Benjamini and Hochberg multiple test correction to control the false discovery rate to 5%.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Analysis of UGT2B4 and SULT2A1 Expression. Total RNA was prepared from cultured

HepG2 clones using the Purelink RNA Mini Kit (Ambion/Life Technologies). RNA (1.5 μg) was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit according to the manufacturer’s instructions (Life Technologies). UGT2B4 and SULT2A1 mRNA levels were measured using TaqMan Gene Expression Assays Hs00607514_mH and Downloaded from

Hs00234219_m1, respectively (Life Technologies) and a StepOnePlus Real Time PCR System

(Applied Biosystems/Life Technologies) as previously described (Barrett et al., 2013). dmd.aspetjournals.org Western Blot Analysis of SULT2A1 and UGT2B4. Whole cell lysates were prepared as previously described (Rondini et al., 2014). concentrations were quantified using the bicinchoninic acid (BCA) protein assay according to the manufacturer’s instructions (Thermo at ASPET Journals on October 2, 2021 Fisher, Rockford, IL). Lysate (15-30 μg) were resolved by SDS-PAGE, using 10 or

12.5% acrylamide gels and transferred onto polyvinylidene difluoride membranes. For

SULT2A1, membranes were developed using a mouse monoclonal anti-SULT2A1 antibody

(clone 4D7; Origene, Rockville, MD) diluted 1:5000 followed by horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA) diluted

1:20,000. The blots were reprobed with goat polyclonal β- antibody (ab21057; Abcam,

Cambridge, MA) diluted 1:1000 followed by HRP-conjugated donkey anti-goat IgG (sc-2020;

Santa Cruz Biotechnology) diluted 1:8000 or with mouse monoclonal α-tubulin antibody (sc-

5286, Santa Cruz Biotechnology) diluted 1:250 followed by HRP-conjugated goat anti-mouse

IgG diluted 1:5000. UGT2B4 was detected using rabbit polyclonal anti-UGT2B4 antibody (15425

1-AP; Proteintech, Chicago, IL) diluted 1:750 followed by HRP-conjugated goat anti-rabbit IgG

(sc-2030; Santa Cruz Biotechnology) diluted 1:20,000. The blot was reprobed with mouse monoclonal anti-β-actin (clone AC15; Sigma-Aldrich) diluted 1:30,000 or 1:40,000 followed by

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HRP-conjugated goat anti-mouse IgG (sc-2005; Santa Cruz Biotechnology) diluted 1:75,000.

Immunoreactive proteins were detected by enhanced chemiluminescence and visualized on X- ray film. Band densities were determined using AlphaView software (Protein Simple; San Jose,

CA). Data are reported as mean ratios of UGT2B4/β-actin ± S.E.M.

Preparation of UGT2B4 Reporter Plasmids. A fragment spanning from ~2 Kb upstream to 13 nt downstream of the UGT2B4 transcription start site (considered as the 5’ position of NCBI RefSeq NM_021139.2) was amplified by PCR using Herculase II Fusion

Enzyme (Agilent Technologies) and genomic DNA from MCF10A cells as template. The primer Downloaded from set utilized is listed in Supplemental Table 1. The amplified ~2 Kb fragment was digested with

XhoI and BglII and ligated into the promoterless pGL4.10 [luc2] luciferase reporter vector dmd.aspetjournals.org (Promega Corporation, Madison, WI) resulting in the (-1991:+13)-UGT2B4-Luc reporter plasmid. This plasmid served as the template to create a series of deletion constructs containing nt -1648:+13, -1119:+13, -804:+13, -484:+13, and -112:+13 (primer sets are listed in at ASPET Journals on October 2, 2021 Supplemental Table 1).

Reporter constructs containing a series of upstream fragments (~2000 nt each) of the

UGT2B4 gene 5’-flanking region extending ~16 Kb upstream from the transcription start site were prepared from genomic DNA using PCR (primer sets listed in Supplemental Table 1). After

PCR, fragments were digested with SacI and XhoI and ligated into the construct (-112:+13)-

UGT2B4-Luc, which provided UGT2B4 core promoter elements. Additional deletion constructs were prepared using the primer sets, plasmid templates, and oligonucleotide pairs indicated in

Supplemental Table 1.

Preparation of an FXR-responsive reporter plasmid (FXRE-Luc) has been described previously (Kocarek and Mercer-Haines, 2002).

Site-Directed Mutagenesis of FXR Response Element (FXRE). An FXRE located at nt -1193 to -1187 of the UGT2B4 5’-flanking region (Barbier et al., 2003) was mutated using (-

1991:+13)-UGT2B4-Luc as template and the QuikChange II XL site directed mutagenesis kit

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(Agilent Technologies) according to the manufacturer’s instructions. The mutagenic primers are listed in Supplemental Table 1.

Preparation of Linker Scanning (LS) Mutants of the (-10503: -10037)(-112:+13)-

UGT2B4-Luc plasmid. Linker scanning mutants spanning nt -10090 to -10037 in fragment (-

10503:-10037) (Supplemental Figure 1) were prepared using the method described by Gustin and Burk (2000), replacing successive 6 nt sections with BamHI sites (GGATCC). Briefly, three

PCR reactions were performed for each mutant. Initially PCR reactions 1 and 2 were performed using LS primers 1 and 2 or LS primers 3 and 4 (Supplemental Table 1) respectively, the (- Downloaded from

10503:-10037)(-112:+13)-UGT2B4-Luc plasmid as template, and HotStar HiFidelity DNA polymerase (Qiagen). Each product from PCR reaction 1 and 2 was gel-purified and digested dmd.aspetjournals.org with BamHI, and the digested products were combined and ligated together. The ligated product then served as template for the third PCR reaction using primers 1 and 4. The final 662 nt fragment was gel-purified, digested with SacI and HindIII, and ligated into the SacI/HindIII- at ASPET Journals on October 2, 2021 digested and gel-purified pGL4.10 [luc2] luciferase reporter vector.

The sequences of all reporter constructs were verified using the services of the Wayne

State University Applied Genomics Technology Center (Detroit, MI).

Preparation of UGT2B4-3’-Untranslated Region (3’-UTR) Reporter Plasmid. The

SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA) was used to clone the

3’-region of the UGT2B4 mRNA sequence including 155 nt of the coding region and the entire

3’-UTR (UGT2B4 gene-specific primer listed in Supplemental Table 1). RNA isolated from shPAPSS1/2 cells served as the template. The blunt ends of the PCR product were modified by

A-Tailing with GoTaq DNA Polymerase according to the manufacturer’s instructions (Promega), and the modified fragment was ligated into pGEM-T Easy (Promega). This vector served as the template to amplify the UGT2B4 3’-UTR specifically using Herculase II Fusion . The primer set utilized is listed in Supplemental Table 1. The fragment was digested with SacI and

XhoI and ligated into the pmirGLO Dual-Luciferase miRNA Target Expression Vector

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(Promega), resulting in the UGT2B4-3’-UTR-Luc reporter plasmid. Computational analysis of the UGT2B4 3’-UTR for predicted microRNA binding sites was performed using 10 algorithms available through miRWalk (Dweep et al., 2011) (http://www.umm.uni- heidelberg.de/apps/zmf/mirwalk/predictedmirnagene.html).

Transient Transfection Analysis. Approximately 250,000 shNT and shPAPSS1/2 cells were seeded into the wells of 12-well plates and cultured in 1 ml of supplemented DMEM. Forty- eight hours later, culture medium was replaced with 1 ml of Opti-MEM I Reduced Serum

Medium (Life Technologies). Then 200 μl was added of a premixed complex of 4 μl Downloaded from

Lipofectamine 2000 (Life Technologies) and plasmid DNA consisting of selected combinations of the following: 1500 ng of FXRE-Luc or a UGT2B4-Luc reporter plasmid, 150 ng pGL3- dmd.aspetjournals.org Promoter (Promega), 50 ng pcDNA3.1 (Life Technologies), 50 ng FXR expression plasmid

(Origene), 1 ng pRL-SV40 (Promega), and sufficient pBluescript II KS+ (Agilent Technologies) to keep the total amount of DNA constant among samples. Six or 24h later, the transfection at ASPET Journals on October 2, 2021 medium was replaced with fresh supplemented DMEM alone or containing 0.1% dimethyl sulfoxide (DMSO), 10 μM 3-(2,6-dichlorophenyl)-4-(3’-carboxy-2-chlorostilben-4-yl)oxymethyl-5- isopropylisoxazole (GW4064), or 100 μM chenodeoxycholic acid (CDCA). The cells were harvested the next day for measurement of firefly and Renilla luciferase activities using the Dual

Luciferase Reporter Assay System and a GloMax Luminometer (Promega). Transfection data from the shNT and shPAPSS1/2 cell lines were normalized and analyzed as previously described (Barrett et al., 2013).

RNA Interference. shNT and shPAPSS1/2 cells were cultured as described above.

Forty-eight hours after seeding, culture medium was replaced with 1 ml of Opti-MEM I Reduced

Serum Medium. Then 200 μl was added of a premixed complex of 4 μl Lipofectamine 2000, plasmid DNA, and siRNA consisting of selected combinations of the following: 10 pmol FXR siRNA or nontargeting siRNA (ON-TARGETplus SMART pools, Thermo Scientific, Waltham,

MA), 1450 ng FXRE-Luc or (-1991:13)-UGT2B4-Luc, 150 ng pGL3-Promoter (Promega), 1 ng

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pRL-SV40 (Promega), and sufficient pBluescript II KS+ (Agilent Technologies) to keep the total amount of nucleic acid constant among samples. The following day, transfection medium was replaced with 1 ml of fresh supplemented DMEM containing 0.1% DMSO or 10 μM GW4064.

Forty-eight hours post-transfection, cells were harvested for measurement of firefly and Renilla luciferase activity.

UGT2B4 mRNA Synthesis and Stability. UGT2B4 mRNA synthesis and stability in shNT and shPAPSS1/2 cells were assessed by pulse-chase analysis, using the Click-iT

Nascent RNA Capture Kit essentially according to the manufacturer’s instructions (Life Downloaded from

Technologies). shNT and shPAPSS1/2 cells were incubated with DMEM containing the uridine analog ethynyl uridine (EU, 0.5 mM) for 1h. A portion of the cells was then harvested and total dmd.aspetjournals.org RNA was isolated for estimation of mRNA synthesis. The remaining cells were then rinsed with

HBSS and medium was replaced with EU-free DMEM. Cells were then harvested during this chase period at time points ranging from 2 to 10h after replacing the medium, and total RNA at ASPET Journals on October 2, 2021 was isolated. Newly synthesized EU-labeled RNA was biotinylated and then captured with streptavidin Dynabeads. cDNA was synthesized from the captured EU-labeled RNA and

UGT2B4 mRNA levels were measured by qRT-PCR. For estimation of UGT2B4 half-life, the relative level of EU-labeled UGT2B4 mRNA at each chase time point was calculated relative to the amount at the first chase time point, and the slope of the ln(UGT2B4 mRNA) vs. time plot was calculated. Assuming first-order decay kinetics, t½ = 0.693/-slope.

Statistical Analysis. qRT-PCR and promoter transfection data were analyzed using

Student’s t-test or one-way analysis of variance (ANOVA) followed by the Neuman-Keuls multiple comparison test. p<0.05 was considered statistically significant.

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Results

UGT2B4 Expression in PAPSS1/2-Deficient HepG2 Cells. Microarray analysis indicated that 2,944 were significantly differentially expressed in shPAPSS1/2 cells as compared to shNT cells. Supplemental Table 2 shows the microarray results for the differentially expressed genes with fold differences of 2 or greater. The microarray analysis found no significant difference in SULT2A1 mRNA levels between the cell lines; however, UGT2B4 mRNA levels were 23.8-fold higher in shPAPSS1/2 cells than in shNT cells. The only other Downloaded from significant differences in UGT mRNA levels that were observed in the shPAPSS1/2 relative to the shNT cells were (1) an 18.7-fold increase in a sequence described as “UGT2B28 precursor,” dmd.aspetjournals.org (2) a 6.95-fold increase in UGT2A1, and (3) a 1.31-fold decrease in UGT2A3. In agreement with the microarray data, qRT-PCR and western blot analyses indicated no difference in SULT2A1 mRNA (Figure 1A) or protein (Figure 1B) content between the shPAPSS1/2 and shNT cells. at ASPET Journals on October 2, 2021 Figure 2A shows that UGT2B4 mRNA content was ~41-fold (40.7 ± 22.7) higher in shPAPSS1/2 cells than in shNT cells, while Figure 2B shows that the amount of UGT2B4 protein was also significantly increased in the shPAPSS1/2 cells.

UGT2B4 Expression in SULT2A1-Deficient HepG2 Cells. SULT2A1 is the only human

SULT that catalyzes bile acid sulfonation (Comer et al., 1993) and along with UGT2B4 plays an important role in hepatic bile acid homeostasis (Pillot et al., 1993). To determine whether knocking down SULT2A1 specifically would recapitulate the effect of global sulfotransferase suppression on UGT2B4, UGT2B4 expression was assessed in a HepG2 clone stably expressing a SULT2A1-targeting shRNA (shSULT2A1, described previously Barrett et al.,

2013). UGT2B4 mRNA content was ~7-fold (6.6 ± 6.1) higher in shSULT2A1 cells compared to control cells (shNT) (Figure 3A) and UGT2B4 protein content was also significantly higher

(Figure 3B).

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Lack of a Role for FXR in PAPSS Knockdown-Mediated Upregulation of UGT2B4.

UGT2B4 is a known target gene of FXR (Barbier et al., 2003) and we previously found that FXR activation was ~2.5-fold higher in shPAPSS1/2 cells compared to control cells (Barrett et al.,

2013). To examine whether FXR activation plays a role in the significant upregulation of

UGT2B4 expression in shPAPSS1/2 cells, a luciferase reporter plasmid containing ~2 Kb of the

UGT2B4 5’-flanking region, which includes an FXRE at nucleotides -1193 to -1187 (Barbier et al., 2003), was transiently transfected into shNT and shPAPSS1/2 cells. Figure 4 shows that (-

1991:+13)-UGT2B4-Luc activity was ~4-fold higher when transiently transfected into Downloaded from shPAPSS1/2 cells than when transfected into shNT cells. Treatment of transfected shPAPSS1/2 cells with an FXR agonist, GW4064 or CDCA, for 24h increased UGT2B4 promoter activity ~4- dmd.aspetjournals.org fold and ~2-fold, respectively, compared to DMSO-treated shPAPSS1/2 cells (Figure 5A). Also, co-transfection of shPAPSS1/2 cells with an FXR expression plasmid increased (-1991:+13)-

UGT2B4-Luc activity ~4-fold compared to co-transfection with empty expression vector (Figure at ASPET Journals on October 2, 2021 5B).

To determine whether UGT2B4 transcriptional activation in HepG2 cells with limited sulfonation capacity is mediated through FXR, FXR was knocked down in shPAPSS1/2 cells.

Co-transfection of shPAPSS1/2 cells with an siRNA targeting FXR reduced GW4064-mediated activation of an FXR-responsive reporter plasmid (FXRE-Luc) by 97% (Figure 6A), demonstrating effective knockdown of FXR. However, transient silencing of FXR did not diminish UGT2B4 reporter expression (Figure 6B). Mutation of the UGT2B4 FXRE (Mut FXRE) caused a non-significant reduction in luciferase activity of 22%, relative to UGT2B4 reporter containing the wild-type FXRE (WT FXRE) (Figure 7). However, Mut FXRE reporter activity in shPAPSS1/2 cells was still 3-fold higher than was WT-FXRE activity in shNT cells. These data indicate that FXR agonist treatment and FXR overexpression can activate the (-1991:+13)-

UGT2B4-Luc reporter. However, FXR does not appear to be responsible for the up-regulation of

UGT2B4 that occurs in shPAPSS1/2 cells.

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Transcriptional Regulation of UGT2B4 in PAPSS1/2-Deficient HepG2 Cells. To search for other possible cis-acting elements in the (-1991:+13)-UGT2B4-Luc reporter that could be responsible for conferring PAPSS1/2 deficiency-mediated UGT2B4 upregulation, a series of luciferase reporter plasmids containing UGT2B4 promoter fragments ranging in size from 1661 to 126 nt was evaluated in shPAPSS1/2 cells and shNT cells (Figure 8). The pattern of activity for all 5 constructs was the same in both cell lines, although the luciferase activity of each reporter was ~3-fold higher in shPAPSS1/2 cells than in shNT cells. Deletion of the FXRE, which resides in the 529 nt sequence upstream of the (-1119:+13) fragment, did not reduce Downloaded from promoter activity, but rather increased activity. In fact, activity of (-1119:+13)-UGT2B4-Luc was significantly higher compared to all other constructs in shNT and shPAPSS1/2 cells. These data dmd.aspetjournals.org do not clearly implicate any region for the upregulation of UGT2B4 expression in shPAPSS1/2 cells, but suggest that activity may be conferred within the proximal 112 nt.

To evaluate whether regions that contribute to UGT2B4 upregulation in shPAPSS1/2 at ASPET Journals on October 2, 2021 cells are located farther upstream than 2 Kb, we examined up to ~16Kb of the 5’-flanking region of the UGT2B4 gene. Each successive upstream fragment was ~2Kb in size and all upstream fragments were ligated into (-112:+13)-UGT2B4-Luc as a common core promoter. Figure 9 shows the luciferase activities after the reporter series was transiently transfected into shPAPSS1/2 cells or shNT cells. For analysis of these data, luciferase activities were normalized to the activity of the core promoter reporter (-112:+13)-UGT2B4-Luc within each cell line, to eliminate any contribution of the (-112:+13) fragment to differences between cell lines.

When the data were analyzed in that manner, most constructs showed little or no difference in activity between the shNT and shPAPSS1/2 cell lines. The exception was (-12072:-10037)(-

112:+13)-UGT2B4-Luc, which showed ~2-fold higher activity in shPAPSS1/2 cells than in shNT cells. The activity of this construct in shNT cells was also higher than that of other constructs containing more proximal UGT2B4 5’-flanking regions. These data suggest that the (-12072:-

10037) region of the UGT2B4 gene contains information that (1) positively regulates UGT2B4

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transcription and (2) increases transcription to a greater extent in shPAPSS1/2 cells than in shNT cells.

Progressively refined deletion series were prepared to identify the region within the (-

12072:10037) fragment that conferred enhanced UGT2B4 promoter activity in shNT and shPAPSS1/2 cells (Supplemental Figures 2, 3, and 4). The data show that the UGT2B4 information conferring increased luciferase activity is contained within the -10111:-10037 region.

Supplemental Figure 4 also shows that when (-10111:-10037) was shortened by 31 nt, the promoter activity increased ~2-fold, suggesting the presence of a suppressive element between Downloaded from nt -10111 and -10080.

Linker scanning mutagenesis analysis was then used to identify the sequence(s) that dmd.aspetjournals.org conferred enhanced transcriptional activity (Figure 10). Linker scanning mutants LS1 through

LS9 were prepared to span nt -10090 to -10037 using the (-10503:-10037)(-112:+13)-UGT2B4-

Luc reporter as template (schematic representation shown in Supplemental Figure 1). at ASPET Journals on October 2, 2021 shPAPSS1/2 cells and shNT cells were transfected with mutants LS1 through LS9, as well as with constructs (-10503:-10037)(-112:+13)-UGT2B4-Luc, (-10111:-10037)(-112:+13)-UGT2B4-

Luc, and (-10080:-10037)(-112:+13)-UGT2B4-Luc for comparison. Reporter activity was normalized to the activity of the (-112:+13)-UGT2B4-Luc reporter within each cell line to exclude any contribution of the core promoter region to differences between the cell lines.

Replacing nt -10090 to -10085 with a BamHI site (LS1 mutant) resulted in a significant increase in luciferase activity relative to constructs (-10503:-10037) and (-10111:-10037) (Figure

10). The luciferase activity of LS1 was comparable to that of the (-10080:-10037)(-112:+13)-

UGT2B4-Luc deletion construct, and activity was ~2-fold higher in shPAPSS1/2 cells than in shNT cells. Promoter activity was reduced somewhat in mutants LS2 through LS6 compared to constructs (-10503:-10037) and (-10111:-10037), and activity in LS2 through LS6 was ~1.2- to

2-fold higher when transfected into shPAPSS1/2 cells compared to shNT cells. Disrupting any of the last 18 nt of the (-10503:-10037) fragment (mutants LS7, LS8 or LS9) reduced luciferase

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activity to core promoter reporter levels. Therefore, these data identified two distal cis-regulatory regions, (1) between nt -10090 to -10085 and (2) between nt -10054 to -10037, in the 5’-flanking region of the UGT2B4 gene that negatively and positively regulated transcription. Computational analysis was performed on nt -10090 to -10037 to identify putative binding sites (MatInspector software; Genomatrix, Inc., Cincinnati, OH) (Quandt et al., 1995; Cartharius et al., 2005). Supplemental Table 3 lists the matrix, description, strand, core and matrix similarity, sequence, and core sequence location in the LS mutants. Twenty-four of the 32 predicted binding sites are for homeodomain (HD) proteins, and several of these are located Downloaded from within the LS1 mutation that marked a suppressive element or within the LS7-9 mutations that indicated an enhancer(s). The LS2-6 mutations, which all partially reduced reporter expression, dmd.aspetjournals.org also contained predicted binding sites for HD proteins, such as the POU class 2 1

(OCT1) (in LS2 and 3), which has been implicated in the regulation of UGT2B7 (Ishii et al.,

2000). The LS2-6 region also contained sites for other transcription factors that could be at ASPET Journals on October 2, 2021 expected to contribute to UGT2B4 transcription, such as the liver-enriched transcription factors, forkhead box factors (in LS3) and CCAAT/enhancer binding protein α (in LS3 and 4), and the lipid-activated nuclear receptor peroxisome proliferator-activated receptor γ (in LS4 and 5).

Regulation of UGT2B4 mRNA Synthesis and Stability in PAPSS1/2-Deficient

HepG2 Cells. To determine whether increased transcript stability contributes to the significant upregulation of UGT2B4 mRNA in HepG2 cells with diminished sulfonation capacity we analyzed de novo mRNA synthesis and decay by pulse-chase analysis. Following EU pulse labeling, UGT2B4 mRNA synthesis was 15.6 ± 4.8-fold higher in shPAPSS1/2 cells than in shNT cells (mean ± S.E.M. of 7 independent experiments). The decreases in EU-labeled mRNA levels during the chase period indicated that the half-life of UGT2B4 mRNA in shNT cells was

2.23 ± 0.35h whereas the half-life in shPAPSS1/2 cells was 9.80 ± 4.34 (means ± S.E.M. of 4 experiments). Although the data were somewhat variable across multiple independent experiments, overall they suggest that the accumulation of UGT2B4 mRNA in shPAPSS1/2

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cells is likely attributable to a combination of a higher de novo mRNA synthesis rate and longer half-life.

The 3’-UTR is known to regulate mRNA translation efficiency, stability, and localization

(Guhaniyogi and Brewer, 2001). To determine whether the 3’-UTR of UGT2B4 influences mRNA levels in shPAPSS1/2 cells, we ligated the UGT2B4 3’-UTR region downstream of the firefly luciferase coding region and transfected this reporter or the empty vector into shPAPSS1/2 and shNT cells. Figure 11 shows that the UGT2B4 3’-UTR reduced luciferase activity more in shNT cells (31% reduction relative to empty vector) than in shPAPSS1/2 cells Downloaded from

(21% reduction), indicating that the UGT2B4 3’-UTR exerted a stabilizing effect in the shPAPSS1/2 cells relative to the shNT cells. Taken together with the pulse-chase data, these dmd.aspetjournals.org results suggest that PAPSS knockdown increases the stability of UGT2B4 mRNA through a mechanism that is mediated through its 3’-UTR.

We performed a computational analysis of predicted microRNA binding sites in the 3’- at ASPET Journals on October 2, 2021 UTR of human UGT2B4. To reduce false positives we used a consensus of ten prediction algorithms available through miRWalk (Dweep et al., 2011). This analysis identified 23 that were predicted by at least five algorithms to target UGT2B4 (Supplemental

Table 4).

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Discussion

Dawson et al. (Dawson et al., 2006) and we (Barrett et al., 2013) previously reported that hyposulfatemic mice lacking the renal NaS1 transporter had increased hepatic Sult2a1 expression in an apparent attempt to compensate for reduced sulfonation capacity. Consistent with that interpretation, knockdown of the enzymes responsible for synthesis of PAPS (PAPSS1 and 2), the obligate cofactor for all sulfonation reactions, activated transfected Sult2a1 promoter constructs in HepG2 cells. Specific knockdown of SULT2A1 in HepG2 cells also caused Downloaded from activation of the mouse Sult2a1 promoter, suggesting that transcriptional activation of mouse

Sult2a1 occurs specifically in response to loss of SULT2A1-mediated metabolism of some dmd.aspetjournals.org endogenous molecule. However, a major species difference between mice and was observed because PAPSS1/2 knockdown did not cause up-regulation of endogenous SULT2A1 in HepG2 cells but instead caused marked up-regulation of UGT2B4. These results at ASPET Journals on October 2, 2021 demonstrate a physiological linkage between SULT2A1 and UGT2B4 regulation in humans.

The concept of overlap between SULTs and UGTs is well established since these enzyme systems share many substrates and mechanisms of regulation. Sulfonation is generally considered to be a high-affinity, low-capacity reaction while glucuronidation is a low-affinity, high-capacity reaction (Parkinson et al., 2013). As substrate concentrations increase and

SULTs become saturated or PAPS is depleted, glucuronidation increases (Morris and Pang,

1987; Zamek-Gliszczynski et al., 2006).

Redundancy between conjugation pathways occurs as a protective mechanism during cholestasis. In healthy adults, the biliary pathway is the preferred route for bile acid excretion, and sulfonation is the predominant detoxification pathway (Radominska et al., 1990; Comer et al., 1993). However, under cholestatic conditions serum and urinary bile acid sulfonates and glucuronides increase (Frohling and Stiehl, 1976; van Berge Henegouwen et al., 1976; Stiehl et al., 1980; Meng et al., 1997). The shift to the urinary pathway for bile acid excretion acts as a

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defense mechanism to protect the hepatocytes from the harmful effects of bile acid accumulation. Increased glucuronidation appears to act as a back-up mechanism to detoxify excess bile acids when the sulfonation pathway becomes saturated.

A functional relationship between SULT2A1 and UGT2B4 in bile acid detoxification has been established by their roles in metabolism of the toxic secondary bile acid lithocholic acid

(LCA) (Miyal et al., 1975). SULT2A1 has high activity toward LCA, and catalyzes this reaction with an apparent Km of ~1.5 μM (Radominska et al., 1990). However, SULT2A1 has relatively low activity toward the 6α-hydroxylated metabolite of LCA, hyodeoxycholic acid (HDCA) Downloaded from

(Radominska et al., 1990). By contrast, UGT2B4 has little activity toward LCA but efficiently glucuronidates HDCA (Radominska-Pyrek et al., 1987; Pillot et al., 1993) with a Km of ~25 μM dmd.aspetjournals.org (Barre et al., 2007). The available evidence suggests that LCA 6α-hydroxylation is catalyzed mainly by CYP3A4 with a Km of ~45 μM (Araya et al., 1999; Deo and Bandiera, 2009).

Therefore, under normal physiological conditions, LCA is mainly detoxified by SULT2A1- at ASPET Journals on October 2, 2021 mediated sulfonation, and 6α-hydroxlyated bile acid levels remain low (Summerfield et al., 1976;

Shoda et al., 1990; Wietholtz et al., 1996). Elevation of LCA levels results in increased HDCA formation by CYP3A4-mediated 6α-hydroxylation, a reaction that is facilitated by LCA-mediated activation of PXR (Stedman et al., 2004; Staudinger et al., 2001). The HDCA that is formed is then glucuronidated by UGT2B4.

The bile acid-sensing nuclear receptor FXR controls expression of several genes that are involved in bile acid synthesis, transport, and metabolism (Makishima et al., 1999; Wang et al., 1999; Goodwin et al., 2000; Sinal et al., 2000; Landrier et al., 2006; Kim et al., 2007; Barbier et al., 2003; Song et al, 2001), and we previously reported higher FXR activity in PAPSS1/2- deficient HepG2 cells than in control cells (Barrett et al., 2013). Additionally we attributed activation of murine Sult2a1 in shPAPSS1/2 cells to an FXR-mediated mechanism (Barrett et al., 2013). UGT2B4 has been reported to contain a functional FXRE, although this FXRE is atypical in that it binds FXR as a monomer (Barbier et al., 2003). We confirmed the FXR

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responsiveness of the UGT2B4 promoter by showing that treatment with an FXR agonist or

FXR overexpression activated UGT2B4 promoter activity. However, activation through the reported FXRE could not explain the marked up-regulation of UGT2B4 that occurred in shPAPSS1/2 cells, since neither knocking down FXR nor mutating or deleting the FXRE reduced UGT2B4 promoter activity in shPAPSS1/2 to the level that was observed in control cells.

Our microarray analysis did not indicate that PAPSS knockdown caused widespread regulation of genes that are involved in bile acid homeostasis, although CYP7A1 mRNA content Downloaded from was 2.2-fold higher and ATP-binding cassette, sub-family C, member 2 mRNA content was 1.9- fold lower in the PAPSS1/2 knockdown cells. This suggests that UGT2B4 up-regulation by dmd.aspetjournals.org sulfonation deficiency is not part of the paradigmatic mechanism for controlling bile acid homeostasis but is rather part of a more restricted portion of bile acid regulation that specifically controls conjugation as a detoxifying mechanism. In this regard, several genes involved in bile at ASPET Journals on October 2, 2021 acid conjugation were significantly regulated by PAPSS knockdown (Supplemental Table 2). For example, UGT2A1, which also catalyzes bile acid conjugation (Perreault et al., 2013), was up- regulated 6.95-fold by PAPSS knockdown. Also, CYP3A5 and CYP3A7 were up-regulated (~3- fold). As noted above, CYP3A-mediated catalysis is necessary to convert the bile acids that are good substrates for sulfonation into the 6α-hydroxylated metabolites that are the preferred substrates for glucuronidation. Altogether these gene expression changes suggest that loss of sulfonation causes a shift toward a phenotype that favors bile acid glucuronidation in an attempt to prevent hepatotoxicity.

Evaluation of UGT2B4 5’-flanking sequences upstream of the reported FXRE identified a region approximately 10-12Kb upstream of the transcription start site that augmented UGT2B4 promoter activity in shPAPSS1/2 cells. Deletion and linker scanning mutagenesis analyses identified two closely localized regions, from nt -10090 to -10085 and nt -10054 to -10037, that had opposing effects on UGT2B4 transcription. The more upstream region negatively regulated

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UGT2B4 transcription while the more downstream region positively regulated transcription.

Computational analysis identified a large number of putative HD binding sites within these regions (Supplemental Table 3). We hypothesize that these two regions function together as a composite regulatory module and that at least some of the proteins that interact with this module are sensitive to the sulfonation status of the cell. Detailed analysis is required to identify the specific regulatory proteins that interact with this module and to determine how sulfonation deficiency alters the activities of these factors.

An increase in steady state mRNA level could occur through an increase in the rate of Downloaded from mRNA synthesis, a decrease in the rate of degradation, or a combination of both. Pulse-chase analysis confirmed that increased mRNA synthesis was an important determinant of increased dmd.aspetjournals.org UGT2B4 expression in shPAPSS1/2 cells but also indicated that the half-life of UGT2B4 mRNA was longer in shPAPSS1/2 cells than it was in control cells. This increase in UGT2B4 mRNA stability is likely mediated through the 3’-UTR since reporter assays demonstrated that the at ASPET Journals on October 2, 2021 suppressive effect of the UGT2B4 3’-UTR was less in shPAPSS1/2 cells than in control cells. 3’-

UTRs frequently contain regulatory sequences that can be bound by trans-acting factors, such as RNA-binding proteins and microRNAs, that positively or negatively modulate mRNA stability, localization, or translation efficiency (Guhaniyogi and Brewer, 2001; Mata et al., 2005; Wu and

Brewer, 2012). Little is known about the regulation of UGT2B4 by microRNAs. Of the miRNAs that were computationally predicted to target the UGT2B4 3’-UTR (Supplemental Table 4), at least 5 (miR-629, miR-101, miR-203, miR-223, and miR-216b) have been reported to be expressed in HepG2 cells and regulate such processes as cell proliferation, DNA methylation, hepatocyte nuclear factor 4α activity, cytochrome P450 activities, multidrug resistance, and lipoprotein cholesterol uptake (Hatziapostolou et al., 2011; Huang et al., 2012; Wei W et al.,

2013; Wei X et al., 2013; Takahashi et al., 2014; Yang et al., 2013; Wang et al., 2013; Liu et al.,

2015). Some evidence involving single nucleotide polymorphisms (SNPs) that have been identified in the 3’-UTRs of UGTs suggests that posttranscriptional regulation might contribute to

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interindividual variability in glucuronidation. For example, SNP rs3100 of the UGT2B15 3’-UTR increased expression from a luciferase 3’-UTR reporter, suggesting that this SNP might increase UGT2B15 expression and activity (Sun et al., 2011).

In summary, knocking down sulfonation capacity in a human liver cell model increases

UGT2B4 expression by increasing transcriptional activation and enhancing mRNA stability. The combined effects of increasing both transcription and mRNA stability would result in a greater accumulation of intracellular UGT2B4 mRNA that is available for translation. The upregulation of this important bile acid-conjugating UGT may occur as a compensatory mechanism in response Downloaded from to the loss of sulfotransferase activity to prevent the build-up of bile acids. Further studies are required to elucidate the specific activating and suppressive elements within the 5’-flanking dmd.aspetjournals.org region of the UGT2B4 gene and the 3’-UTR of the UGT2B4 transcript, as well as their respective binding factors, that operate as low sulfate sensors in the upregulation of UGT2B4. at ASPET Journals on October 2, 2021

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Authorship Contributions

Participated in research design: Barrett, Kocarek, Dombkowski, and Runge-Morris

Conducted experiments: Barrett, Fang, and Cukovic

Performed data analysis: Barrett, Dombkowski, and Kocarek

Wrote or contributed to the writing of the manuscript: Barrett, Kocarek, and Runge-Morris Downloaded from dmd.aspetjournals.org at ASPET Journals on October 2, 2021

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Footnotes

This research was supported by the National Institutes of Health National Institute of

Environmental Health Sciences [Grants R01 ES005823 (to M.R.-M.) and Center Grant P30

ES020957]. Downloaded from dmd.aspetjournals.org at ASPET Journals on October 2, 2021

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Figure Legends

Fig. 1. Lack of effect of PAPSS1 and PAPSS2 knockdown on SULT2A1 expression in HepG2 cells. SULT2A1 mRNA (A) and protein (B) levels were measured in shNT and shPAPSS1/2 cells using TaqMan Gene Expression Assays and Western blot analysis, respectively. For panel

A, each bar represents the mean mRNA level ± S.E.M. (relative to shNT cells) from 3 independent experiments. Panel B shows the immunoreactive bands from two independent experiments. Downloaded from

Fig. 2. PAPSS1 and PAPSS2 knockdown increases UGT2B4 expression in HepG2 cells. dmd.aspetjournals.org UGT2B4 mRNA (A) and protein (B, C) levels were measured in shNT and shPAPSS1/2 cells using TaqMan Gene Expression Assays and Western blot analysis, respectively. For panel A, each bar represents the mean mRNA level ± S.E.M. (relative to shNT cells) from 4 independent at ASPET Journals on October 2, 2021 experiments. ***Significantly different from shNT, p<0.001 using ratio paired t-test. Panel B shows the immunoreactive bands from one representative experiment, and panel C shows the densitometrically quantified data from 4 independent experiments; each bar represents the mean ratio of UGT2B4/β-actin ± S.E.M. (relative to shNT cells). **Significantly different from shNT, p<0.01 by paired t-test.

Fig. 3. SULT2A1 knockdown increases UGT2B4 expression in HepG2 cells. UGT2B4 mRNA

(A) and protein (B, C) levels were measured in shNT and shSULT2A1 cells using TaqMan Gene

Expression Assays and Western blot analysis, respectively. For panel A, each bar represents the mean relative mRNA level ± S.E.M. from 3 independent experiments. *Significantly different from shNT, p<0.05 using ratio paired t-test. Panel B shows the immunoreactive bands from one representative experiment, and panel C shows the densitometrically quantified data from 3

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independent experiments; each bar represents the mean ratio of UGT2B4/β-actin ± S.E.M.

(relative to shNT cells). *Significantly different from shNT, p<0.05 by paired t-test.

Fig. 4. UGT2B4 promoter activity is greater in shPAPSS1/2 than shNT HepG2 cells. shNT and shPAPSS1/2 cells were transfected with a luciferase reporter construct containing 1991 nt of the

5’-flanking region of the UGT2B4 gene. Forty-eight hours after transfection, cells were harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. normalized (firefly/Renilla) luciferase activity relative to the activity that was measured for the Downloaded from shNT cells (n=9 wells per group, derived from combining data from three independent experiments with triplicate transfection). ***Significantly different from shNT, p<0.001 by unpaired dmd.aspetjournals.org t-test.

Fig. 5. UGT2B4 promoter response to FXR agonists and FXR overexpression in shPAPSS1/2 at ASPET Journals on October 2, 2021 cells. (A) shPAPSS1/2 cells were transfected with a reporter construct containing 1991 nt of the

5’-flanking region of the UGT2B4 gene. Twenty-four hours after transfection, cells were treated with 0.1% DMSO, 10 μM GW4064, or 100 μM chenodeoxycholic acid (CDCA). (B) shPAPSS1/2 cells were co-transfected with (-1991:+13)-UGT2B4-Luc and FXR expression plasmid or the empty expression vector, pcDNA3.1. Forty eight hours after transfection, cells were harvested and luciferase activities were measured. Each bar represents the mean ± S.D. normalized

(firefly/Renilla) luciferase activity relative to the activity measured in DMSO treated or pcDNA3.1 transfected cells (n=6 wells per group, derived from combining data from two independent experiments with triplicate transfection). **Significantly different from DMSO, p<0.01;

***Significantly different from DMSO or pcDNA3.1, p<0.001 by one-way ANOVA and Neuman-

Keuls test (A) or unpaired t-test (B).

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Fig. 6. Effect of knocking down FXR on UGT2B4 promoter activity in shPAPSS1/2 cells. (A) shPAPSS1/2 cells were transfected with an FXR-responsive reporter plasmid. Half of the cells were co-transfected with nontargeting (NT) siRNA, the other half with siRNA targeting FXR.

Twenty-four hours after transfection cells were treated with 0.1% DMSO or 10μM GW4064. (B) shPAPSS1/2 cells were transfected with a reporter construct containing 1991 nt of the 5’- flanking region of the UGT2B4 gene; designated (-1991:+13)-UGT2B4-Luc. Half of the cells were co-transfected with NT siRNA, the other half with FXR siRNA. All cells were treated with

0.1% DMSO twenty-four hours after transfection. Forty eight hours after transfection, cells were Downloaded from harvested and luciferase activities were measured. Each bar represents the mean ± S.D. normalized (firefly/Renilla) luciferase activity (n=6 wells per group, derived from combining data dmd.aspetjournals.org from two independent experiments with triplicate transfection). In each panel, the mean value for DMSO-treated, NT siRNA-transfected shPAPSS1/2 cell group is defined as 1. Groups not sharing an upper case letter are significantly different from each other, p<0.05 (by one-way at ASPET Journals on October 2, 2021 ANOVA and Neuman-Keuls test).

Fig. 7. Effect of mutating an FXR response element (FXRE) on UGT2B4 promoter activity in shPAPSS1/2 cells. shPAPSS1/2 cells were transfected with a reporter construct containing

1991 nt of the 5’-flanking region of the UGT2B4 gene, designated (-1991:+13)-UGT2B4-Luc, with either the wild type (WT FXRE) or mutated (Mut FXRE) FXRE. shNT cells were transfected with the reporter containing the WT FXRE. Forty eight hours after transfection, cells were harvested and luciferase activities were measured. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements relative to the activity measured in WT

FXRE-transfected shPAPSS1/2 cells (n=6 wells per group, derived from combining data from two independent experiments with triplicate transfection). Groups not sharing an upper case letter are significantly different from each other, p<0.05 (by one-way ANOVA and Neuman-

Keuls test).

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Fig. 8. Promoter activities of a nested deletion series of reporter plasmids constructed from (-

1991:+13)-UGT2B4-Luc. shNT and shPAPSS1/2 cells were transfected with the indicated reporter plasmids. Forty-eight hours after transfection, cells were harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements relative to the activity measure in shNT cells transfected with (-

112:+13)-UGT2B4-Luc (n=6 wells per group, derived from combining data from two independent experiments with triplicate transfection). For shNT cells, groups not sharing an Downloaded from upper case letter are significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by dmd.aspetjournals.org one-way ANOVA and Neuman-Keuls test).

Fig. 9. Transient transfection analysis of the effects of upstream 5’-flanking regions of the at ASPET Journals on October 2, 2021 UGT2B4 gene on promoter activity in shPAPSS1/2 cells and shNT cells. The indicated 5’- flanking regions of the UGT2B4 gene were ligated into the pGL4.10 plasmid upstream of nucleotides -112 to +13, which served as the common proximal promoter for all constructs.

Reporter activities of (-1991:+13)-UGT2B4-Luc, used in previous experiments (labeled -1991),

(-112:+13)-UGT2B4-Luc, and pGL4.10 empty vector are shown for comparison. shNT and shPAPSS1/2 cells were transfected with the reporters and 48 hours later were harvested for measurement of luciferase activities. Each bar represents mean ± S.D. of normalized

(firefly/Renilla) luciferase measurements relative to the activity measured in cells transfected with (-112:+13)-UGT2B4-Luc within each cell line (n=12 wells per group, derived from combining data from four independent experiments with triplicate transfection). For shNT cells, groups not sharing an upper case letter are significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by one-way ANOVA and Neuman-Keuls test).

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Figure 10. Impact of linker scanning mutagenesis on the activity of the (-10503:-10037)(-

112:+13)-UGT2B4-Luc reporter. Linker scanning mutants and two deletion constructs were constructed from (-10503:-10037)(-112:+13)-UGT2B4-Luc. LS1 through LS9 mutants span nt -

10090 to nt -10037. shNT and shPAPSS1/2 cells were transfected with the reporters as indicated. Forty-eight hours later, cells were harvested for measurement of luciferase activities.

Each bar represents mean ± S.D. of normalized (firefly/Renilla) luciferase measurements relative to the activity measured in cells transfected with (-112:+13)-UGT2B4-Luc within each Downloaded from cell line (n=12 wells per group, derived from combining data from four independent experiments with triplicate transfection). For shNT cells, groups not sharing an upper case letter are dmd.aspetjournals.org significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by one-way ANOVA and

Neuman-Keuls test). at ASPET Journals on October 2, 2021

Figure 11. Impact of UGT2B4 3’-untranslated region (3’-UTR) on luciferase activity in shNT and shPAPSS1/2 cells. The UGT2B4 3’-UTR was ligated downstream of the firefly luciferase gene in the pmirGLO luciferase reporter vector. UGT2B4-3’-UTR/pmirGLO or empty vector was transfected into shNT or shPAPSS1/2 cells, and 48 hours later cells were harvested for measurement of luciferase activities. Each bar represents the mean ratio ± S.E.M. (n=8 independent experiments) of normalized (firefly/Renilla) UGT2B4 3’-UTR luciferase measurements relative to the activity measured in cells transfected with the empty vector within each cell line. *Significantly different from shNT, p<0.05 by paired t-test.

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Supplemental Data

Upregulation of UGT2B4 Expression by 3’-Phosphoadenosine 5’-Phosphosulfate Synthase

Knockdown: Implications for Coordinated Control of Bile Acid Conjugation

Kathleen G. Barrett, Hailin Fang, Daniela Cukovic, Alan A. Dombkowski, Thomas A. Kocarek,

and Melissa Runge-Morris

Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (K.G.B.,

H.F., T.A.K, M.R.-M.) and Department of Pediatrics, Wayne State University, Detroit, Michigan

(D.C. and A.A.D.).

Supplemental Table 1: PCR Primers and Oligonucleotides used to prepare constructs in this study

Name Sequence Comments Primers to prepare 2 Kb UGT2B4 Deletion Series:

R 5’ GCGAGATCTGCTCCTTTCTGTCATTTCTCATGGTG 3’ Common to all (-1991:+13) deletions; Bglll site underscored (-1991:+13) F 5’ GCCCTCGAGATGCCACATCCACTAACCCTT 3’ XhoI site underscored (-1648:+13) F 5’ GCCCTCGAGCCACCACATCTGCAGTTATTTCC 3’ (-1119:+13) F 5’ GCCCTCGAGGAAGTGAGTCAGAGAGCAAGC 3’ (-804:+13) F 5’ GCCCTCGAGTAGCAGTTGCCCAGGATAGGA 3’ (-484:+13) F 5’ GCCCTCGAGACATGAGTCAGGTGATGAGTCCA 3’ (-112:+13) F 5’ GCCCTCGAGTGTCTCTTTGTCATCCACATGC 3’

Primers to amplify regions in UGT2B4 5’-flanking regiona:

(-16056:-13956) F 5’ GGGGAGCTCAGCCTACTATGAACAGTTATTAACCA 3’ SacI site underscored R 5’ GGGCTCGAGAATTCCACCTGTTTGTATTTGCT 3’ XhoI site underscored (-14083:-11887) F 5’ GGGGAGCTCAGGACTTGAAACTATAGACTTCCCA 3’ R 5’ GGGCTCGAGACGGGCAAATATTTCATGACGAA 3’ (-12072:-10037) F 5’ GGGGAGCTCATGAAAGTTAGATTTTAGCCAGA 3’ R 5’ GGGCTCGAGTGGGCAAATAATGACTACTCC 3’ (-10075:-7894) F 5’ GGGGAGCTCTTGCCCAAAAGTCATTCAGG 3’ R 5’ GGGCTCGAGCCATAGTGAGGCAGACACC 3’ (-7993:-5496) F 5’ GGGGAGCTCTCTGGCTCCTCAAGGTTT 3’ R 5’ GGGCTCGAGACTAGAGATTTGAAAACACCCT 3’ (-5590:-3453) F 5’ GGGGAGCTCGCCTCCAAGTTGAAATTGCT 3’ R 5’ GGGCTCGAGGTAGATATCTGTCACGTCCAC 3’ (-3486:-1447) F 5’ GGGGAGCTCAACTCTGGATCAAGTGGACG 3’ R 5’ GGGCTCGAGACTCCAGCCCAGATTTCC 3’

Primers and oligos to prepare deletions series within the (-12072:-10037) fragmenta:

R 5’ GGGCTCGAGTGGGCAAATAATGACTACTCC 3’ Common to all deletions; XhoI site underscored (-11524:-10037)b F 5’ GGGGAGCTCTTGGCTCTTGCTGGTGCATA 3’ SacI site underscored (-11114:-10037)b F 5’ GGGGAGCTCGCTGAATTTTATCGAAGGC 3’ (-10767:-10037)b F 5’ GGGGAGCTCGTTTAGTAGAAATAATACCAGGT 3’ (-10503:-10037)b F 5’ GGGGAGCTCGTCAAGTCTGTGCTAATCTCC 3’ (-10391:-10037)c F 5’ GGGGAGCTCAAAATACCCAGATTCTTGA 3’ (-10276:-10037)c F 5’ GGGGAGCTCTTTTGCTCTTGGTTCTCT 3’ (-10159:-10037)c F 5’ GGGGAGCTCGTTTATCCTAGAGATTTTGGCAT 3’ (-10136:-10037)d F 5’ GGGGAGCTCGTTGTATCTTTGTTCTCATTAGTTT 3’

U 5’ CCAAAGATCTTCTTGATTTCTGTCTTAATTTCATTATTTGCCCAAA SacI site 5’overhang & (-10111: -10037) AGTCATTCAGGAGTAGTCATTATTTGCCCAC 3’ XhoI site 3’ overhang underscored L 3’ TCGAGGTTTCTAGAAGAACTAAAGACAGAATTAAAGTAATAAACG

GGTTTTCAGTAAGTCCTCATCAGTAATAAACGGGTGAGCT 5’ (-10080: -10037) U 5’ CATTATTTGCCCAAAAGTCATTCAGGAGTAGTCATTATTTGCCCAC 3’ L 3’ TCGAGTAATAAACGGGTTTTCAGTAAGTCCTCATCAGTAATAAACG

GGTGAGCT 5’

Primers to prepare FXRE mutation:

F 5’ ATGTTTAAGTTATTATCTATAGAACAGTTAAGATaAAaTTTAATCTT GTAACAATGTCTGCATAATTCTAAG 3’ Mutated bases lower case R 5’ CTTAGAATTATGCAGACATTGTTACAAGATTAAAtTTtATCTTAACT GTTCTATAGATAATAACTTAAACAT 3’

Primers to prepare 3’UTR:

(+1480:+2119) F 5’ TTGCAGCCCACGACCTCACCTGGTT 3’ GSP for 3’ RACE (+1635:+2102) F 5’ GGGGAGCTC TTACGTCTGAGGCTGGAAGCTG 3’ SacI site underscored R 5’ GGGCTCGAGACAATTTAATTATTTATTAGCT 3’ XhoI site underscored

Primers to prepare Linker scanning (LS) mutants:

Primer 1 F 5’ GGGGAGCTCGTCAAGTCTGTGCTAATCTCC 3’ Common to all LS mutants; SacI site underscored Primer 4 R 5’ ATCTTCCATGGTGGCTTTAC 3’ Common to all LS mutants

LS1 Primer 2 R 5’ GGGGGATCCCAGAAATCAAGAAGATCTTTGAAACTA 3’ BamHI site underscored LS1 Primer 3 F 5’ GGGGGATCCTTTCATTATTTGCCCAAAAGTCA 3’ LS2 Primer 2 R 5’ GGGGGATCCTTAAGACAGAAATCAAGAAGATCTTT 3’ LS2 Primer 3 F 5’ GGGGGATCCTATTTGCCCAAAAGTCATTC 3’ LS3 Primer 2 R 5’ GGGGGATCCATGAAATTAAGACAGAAATCAAGAAGA 3’ LS3 Primer 3 F 5’ GGGGGATCCCCCAAAAGTCATTCAGGAGT 3’ LS4 Primer 2 R 5’ GGGGGATCCCAAATAATGAAATTAAGACAGAAATCAAG 3’ LS4 Primer 3 F 5’ GGGGGATCCAGTCATTCAGGAGTAGTCAT 3’ LS5 Primer 2 R 5’ GGGGGATCCTTTGGGCAAATAATGAAATTAAGAC 3’ LS5 Primer 3 F 5’ GGGGGATCCTCAGGAGTAGTCATTATTTGCC 3’ LS6 Primer 2 R 5’ GGGGGATCCATGACTTTTGGGCAAATAATGAA 3’ LS6 Primer 3 F 5’ GGGGGATCCGTAGTCATTATTTGCCCACT 3’ LS7 Primer 2 R 5’ GGGGGATCCTCCTGAATGACTTTTGGGC 3’ LS7 Primer 3 F 5’ GGGGGATCCATTATTTGCCCACTCG 3’ LS8 Primer 2 R 5’ GGGGGATCCGACTACTCCTGAATGACTTTTGG 3’ LS8 Primer 3 F 5’ GGGGGATCCTGCCCACTCGAGTGTCT 3’ LS9 Primer 2 R 5’ GGGGGATCCAATAATGACTACTCCTGAATGACTT 3’ LS9 Primer 3 F 5’ GGGGGATCCCTCGAGTGTCTCTTTGTCAT 3’

F, Forward primer; R, Reverse primer; U, Upper oligonucleotide; L, Lower oligonucleotide; GSP, Gene Specific Primer. a. Fragments were ligated into (-112:+13)-UGT2B4-Luc. b. (-12072:-10037)(-112:+13)-UGT2B4-Luc served as the template for this deletion series. c. (-10503:-10037)(-112:+13)-UGT2B4-Luc served as the template for this deletion series. d. (-10159:-10037)( 112:+13)-UGT2B4-Luc served as the template for this deletion series.

Supplemental Table 2. Microarray analysis of gene expression in shPAPSS1/2 cells relative to shNT cells. Annotated genes showing changes of a least 2‐fold are listed. Genes highlighted in yellow are discussed in the text. GeneSymbol GeneName Description GenbankAccession Fold Change Direction Corrected p‐value AMOT angiomotin Homo sapiens angiomotin (AMOT), transcript variant 2, mRNA [NM_133265] NM_133265 77.95 up 0.0149 LOC100130179 similar to env protein PREDICTED: Homo sapiens similar to env protein (LOC100130179), miscRNA [XR_038353 XR_038353 76.77 up 0.0295 AQP9 aquaporin 9 Homo sapiens aquaporin 9 (AQP9), mRNA [NM_020980] NM_020980 76.53 up 0.0081 PNMAL1 PNMA‐like 1 Homo sapiens PNMA‐like 1 (PNMAL1), transcript variant 1, mRNA [NM_018215] NM_018215 72.26 up 0.0101 DPYD dihydropyrimidine dehydrogenase Homo sapiens dihydropyrimidine dehydrogenase (DPYD), transcript variant 1, mRNA [NM_000 NM_000110 71.33 up 0.0235 QPCT glutaminyl‐peptide cyclotransferase Homo sapiens glutaminyl‐peptide cyclotransferase (QPCT), mRNA [NM_012413] NM_012413 67.41 up 0.0081 LOC399815 10 open reading frame 88 pseudogene Homo sapiens open reading frame 88 pseudogene (LOC399815), non‐coding RNR_027282 39.88 down 0.0160 NRCAM neuronal cell adhesion molecule Homo sapiens neuronal cell adhesion molecule (NRCAM), transcript variant 1, mRNA [NM_001 NM_001037132 36.00 up 0.0188 DPYSL4 dihydropyrimidinase‐like 4 Homo sapiens dihydropyrimidinase‐like 4 (DPYSL4), mRNA [NM_006426] NM_006426 30.62 down 0.0182 KCNMB4 potassium large conductance calcium‐activated channel, subfamily M, beta member 4 Homo sapiens potassium large conductance calcium‐activated channel, subfamily M, beta memNM_014505 30.47 up 0.0081 FAM50B family with sequence similarity 50, member B Homo sapiens family with sequence similarity 50, member B (FAM50B), mRNA [NM_012135] NM_012135 27.10 down 0.0120 QPCT glutaminyl‐peptide cyclotransferase Homo sapiens glutaminyl‐peptide cyclotransferase (QPCT), mRNA [NM_012413] NM_012413 27.07 up 0.0091 FAM19A4 family with sequence similarity 19 (chemokine (C‐C motif)‐like), member A4 Homo sapiens family with sequence similarity 19 (chemokine (C‐C motif)‐like), member A4 (FAMNM_182522 26.56 up 0.0185 ST3GAL5 ST3 beta‐galactoside alpha‐2,3‐ 5 Homo sapiens ST3 beta‐galactoside alpha‐2,3‐sialyltransferase 5 (ST3GAL5), transcript variant 1NM_003896 26.06 up 0.0215 FUT11 11 (alpha (1,3) fucosyltransferase) Homo sapiens fucosyltransferase 11 (alpha (1,3) fucosyltransferase) (FUT11), mRNA [NM_1735NM_173540 25.69 down 0.0120 KLHL13 kelch‐like 13 (Drosophila) Homo sapiens kelch‐like 13 (Drosophila) (KLHL13), mRNA [NM_033495] NM_033495 25.39 up 0.0442 SULF2 sulfatase 2 Homo sapiens sulfatase 2 (SULF2), transcript variant 1, mRNA [NM_018837] NM_018837 24.84 up 0.0198 SPG20 spastic paraplegia 20 (Troyer syndrome) Homo sapiens spastic paraplegia 20 (Troyer syndrome) (SPG20), transcript variant 1, mRNA [NMNM_015087 24.39 down 0.0185 NEB nebulin Homo sapiens nebulin (NEB), transcript variant 3, mRNA [NM_004543] NM_004543 24.27 up 0.0391 HECTD2 HECT domain containing 2 Homo sapiens HECT domain containing 2 (HECTD2), transcript variant 1, mRNA [NM_182765 NM_182765 24.26 down 0.0114 UGT2B4 UDP glucuronosyltransferase 2 family, polypeptide B4 Homo sapiens UDP glucuronosyltransferase 2 family, polypeptide B4 (UGT2B4), mRNA [NM_02NM_021139 23.77 up 0.0130 LOC400043 hypothetical LOC400043 Homo sapiens hypothetical LOC400043 (LOC400043), non‐coding RNA [NR_026656 NR_026656 22.73 up 0.0165 OXCT1 3‐oxoacid CoA 1 Homo sapiens 3‐oxoacid CoA transferase 1 (OXCT1), nuclear gene encoding mitochondrial protNM_000436 19.89 down 0.0389 JAKMIP2 janus and microtubule interacting protein 2 Homo sapiens janus kinase and microtubule interacting protein 2 (JAKMIP2), mRNA [NM_0147 NM_014790 19.66 up 0.0318 ISM1 isthmin 1 homolog () Homo sapiens isthmin 1 homolog (zebrafish) (ISM1), mRNA [NM_080826] NM_080826 19.37 up 0.0396 GPR161 ‐coupled receptor 161 Homo sapiens G protein‐coupled receptor 161 (GPR161), transcript variant 2, mRNA [NM_1538NM_153832 19.14 up 0.0322 NCRNA00162 non‐protein coding RNA 162 Homo sapiens non‐protein coding RNA 162 (NCRNA00162), non‐coding RNA [NR_024089 NR_024089 18.86 down 0.0131 UDP‐glucuronosyltransferase 2B28 Precursor (UDPGT 2B28)(EC 2.4.1.17) [Source:UniProtKB/SwBG192243 18.73 up 0.0379 JAKMIP2 janus kinase and microtubule interacting protein 2 Homo sapiens janus kinase and microtubule interacting protein 2 (JAKMIP2), mRNA [NM_0147 NM_014790 18.50 up 0.0286 ZNF558 protein 558 Homo sapiens zinc finger protein 558 (ZNF558), mRNA [NM_144693] NM_144693 17.62 down 0.0392 TMEM195 transmembrane protein 195 Homo sapiens transmembrane protein 195 (TMEM195), mRNA [NM_001004320] NM_001004320 17.61 up 0.0101 SPINK1 serine peptidase inhibitor, Kazal type 1 Homo sapiens serine peptidase inhibitor, Kazal type 1 (SPINK1), mRNA [NM_003122] NM_003122 16.44 up 0.0154 NRCAM neuronal cell adhesion molecule Homo sapiens neuronal cell adhesion molecule (NRCAM), transcript variant 1, mRNA [NM_001 NM_001037132 16.11 up 0.0130 CNTNAP3 contactin associated protein‐like 3 Homo sapiens contactin associated protein‐like 3 (CNTNAP3), mRNA [NM_033655] NM_033655 15.56 down 0.0473 MAM and LDL‐receptor class A domain‐containing protein C10orf112 Precursor [Source:UniProXM_295865 15.19 down 0.0232 CD163 CD163 molecule Homo sapiens CD163 molecule (CD163), transcript variant 1, mRNA [NM_004244] NM_004244 14.49 up 0.0473 CCNJL cyclin J‐like Homo sapiens cyclin J‐like (CCNJL), mRNA [NM_024565] NM_024565 14.38 down 0.0107 RAB31 RAB31, member RAS oncogene family Homo sapiens RAB31, member RAS oncogene family (RAB31), mRNA [NM_006868] NM_006868 14.08 up 0.0219 DACT2 dapper, antagonist of beta‐catenin, homolog 2 (Xenopus laevis) Homo sapiens dapper, antagonist of beta‐catenin, homolog 2 (Xenopus laevis) (DACT2), mRNA NM_214462 13.88 up 0.0205 CCL16 chemokine (C‐C motif) ligand 16 Homo sapiens chemokine (C‐C motif) ligand 16 (CCL16), mRNA [NM_004590] NM_004590 13.85 down 0.0138 BHMT2 betaine‐homocysteine methyltransferase 2 Homo sapiens betaine‐homocysteine methyltransferase 2 (BHMT2), mRNA [NM_017614] NM_017614 13.76 down 0.0093 DMGDH dimethylglycine dehydrogenase Homo sapiens dimethylglycine dehydrogenase (DMGDH), nuclear gene encoding mitochondria NM_013391 13.43 down 0.0130 CAGE1 cancer antigen 1 Homo sapiens cancer antigen 1 (CAGE1), mRNA [NM_205864] NM_205864 13.21 up 0.0107 PRR20B proline rich 20B Homo sapiens proline rich 20B (PRR20B), mRNA [NM_001130404] NM_001130404 13.03 up 0.0401 MACROD2 MACRO domain containing 2 Homo sapiens MACRO domain containing 2 (MACROD2), transcript variant 1, mRNA [NM_0806NM_080676 12.52 up 0.0407 PTER phosphotriesterase related Homo sapiens phosphotriesterase related (PTER), transcript variant 1, mRNA [NM_001001484]NM_001001484 12.01 down 0.0276 FLRT2 fibronectin rich transmembrane protein 2 Homo sapiens fibronectin leucine rich transmembrane protein 2 (FLRT2), mRNA [NM_013231 NM_013231 11.88 up 0.0351 HULC highly up‐regulated in liver cancer (non‐protein coding) Homo sapiens highly up‐regulated in liver cancer (non‐protein coding) (HULC), non‐coding RNANR_004855 11.88 up 0.0193 SLC16A9 solute carrier family 16, member 9 (monocarboxylic acid transporter 9 Homo sapiens solute carrier family 16, member 9 (monocarboxylic acid transporter 9) (SLC16A NM_194298 11.87 up 0.0118 LOC153469 hypothetical protein LOC153469 Homo sapiens cDNA FLJ14672 fis, clone NT2RP2003687 [AK027578] AK027578 11.75 up 0.0262 ATP10A ATPase, class V, type 10A Homo sapiens ATPase, class V, type 10A (ATP10A), mRNA [NM_024490] NM_024490 11.70 up 0.0183 RBM43 RNA binding motif protein 43 Homo sapiens RNA binding motif protein 43 (RBM43), mRNA [NM_198557] NM_198557 11.64 up 0.0138 DPYD dihydropyrimidine dehydrogenase Homo sapiens dihydropyrimidine dehydrogenase (DPYD), transcript variant 2, mRNA [NM_001 NM_001160301 11.60 up 0.0170 PRLR prolactin receptor Homo sapiens prolactin receptor (PRLR), mRNA [NM_000949] NM_000949 11.56 up 0.0181 CRYAA crystallin, alpha A Homo sapiens crystallin, alpha A (CRYAA), mRNA [NM_000394] NM_000394 11.45 down 0.0205 EMX1 empty spiracles homeobox 1 Homo sapiens empty spiracles homeobox 1 (EMX1), mRNA [NM_004097] NM_004097 11.44 up 0.0081 LIPG lipase, endothelial Homo sapiens lipase, endothelial (LIPG), mRNA [NM_006033] NM_006033 11.43 up 0.0155 ALPK1 alpha‐kinase 1 Homo sapiens alpha‐kinase 1 (ALPK1), transcript variant 1, mRNA [NM_025144] NM_025144 11.43 up 0.0368 PLP1 proteolipid protein 1 Homo sapiens proteolipid protein 1 (PLP1), transcript variant 3, mRNA [NM_001128834 NM_001128834 11.37 up 0.0185 CUZD1 CUB and zona pellucida‐like domains 1 Homo sapiens CUB and zona pellucida‐like domains 1 (CUZD1), mRNA [NM_022034] NM_022034 11.34 down 0.0333 FZD3 frizzled homolog 3 (Drosophila) Homo sapiens frizzled homolog 3 (Drosophila) (FZD3), mRNA [NM_017412] NM_017412 11.03 up 0.0314 SPARC secreted protein, acidic, cysteine‐rich () Homo sapiens secreted protein, acidic, cysteine‐rich (osteonectin) (SPARC), mRNA [NM_00311 NM_003118 10.79 up 0.0232 FBXL21 F‐box and leucine‐rich repeat protein 21 (gene/pseudogene) Homo sapiens F‐box and leucine‐rich repeat protein 21 (gene/pseudogene) (FBXL21), mRNA [NNM_012159 10.77 up 0.0354 GIMAP2 GTPase, IMAP family member 2 Homo sapiens GTPase, IMAP family member 2 (GIMAP2), mRNA [NM_015660] NM_015660 10.69 up 0.0130 FAM19A4 family with sequence similarity 19 (chemokine (C‐C motif)‐like), member A4 Homo sapiens family with sequence similarity 19 (chemokine (C‐C motif)‐like), member A4 (FAMNM_182522 10.68 up 0.0125 KLF9 Kruppel‐like factor 9 Homo sapiens Kruppel‐like factor 9 (KLF9), mRNA [NM_001206] NM_001206 10.62 up 0.0243 TSPAN13 tetraspanin 13 Homo sapiens tetraspanin 13 (TSPAN13), mRNA [NM_014399] NM_014399 10.50 up 0.0124 ARHGEF5 Rho guanine nucleotide exchange factor (GEF) 5 Homo sapiens Rho guanine nucleotide exchange factor (GEF) 5 (ARHGEF5), mRNA [NM_005435NM_005435 10.30 up 0.0174 TUB tubby homolog (mouse) Homo sapiens tubby homolog (mouse) (TUB), transcript variant 1, mRNA [NM_003320] NM_003320 10.20 up 0.0498 PTPRJ protein tyrosine phosphatase, receptor type, J Homo sapiens protein tyrosine phosphatase, receptor type, J (PTPRJ), transcript variant 1, mRNNM_002843 10.13 up 0.0473 C1orf97 open reading frame 97 Homo sapiens chromosome 1 open reading frame 97 (C1orf97), non‐coding RNA [NR_026761 NR_026761 10.10 up 0.0103 GC group‐specific component (vitamin D binding protein) Homo sapiens group‐specific component (vitamin D binding protein) (GC), mRNA [NM_000583 NM_000583 10.00 up 0.0117 CNTNAP3 contactin associated protein‐like 3 Homo sapiens contactin associated protein‐like 3 (CNTNAP3), mRNA [NM_033655] NM_033655 9.79 down 0.0410 DZIP1 DAZ interacting protein 1 Homo sapiens DAZ interacting protein 1 (DZIP1), transcript variant 2, mRNA [NM_198968] NM_198968 9.75 up 0.0490 EOMES homolog (Xenopus laevis) Homo sapiens eomesodermin homolog (Xenopus laevis) (EOMES), mRNA [NM_005442] NM_005442 9.69 up 0.0407 LOC550643 hypothetical LOC550643 Homo sapiens hypothetical LOC550643 (LOC550643), non‐coding RNA [NR_015367 NR_015367 9.59 up 0.0328 CSGALNACT1 chondroitin sulfate N‐acetylgalactosaminyltransferase 1 Homo sapiens chondroitin sulfate N‐acetylgalactosaminyltransferase 1 (CSGALNACT1), transcri NM_018371 9.51 down 0.0433 ATP‐dependent DNA 2 subunit 2 (EC 3.6.1.‐)(ATP‐dependent DNA helicase II 80 kDa subunit)(Lupus Ku autoant 9.19 down 0.0492 C1orf97 chromosome 1 open reading frame 97 Homo sapiens chromosome 1 open reading frame 97 (C1orf97), non‐coding RNA [NR_026761 NR_026761 9.06 up 0.0100 LAMB3 , beta 3 Homo sapiens laminin, beta 3 (LAMB3), transcript variant 2, mRNA [NM_001017402 NM_001017402 8.97 up 0.0282 TUBBP5 tubulin, beta pseudogene 5 Homo sapiens tubulin, beta pseudogene 5 (TUBBP5), non‐coding RNA [NR_027156 NR_027156 8.96 up 0.0305 C8orf68 open reading frame 68 Homo sapiens cDNA clone IMAGE:4824304 [BC022082] BC022082 8.94 up 0.0191 ZAK sterile alpha motif and containing kinase AZK Homo sapiens sterile alpha motif and leucine zipper containing kinase AZK (ZAK), transcript varNM_016653 8.92 down 0.0435 SOHLH1 spermatogenesis and oogenesis specific basic helix‐loop‐helix 1 Homo sapiens spermatogenesis and oogenesis specific basic helix‐loop‐helix 1 (SOHLH1), trans NM_001012415 8.78 up 0.0255 FAM24B family with sequence similarity 24, member B Homo sapiens family with sequence similarity 24, member B (FAM24B), mRNA [NM_152644] NM_152644 8.75 down 0.0077 Dimethylglycine dehydrogenase, mitochondrial Precursor (EC 1.5.99.2)(ME2GLYDH) [Source:UniProtKB/Swiss‐Prot;Acc 8.52 down 0.0311 CSTA cystatin A (stefin A) Homo sapiens cystatin A (stefin A) (CSTA), mRNA [NM_005213] NM_005213 8.27 up 0.0290 EML6 microtubule associated protein like 6 Homo sapiens echinoderm microtubule associated protein like 6 (EML6), mRNA [NM_0010397 NM_001039753 8.27 up 0.0382 FRMD4B FERM domain containing 4B Homo sapiens FERM domain containing 4B (FRMD4B), mRNA [NM_015123] NM_015123 8.25 up 0.0224 CXCL1 chemokine (C‐X‐C motif) ligand 1 (melanoma growth stimulating activity, alpha) Homo sapiens chemokine (C‐X‐C motif) ligand 1 (melanoma growth stimulating activity, alpha) NM_001511 8.19 up 0.0202 MX1 myxovirus (influenza virus) resistance 1, interferon‐inducible protein p78 (mouse Homo sapiens myxovirus (influenza virus) resistance 1, interferon‐inducible protein p78 (mouseNM_002462 8.14 up 0.0173 TGFA transforming growth factor, alpha Homo sapiens transforming growth factor, alpha (TGFA), transcript variant 1, mRNA [NM_0032NM_003236 8.10 up 0.0239 ANTXR1 anthrax toxin receptor 1 Homo sapiens anthrax toxin receptor 1 (ANTXR1), transcript variant 1, mRNA [NM_032208 NM_032208 7.97 up 0.0271 SGK1 serum/glucocorticoid regulated kinase 1 Homo sapiens serum/glucocorticoid regulated kinase 1 (SGK1), transcript variant 1, mRNA [NMNM_005627 7.95 up 0.0185 C3orf57 open reading frame 57 Homo sapiens chromosome 3 open reading frame 57 (C3orf57), mRNA [NM_001040100 NM_001040100 7.90 up 0.0247 KCNK13 potassium channel, subfamily K, member 13 Homo sapiens potassium channel, subfamily K, member 13 (KCNK13), mRNA [NM_022054] NM_022054 7.85 up 0.0313 SLC25A18 solute carrier family 25 (mitochondrial carrier), member 18 Homo sapiens solute carrier family 25 (mitochondrial carrier), member 18 (SLC25A18), nuclear NM_031481 7.73 up 0.0262 CNTNAP3 contactin associated protein‐like 3 Homo sapiens contactin associated protein‐like 3 (CNTNAP3), mRNA [NM_033655] NM_033655 7.50 down 0.0236 VAX2 ventral anterior homeobox 2 Homo sapiens ventral anterior homeobox 2 (VAX2), mRNA [NM_012476] NM_012476 7.42 up 0.0077 CSAG2 CSAG family, member 2 Homo sapiens CSAG family, member 2 (CSAG2), transcript variant 2, mRNA [NM_004909] NM_004909 7.41 up 0.0272 CC2D2A coiled‐coil and C2 domain containing 2A Homo sapiens coiled‐coil and C2 domain containing 2A (CC2D2A), transcript variant 1, mRNA [NNM_001080522 7.36 up 0.0435 RTP4 receptor (chemosensory) transporter protein 4 Homo sapiens receptor (chemosensory) transporter protein 4 (RTP4), mRNA [NM_022147] NM_022147 7.36 up 0.0244 ACSS1 acyl‐CoA synthetase short‐chain family member 1 Homo sapiens acyl‐CoA synthetase short‐chain family member 1 (ACSS1), nuclear gene encodinNM_032501 7.33 up 0.0149 LOC100130692 hypothetical LOC100130692 PREDICTED: Homo sapiens hypothetical LOC100130692 (LOC100130692), mRNA [XM_0017178XM_001717855 7.33 up 0.0326 SLCO4C1 solute carrier organic anion transporter family, member 4C1 Homo sapiens solute carrier organic anion transporter family, member 4C1 (SLCO4C1), mRNA [NM_180991 7.30 up 0.0403 PLXDC2 plexin domain containing 2 Homo sapiens plexin domain containing 2 (PLXDC2), mRNA [NM_032812] NM_032812 7.20 down 0.0189 GBP1 guanylate binding protein 1, interferon‐inducible, 67kDa Homo sapiens guanylate binding protein 1, interferon‐inducible, 67kDa (GBP1), mRNA [NM_00NM_002053 7.18 up 0.0229 C2orf54 chromosome 2 open reading frame 54 Homo sapiens chromosome 2 open reading frame 54 (C2orf54), transcript variant 2, mRNA [NMNM_024861 7.17 up 0.0244 OR2A9P olfactory receptor, family 2, subfamily A, member 9 pseudogene Homo sapiens olfactory receptor, family 2, subfamily A, member 9 pseudogene (OR2A9P), non NR_002157 7.11 up 0.0246 DLG3 discs, large homolog 3 (Drosophila) Homo sapiens discs, large homolog 3 (Drosophila) (DLG3), transcript variant 1, mRNA [NM_021NM_021120 7.06 up 0.0209 NCRNA00087 non‐protein coding RNA 87 Homo sapiens non‐protein coding RNA 87 (NCRNA00087), non‐coding RNA [NR_024493 NR_024493 7.04 up 0.0202 NRP1 neuropilin 1 Homo sapiens neuropilin 1 (NRP1), transcript variant 1, mRNA [NM_003873 NM_003873 6.99 up 0.0263 SCGN secretagogin, EF‐hand calcium binding protein Homo sapiens secretagogin, EF‐hand calcium binding protein (SCGN), mRNA [NM_006998] NM_006998 6.96 up 0.0191 UGT2A1 UDP glucuronosyltransferase 2 family, polypeptide A1 Homo sapiens UDP glucuronosyltransferase 2 family, polypeptide A1 (UGT2A1), mRNA [NM_00NM_006798 6.95 up 0.0363 FAM81A family with sequence similarity 81, member A Homo sapiens family with sequence similarity 81, member A (FAM81A), mRNA [NM_152450] NM_152450 6.94 up 0.0262 LOC100127909 hypothetical protein LOC100127909 PREDICTED: Homo sapiens hypothetical protein LOC100127909 (LOC100127909), miscRNA [XR XR_078849 6.93 up 0.0301 C6orf115 chromosome 6 open reading frame 115 Homo sapiens chromosome 6 open reading frame 115 (C6orf115), mRNA [NM_021243 NM_021243 6.89 up 0.0196 NDUFA4L2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4‐like 2 Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4‐like 2 (NDUFA4L2), mNM_020142 6.81 down 0.0458 PPP1R3B protein phosphatase 1, regulatory (inhibitor) subunit 3B Homo sapiens protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B), mRNA [NM_NM_024607 6.81 up 0.0329 SLC26A8 solute carrier family 26, member 8 Homo sapiens solute carrier family 26, member 8 (SLC26A8), transcript variant 1, mRNA [NM_0NM_052961 6.76 down 0.0271 NOTCH2 Notch homolog 2 (Drosophila) Homo sapiens Notch homolog 2 (Drosophila) (NOTCH2), mRNA [NM_024408] NM_024408 6.65 up 0.0131 SGEF Src homology 3 domain‐containing guanine nucleotide exchange factor Homo sapiens Src homology 3 domain‐containing guanine nucleotide exchange factor (SGEF), mNM_015595 6.49 up 0.0461 CLMN calmin (calponin‐like, transmembrane) Homo sapiens calmin (calponin‐like, transmembrane) (CLMN), mRNA [NM_024734] NM_024734 6.45 up 0.0077 CXorf50 chromosome X open reading frame 50 Homo sapiens chromosome X open reading frame 50 (CXorf50), non‐coding RNA [NR_026595 NR_026595 6.41 up 0.0344 LOC201617 hypothetical protein LOC201617 Homo sapiens cDNA FLJ31269 fis, clone KIDNE2006092 [AK055831] AK055831 6.38 up 0.0160 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 Homo sapiens serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1), transcr NM_000062 6.38 up 0.0414 Neuropilin‐1 Precursor (Vascular endothelial cell growth factor 165 receptor)(CD304 antigen) [Source:UniProtKB/Swiss 6.33 up 0.0219 GRB10 growth factor receptor‐bound protein 10 Homo sapiens growth factor receptor‐bound protein 10 (GRB10), transcript variant 4, mRNA [NNM_001001555 6.30 up 0.0270 HPX hemopexin Homo sapiens hemopexin (HPX), mRNA [NM_000613] NM_000613 6.23 up 0.0081 TMPRSS6 transmembrane protease, serine 6 Homo sapiens transmembrane protease, serine 6 (TMPRSS6), mRNA [NM_153609] NM_153609 6.22 up 0.0173 ANXA13 A13 Homo sapiens annexin A13 (ANXA13), transcript variant 2, mRNA [NM_001003954] NM_001003954 6.21 down 0.0232 PAOX polyamine oxidase (exo‐N4‐amino) Homo sapiens polyamine oxidase (exo‐N4‐amino) (PAOX), transcript variant 5, mRNA [NM_207NM_207128 6.20 down 0.0225 FAM65C family with sequence similarity 65, member C Homo sapiens family with sequence similarity 65, member C (FAM65C), mRNA [NM_080829] NM_080829 6.18 up 0.0499 CLMN calmin (calponin‐like, transmembrane) Homo sapiens calmin (calponin‐like, transmembrane) (CLMN), mRNA [NM_024734] NM_024734 6.15 up 0.0107 RNASE2 ribonuclease, RNase A family, 2 (liver, eosinophil‐derived neurotoxin Homo sapiens ribonuclease, RNase A family, 2 (liver, eosinophil‐derived neurotoxin) (RNASE2), NM_002934 6.15 up 0.0350 ACSS1 acyl‐CoA synthetase short‐chain family member 1 Homo sapiens acyl‐CoA synthetase short‐chain family member 1 (ACSS1), nuclear gene encodinNM_032501 6.15 up 0.0312 IGSF1 immunoglobulin superfamily, member 1 Homo sapiens immunoglobulin superfamily, member 1 (IGSF1), transcript variant 2, mRNA [NMNM_205833 6.13 up 0.0252 EGFR epidermal growth factor receptor (erythroblastic leukemia viral (v‐erb‐b) oncogene homolog, a Homo sapiens epidermal growth factor receptor (erythroblastic leukemia viral (v‐erb‐b) oncogeNM_201283 6.09 up 0.0307 BMP2 bone morphogenetic protein 2 Homo sapiens bone morphogenetic protein 2 (BMP2), mRNA [NM_001200] NM_001200 6.08 up 0.0118 BCL11B B‐cell CLL/lymphoma 11B (zinc finger protein) Homo sapiens B‐cell CLL/lymphoma 11B (zinc finger protein) (BCL11B), transcript variant 1, mRNM_138576 6.06 down 0.0224 FHL2 four and a half LIM domains 2 Homo sapiens four and a half LIM domains 2 (FHL2), transcript variant 5, mRNA [NM_00103949NM_001039492 6.06 down 0.0244 ANXA6 annexin A6 Homo sapiens annexin A6 (ANXA6), transcript variant 1, mRNA [NM_001155] NM_001155 6.03 up 0.0185 C21orf111 chromosome 21 open reading frame 111 RST41565 Athersys RAGE Library Homo sapiens cDNA, mRNA sequence [BG221750] BG221750 5.96 up 0.0124 S100A1 S100 calcium binding protein A1 Homo sapiens S100 calcium binding protein A1 (S100A1), mRNA [NM_006271 NM_006271 5.92 down 0.0160 LGALS2 lectin, galactoside‐binding, soluble, 2 Homo sapiens lectin, galactoside‐binding, soluble, 2 (LGALS2), mRNA [NM_006498] NM_006498 5.90 up 0.0138 KIRREL2 kin of IRRE like 2 (Drosophila) Homo sapiens kin of IRRE like 2 (Drosophila) (KIRREL2), transcript variant 3, mRNA [NM_199180NM_199180 5.87 up 0.0193 HRCT1 histidine rich carboxyl terminus 1 Homo sapiens histidine rich carboxyl terminus 1 (HRCT1), mRNA [NM_001039792 NM_001039792 5.82 down 0.0101 AGR2 anterior gradient homolog 2 (Xenopus laevis) Homo sapiens anterior gradient homolog 2 (Xenopus laevis) (AGR2), mRNA [NM_006408] NM_006408 5.82 up 0.0318 EML6 echinoderm microtubule associated protein like 6 Homo sapiens echinoderm microtubule associated protein like 6 (EML6), mRNA [NM_0010397 NM_001039753 5.81 up 0.0313 HOXC13 homeobox C13 Homo sapiens homeobox C13 (HOXC13), mRNA [NM_017410] NM_017410 5.78 up 0.0429 GNG4 guanine nucleotide binding protein (G protein), gamma 4 Homo sapiens guanine nucleotide binding protein (G protein), gamma 4 (GNG4), transcript var NM_001098722 5.71 up 0.0328 MFSD6 major facilitator superfamily domain containing 6 Homo sapiens major facilitator superfamily domain containing 6 (MFSD6), mRNA [NM_017694 NM_017694 5.69 up 0.0109 TMEM133 transmembrane protein 133 Homo sapiens transmembrane protein 133 (TMEM133), mRNA [NM_032021] NM_032021 5.69 up 0.0297 CA4 carbonic anhydrase IV Homo sapiens carbonic anhydrase IV (CA4), mRNA [NM_000717] NM_000717 5.64 up 0.0403 CC2D2A coiled‐coil and C2 domain containing 2A Homo sapiens coiled‐coil and C2 domain containing 2A (CC2D2A), transcript variant 1, mRNA [NNM_001080522 5.50 up 0.0239 PRLR prolactin receptor Homo sapiens prolactin receptor (PRLR), mRNA [NM_000949] NM_000949 5.48 up 0.0202 SETD7 SET domain containing ( methyltransferase) 7 Homo sapiens SET domain containing (lysine methyltransferase) 7 (SETD7), mRNA [NM_030648NM_030648 5.44 up 0.0457 LAMP3 lysosomal‐associated membrane protein 3 Homo sapiens lysosomal‐associated membrane protein 3 (LAMP3), mRNA [NM_014398] NM_014398 5.36 up 0.0346 HIST1H2AL histone cluster 1, H2al Homo sapiens histone cluster 1, H2al (HIST1H2AL), mRNA [NM_003511] NM_003511 5.36 down 0.0469 USH1C Usher syndrome 1C (autosomal recessive, severe) Homo sapiens Usher syndrome 1C (autosomal recessive, severe) (USH1C), transcript variant 1, NM_005709 5.30 up 0.0369 ZNF204 zinc finger protein 204 pseudogene Homo sapiens zinc finger protein 204 pseudogene (ZNF204), non‐coding RNA [NR_002722 NR_002722 5.28 up 0.0244 UNC5B unc‐5 homolog B (C. elegans) Homo sapiens unc‐5 homolog B (C. elegans) (UNC5B), mRNA [NM_170744] NM_170744 5.22 up 0.0133 IGSF1 immunoglobulin superfamily, member 1 Homo sapiens immunoglobulin superfamily, member 1 (IGSF1), transcript variant 1, mRNA [NMNM_001555 5.19 up 0.0308 CNTNAP3 contactin associated protein‐like 3 Homo sapiens contactin associated protein‐like 3 (CNTNAP3), mRNA [NM_033655] NM_033655 5.16 down 0.0391 MRAP2 melanocortin 2 receptor accessory protein 2 Homo sapiens melanocortin 2 receptor accessory protein 2 (MRAP2), mRNA [NM_138409] NM_138409 5.15 up 0.0300 PPP1R1C protein phosphatase 1, regulatory (inhibitor) subunit 1C Homo sapiens protein phosphatase 1, regulatory (inhibitor) subunit 1C (PPP1R1C), mRNA [NM_NM_001080545 5.15 up 0.0168 CASZ1 castor zinc finger 1 Homo sapiens castor zinc finger 1 (CASZ1), transcript variant 1, mRNA [NM_001079843 NM_001079843 5.12 up 0.0288 C2orf82 chromosome 2 open reading frame 82 Homo sapiens chromosome 2 open reading frame 82 (C2orf82), mRNA [NM_206895 NM_206895 5.10 down 0.0366 SLC5A11 solute carrier family 5 (sodium/glucose cotransporter), member 11 Homo sapiens solute carrier family 5 (sodium/glucose cotransporter), member 11 (SLC5A11), mNM_052944 5.08 down 0.0109 MACROD2 MACRO domain containing 2 Homo sapiens MACRO domain containing 2 (MACROD2), transcript variant 1, mRNA [NM_0806NM_080676 5.07 up 0.0326 GRB10 growth factor receptor‐bound protein 10 Homo sapiens growth factor receptor‐bound protein 10 (GRB10), transcript variant 4, mRNA [NNM_001001555 5.06 up 0.0301 SH2D4A SH2 domain containing 4A Homo sapiens SH2 domain containing 4A (SH2D4A), mRNA [NM_022071] NM_022071 5.05 down 0.0198 SERPINB9 serpin peptidase inhibitor, clade B (ovalbumin), member 9 Homo sapiens serpin peptidase inhibitor, clade B (ovalbumin), member 9 (SERPINB9), mRNA [NNM_004155 5.05 up 0.0368 PFTK1 PFTAIRE protein kinase 1 Homo sapiens PFTAIRE protein kinase 1 (PFTK1), mRNA [NM_012395] NM_012395 4.99 up 0.0298 SLC6A12 solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 12 Homo sapiens solute carrier family 6 (neurotransmitter transporter, betaine/GABA), member 1NM_003044 4.99 up 0.0354 HECTD2 HECT domain containing 2 Homo sapiens HECT domain containing 2 (HECTD2), transcript variant 2, mRNA [NM_173497 NM_173497 4.94 down 0.0308 NXPH4 neurexophilin 4 Homo sapiens neurexophilin 4 (NXPH4), mRNA [NM_007224] NM_007224 4.93 down 0.0343 CKMT2 creatine kinase, mitochondrial 2 (sarcomeric) Homo sapiens creatine kinase, mitochondrial 2 (sarcomeric) (CKMT2), nuclear gene encoding mNM_001825 4.90 down 0.0174 PAPSS1 3'‐phosphoadenosine 5'‐phosphosulfate synthase 1 Homo sapiens 3'‐phosphoadenosine 5'‐phosphosulfate synthase 1 (PAPSS1), mRNA [NM_0054 NM_005443 4.88 down 0.0100 AGXT alanine‐glyoxylate aminotransferase Homo sapiens alanine‐glyoxylate aminotransferase (AGXT), mRNA [NM_000030] NM_000030 4.88 up 0.0344 FAM38B family with sequence similarity 38, member B Homo sapiens family with sequence similarity 38, member B (FAM38B), mRNA [NM_022068] NM_022068 4.87 down 0.0276 NEURL3 neuralized homolog 3 (Drosophila) pseudogene Homo sapiens neuralized homolog 3 (Drosophila) pseudogene (NEURL3), non‐coding RNA [NR_NR_026875 4.83 up 0.0202 MECOM MDS1 and EVI1 complex locus Homo sapiens MDS1 and EVI1 complex locus (MECOM), transcript variant 6, mRNA [NM_00116NM_001164000 4.83 up 0.0480 SFRS13B splicing factor, arginine/serine‐rich 13B Homo sapiens splicing factor, arginine/serine‐rich 13B (SFRS13B), mRNA [NM_080743 NM_080743 4.81 up 0.0373 PRKCQ protein kinase C, theta Homo sapiens protein kinase C, theta (PRKCQ), mRNA [NM_006257] NM_006257 4.79 down 0.0431 PCDHA4 protocadherin alpha 4 Homo sapiens protocadherin alpha 4 (PCDHA4), transcript variant 2, mRNA [NM_031500 NM_031500 4.77 up 0.0400 CMPK2 cytidine monophosphate (UMP‐CMP) kinase 2, mitochondrial Homo sapiens cytidine monophosphate (UMP‐CMP) kinase 2, mitochondrial (CMPK2), nuclear NM_207315 4.76 up 0.0101 JAK3 Janus kinase 3 Homo sapiens Janus kinase 3 (JAK3), mRNA [NM_000215] NM_000215 4.74 down 0.0165 XAF1 XIAP associated factor 1 Homo sapiens XIAP associated factor 1 (XAF1), transcript variant 1, mRNA [NM_017523] NM_017523 4.71 up 0.0479 SULT1C2 sulfotransferase family, cytosolic, 1C, member 2 Homo sapiens sulfotransferase family, cytosolic, 1C, member 2 (SULT1C2), transcript variant 2, NM_176825 4.70 up 0.0205 ERP27 endoplasmic reticulum protein 27 Homo sapiens endoplasmic reticulum protein 27 (ERP27), mRNA [NM_152321 NM_152321 4.69 up 0.0249 Contactin‐associated protein‐like 3B Precursor (Cell recognition molecule Caspr3b) [Source:UniProtKB/Swiss‐Prot;Acc:Q 4.65 down 0.0403 TMEM158 transmembrane protein 158 Homo sapiens transmembrane protein 158 (TMEM158), mRNA [NM_015444] NM_015444 4.63 up 0.0353 RNF144B ring finger protein 144B Homo sapiens ring finger protein 144B (RNF144B), mRNA [NM_182757] NM_182757 4.62 up 0.0269 PAIP2B poly(A) binding protein interacting protein 2B Homo sapiens poly(A) binding protein interacting protein 2B (PAIP2B), mRNA [NM_020459 NM_020459 4.61 up 0.0348 KRT20 20 Homo sapiens keratin 20 (KRT20), mRNA [NM_019010] NM_019010 4.55 up 0.0217 6‐Sep septin 6 Homo sapiens septin 6 (SEPT6), transcript variant V, mRNA [NM_145802] NM_145802 4.54 up 0.0358 BSPRY B‐box and SPRY domain containing Homo sapiens B‐box and SPRY domain containing (BSPRY), mRNA [NM_017688] NM_017688 4.52 up 0.0317 PTP4A3 protein tyrosine phosphatase type IVA, member 3 Homo sapiens protein tyrosine phosphatase type IVA, member 3 (PTP4A3), transcript variant 1 NM_032611 4.48 down 0.0108 LOC157860 hypothetical protein LOC157860 Homo sapiens cDNA: FLJ22090 fis, clone HEP16084 [AK025743] AK025743 4.43 up 0.0183 FHL2 four and a half LIM domains 2 Homo sapiens four and a half LIM domains 2 (FHL2), transcript variant 2, mRNA [NM_201555 NM_201555 4.43 down 0.0247 LOC389332 hypothetical LOC389332 Homo sapiens hypothetical LOC389332 (LOC389332), non‐coding RNA [NR_024418 NR_024418 4.43 up 0.0344 ZNF323 zinc finger protein 323 Homo sapiens zinc finger protein 323 (ZNF323), transcript variant 1, mRNA [NM_030899 NM_030899 4.42 down 0.0174 ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 motif, 2 Homo sapiens ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2), transNM_021599 4.38 down 0.0219 LYSMD2 LysM, putative peptidoglycan‐binding, domain containing 2 Homo sapiens LysM, putative peptidoglycan‐binding, domain containing 2 (LYSMD2), transcriptNM_153374 4.38 up 0.0193 MSMB microseminoprotein, beta‐ Homo sapiens microseminoprotein, beta‐ (MSMB), transcript variant PSP94, mRNA [NM_00244NM_002443 4.36 down 0.0487 GSTA5 glutathione S‐transferase alpha 5 Homo sapiens glutathione S‐transferase alpha 5 (GSTA5), mRNA [NM_153699] NM_153699 4.34 up 0.0243 RUNX3 runt‐related transcription factor 3 Homo sapiens runt‐related transcription factor 3 (RUNX3), transcript variant 1, mRNA [NM_001NM_001031680 4.33 down 0.0260 NOTCH2NL Notch homolog 2 (Drosophila) N‐terminal like Homo sapiens Notch homolog 2 (Drosophila) N‐terminal like (NOTCH2NL), mRNA [NM_203458 NM_203458 4.32 up 0.0149 SULT1C2 sulfotransferase family, cytosolic, 1C, member 2 Homo sapiens sulfotransferase family, cytosolic, 1C, member 2 (SULT1C2), transcript variant 2, NM_176825 4.31 up 0.0344 RBP7 retinol binding protein 7, cellular Homo sapiens retinol binding protein 7, cellular (RBP7), mRNA [NM_052960 NM_052960 4.30 down 0.0217 TRIM38 tripartite motif‐containing 38 Homo sapiens tripartite motif‐containing 38 (TRIM38), mRNA [NM_006355] NM_006355 4.29 down 0.0469 ZNF703 zinc finger protein 703 Homo sapiens zinc finger protein 703 (ZNF703), mRNA [NM_025069] NM_025069 4.29 up 0.0128 SORBS1 sorbin and SH3 domain containing 1 Homo sapiens sorbin and SH3 domain containing 1 (SORBS1), transcript variant 3, mRNA [NM_ NM_001034954 4.28 down 0.0276 RP11‐35N6.1 lipid phosphate phosphatase‐related protein type 1 Homo sapiens lipid phosphate phosphatase‐related protein type 1 (LPPR1), transcript variant 1NM_207299 4.26 up 0.0492 CD83 CD83 molecule Homo sapiens CD83 molecule (CD83), transcript variant 1, mRNA [NM_004233] NM_004233 4.25 up 0.0202 HABP2 hyaluronan binding protein 2 Homo sapiens hyaluronan binding protein 2 (HABP2), mRNA [NM_004132] NM_004132 4.22 up 0.0131 LOC100130935 similar to CSAG family, member 2 PREDICTED: Homo sapiens similar to CSAG family, member 2 (LOC100130935), mRNA [XM_001XM_001717276 4.22 up 0.0286 CASP1 caspase 1, apoptosis‐related cysteine peptidase (interleukin 1, beta, convertase) Homo sapiens caspase 1, apoptosis‐related cysteine peptidase (interleukin 1, beta, convertase)NM_033292 4.22 up 0.0411 C7orf58 chromosome 7 open reading frame 58 Homo sapiens chromosome 7 open reading frame 58 (C7orf58), transcript variant 1, mRNA [NMNM_024913 4.21 down 0.0271 DNAH12 , axonemal, heavy chain 12 Homo sapiens dynein, axonemal, heavy chain 12 (DNAH12), transcript variant 1, mRNA [NM_17NM_178504 4.20 up 0.0081 C7orf58 chromosome 7 open reading frame 58 Homo sapiens chromosome 7 open reading frame 58 (C7orf58), transcript variant 1, mRNA [NMNM_024913 4.17 down 0.0485 PTHLH parathyroid hormone‐like hormone Homo sapiens parathyroid hormone‐like hormone (PTHLH), transcript variant 1, mRNA [NM_19NM_198965 4.15 up 0.0289 TRO trophinin Homo sapiens trophinin (TRO), transcript variant 3, mRNA [NM_016157] NM_016157 4.14 down 0.0131 STK32C serine/threonine kinase 32C Homo sapiens serine/threonine kinase 32C (STK32C), mRNA [NM_173575] NM_173575 4.12 down 0.0128 C19orf69 chromosome 19 open reading frame 69 Homo sapiens chromosome 19 open reading frame 69 (C19orf69), mRNA [NM_001130514 NM_001130514 4.12 up 0.0244 DA734585 NT2RP2 Homo sapiens cDNA clone NT2RP2005523 5', mRNA sequence [DA734585 DA734585 4.12 up 0.0323 6‐Sep septin 6 Homo sapiens septin 6 (SEPT6), transcript variant V, mRNA [NM_145802] NM_145802 4.10 up 0.0306 SERPINI1 serpin peptidase inhibitor, clade I (neuroserpin), member 1 Homo sapiens serpin peptidase inhibitor, clade I (neuroserpin), member 1 (SERPINI1), transcripNM_005025 4.08 down 0.0359 SORBS1 sorbin and SH3 domain containing 1 Homo sapiens sorbin and SH3 domain containing 1 (SORBS1), transcript variant 3, mRNA [NM_ NM_001034954 4.06 down 0.0366 B3GNT3 UDP‐GlcNAc:betaGal beta‐1,3‐N‐acetylglucosaminyltransferase 3 Homo sapiens UDP‐GlcNAc:betaGal beta‐1,3‐N‐acetylglucosaminyltransferase 3 (B3GNT3), mR NM_014256 4.06 down 0.0337 MFAP3L microfibrillar‐associated protein 3‐like Homo sapiens microfibrillar‐associated protein 3‐like (MFAP3L), transcript variant 1, mRNA [NMNM_021647 4.05 down 0.0244 TRIM34 tripartite motif‐containing 34 Homo sapiens tripartite motif‐containing 34 (TRIM34), transcript variant 3, mRNA [NM_130390NM_130390 4.02 up 0.0360 VCAN versican Homo sapiens versican (VCAN), transcript variant 1, mRNA [NM_004385] NM_004385 4.01 down 0.0181 PRKD1 protein kinase D1 Homo sapiens protein kinase D1 (PRKD1), mRNA [NM_002742] NM_002742 4.00 up 0.0447 GALNTL1 UDP‐N‐acetyl‐alpha‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase‐like 1 Homo sapiens UDP‐N‐acetyl‐alpha‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransfeNM_020692 3.98 up 0.0394 LOC100128386 hypothetical protein LOC100128386 PREDICTED: Homo sapiens hypothetical protein LOC100128386 (LOC100128386), mRNA [XM_0XM_001725380 3.98 up 0.0344 DLGAP1 discs, large (Drosophila) homolog‐associated protein 1 Homo sapiens discs, large (Drosophila) homolog‐associated protein 1 (DLGAP1), transcript variaNM_004746 3.97 down 0.0467 TRIM46 tripartite motif‐containing 46 Homo sapiens tripartite motif‐containing 46 (TRIM46), mRNA [NM_025058] NM_025058 3.95 down 0.0271 C7orf58 chromosome 7 open reading frame 58 Homo sapiens chromosome 7 open reading frame 58 (C7orf58), transcript variant 2, mRNA [NMNM_001105533 3.95 down 0.0431 my050 protein [Source:UniProtKB/TrEMBL;Acc:Q9H3H7] [ENST00000455537 AB210045 3.92 down 0.0447 KNG1 kininogen 1 Homo sapiens kininogen 1 (KNG1), transcript variant 2, mRNA [NM_000893] NM_000893 3.92 up 0.0193 CD9 CD9 molecule Homo sapiens CD9 molecule (CD9), mRNA [NM_001769] NM_001769 3.91 down 0.0101 FLJ41309 hypothetical protein LOC645079 Homo sapiens cDNA FLJ41309 fis, clone BRAMY2042641. [AK123303] AK123303 3.90 down 0.0403 PFKP phosphofructokinase, platelet Homo sapiens phosphofructokinase, platelet (PFKP), mRNA [NM_002627] NM_002627 3.88 down 0.0235 SELL selectin L Homo sapiens selectin L (SELL), transcript variant 1, mRNA [NM_000655] NM_000655 3.85 up 0.0118 CXCR4 chemokine (C‐X‐C motif) receptor 4 Homo sapiens chemokine (C‐X‐C motif) receptor 4 (CXCR4), transcript variant 1, mRNA [NM_00NM_001008540 3.84 up 0.0293 CYP2S1 cytochrome P450, family 2, subfamily S, polypeptide 1 Homo sapiens cytochrome P450, family 2, subfamily S, polypeptide 1 (CYP2S1), mRNA [NM_03 NM_030622 3.84 down 0.0237 HAMP hepcidin antimicrobial peptide Homo sapiens hepcidin antimicrobial peptide (HAMP), mRNA [NM_021175] NM_021175 3.82 up 0.0216 AMHR2 anti‐Mullerian , type II Homo sapiens anti‐Mullerian hormone receptor, type II (AMHR2), transcript variant 1, mRNA [NNM_020547 3.82 up 0.0174 TRIM55 tripartite motif‐containing 55 Homo sapiens tripartite motif‐containing 55 (TRIM55), transcript variant 2, mRNA [NM_033058NM_033058 3.81 up 0.0183 C20orf118 chromosome 20 open reading frame 118 Homo sapiens chromosome 20 open reading frame 118 (C20orf118), mRNA [NM_080628 NM_080628 3.80 down 0.0130 LOC115110 hypothetical protein LOC115110 Homo sapiens hypothetical protein LOC115110 (LOC115110), non‐coding RNA [NR_026927 NR_026927 3.79 up 0.0193 MAGEH1 melanoma antigen family H, 1 Homo sapiens melanoma antigen family H, 1 (MAGEH1), mRNA [NM_014061] NM_014061 3.78 up 0.0182 Homo sapiens cDNA clone IMAGE:5302913 [BC039426] BC039426 3.78 up 0.0101 TRIM50 tripartite motif‐containing 50 Homo sapiens tripartite motif‐containing 50 (TRIM50), mRNA [NM_178125] NM_178125 3.77 up 0.0344 ZNF703 zinc finger protein 703 Homo sapiens zinc finger protein 703 (ZNF703), mRNA [NM_025069] NM_025069 3.77 up 0.0219 FBN3 fibrillin 3 Homo sapiens fibrillin 3 (FBN3), mRNA [NM_032447] NM_032447 3.72 down 0.0436 PIGM phosphatidylinositol glycan anchor biosynthesis, class M Homo sapiens phosphatidylinositol glycan anchor biosynthesis, class M (PIGM), mRNA [NM_14NM_145167 3.71 up 0.0183 Protein furry homolog [Source:UniProtKB/Swiss‐Prot;Acc:Q5TBA9] [ENST00000380218 3.70 down 0.0130 BIN1 bridging integrator 1 Homo sapiens bridging integrator 1 (BIN1), transcript variant 4, mRNA [NM_139346] NM_139346 3.70 up 0.0154 LARP6 La ribonucleoprotein domain family, member 6 Homo sapiens La ribonucleoprotein domain family, member 6 (LARP6), transcript variant 1, mRNM_018357 3.68 up 0.0103 PCDHA11 protocadherin alpha 11 Homo sapiens protocadherin alpha 11 (PCDHA11), transcript variant 1, mRNA [NM_018902 NM_018902 3.67 up 0.0174 TMPRSS2 transmembrane protease, serine 2 Homo sapiens transmembrane protease, serine 2 (TMPRSS2), transcript variant 2, mRNA [NM_NM_005656 3.67 up 0.0265 LOC283050 hypothetical LOC283050 Homo sapiens hypothetical LOC283050 (LOC283050), transcript variant 1, non‐coding RNA [NR NR_024431 3.66 up 0.0318 ALPK2 alpha‐kinase 2 Homo sapiens alpha‐kinase 2 (ALPK2), mRNA [NM_052947] NM_052947 3.64 up 0.0174 S100A1 S100 calcium binding protein A1 Homo sapiens S100 calcium binding protein A1 (S100A1), mRNA [NM_006271 NM_006271 3.63 down 0.0183 TNFSF9 tumor necrosis factor (ligand) superfamily, member 9 Homo sapiens tumor necrosis factor (ligand) superfamily, member 9 (TNFSF9), mRNA [NM_003NM_003811 3.62 down 0.0204 TUBB4Q tubulin, beta polypeptide 4, member Q Homo sapiens tubulin, beta polypeptide 4, member Q (TUBB4Q), mRNA [NM_020040] NM_020040 3.62 up 0.0193 ATP8B3 ATPase, class I, type 8B, member 3 Homo sapiens ATPase, class I, type 8B, member 3 (ATP8B3), mRNA [NM_138813] NM_138813 3.62 down 0.0322 PIK3AP1 phosphoinositide‐3‐kinase adaptor protein 1 Homo sapiens phosphoinositide‐3‐kinase adaptor protein 1 (PIK3AP1), mRNA [NM_152309 NM_152309 3.62 up 0.0235 UBD ubiquitin D Homo sapiens ubiquitin D (UBD), mRNA [NM_006398] NM_006398 3.61 up 0.0100 C1orf210 chromosome 1 open reading frame 210 Homo sapiens chromosome 1 open reading frame 210 (C1orf210), transcript variant 1, mRNA [ NM_182517 3.61 up 0.0108 LOC728875 hypothetical LOC728875 Homo sapiens hypothetical LOC728875 (LOC728875), non‐coding RNA [NR_024584 NR_024584 3.60 up 0.0433 BTBD11 BTB (POZ) domain containing 11 Homo sapiens BTB (POZ) domain containing 11 (BTBD11), transcript variant a, mRNA [NM_001 NM_001018072 3.59 down 0.0077 CCND2 cyclin D2 Homo sapiens cyclin D2 (CCND2), mRNA [NM_001759] NM_001759 3.57 up 0.0165 NRP1 neuropilin 1 Homo sapiens neuropilin 1 (NRP1), transcript variant 3, mRNA [NM_001024629 NM_001024629 3.56 up 0.0193 DOCK9 dedicator of cytokinesis 9 Homo sapiens dedicator of cytokinesis 9 (DOCK9), transcript variant 2, mRNA [NM_001130048 NM_001130048 3.56 up 0.0448 FGB beta chain Homo sapiens fibrinogen beta chain (FGB), mRNA [NM_005141] NM_005141 3.56 up 0.0137 MTTP microsomal triglyceride transfer protein Homo sapiens microsomal triglyceride transfer protein (MTTP), mRNA [NM_000253] NM_000253 3.53 down 0.0493 GAST gastrin Homo sapiens gastrin (GAST), mRNA [NM_000805] NM_000805 3.53 up 0.0487 LOC283089 hypothetical protein LOC283089 Homo sapiens, clone IMAGE:5215872, mRNA [BC043378] BC043378 3.53 up 0.0205 C1orf74 chromosome 1 open reading frame 74 Homo sapiens chromosome 1 open reading frame 74 (C1orf74), mRNA [NM_152485 NM_152485 3.52 up 0.0466 GPR56 G protein‐coupled receptor 56 Homo sapiens G protein‐coupled receptor 56 (GPR56), transcript variant 3, mRNA [NM_201525NM_201525 3.50 up 0.0297 MARCKS myristoylated alanine‐rich protein kinase C substrate Homo sapiens myristoylated alanine‐rich protein kinase C substrate (MARCKS), mRNA [NM_00 NM_002356 3.49 up 0.0462 LIPC lipase, hepatic Homo sapiens lipase, hepatic (LIPC), mRNA [NM_000236] NM_000236 3.49 up 0.0130 NMI N‐ (and STAT) interactor Homo sapiens N‐myc (and STAT) interactor (NMI), mRNA [NM_004688] NM_004688 3.48 up 0.0187 MADCAM1 mucosal vascular addressin cell adhesion molecule 1 Homo sapiens mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), transcript varNM_130760 3.47 down 0.0293 ISM2 isthmin 2 homolog (zebrafish) Homo sapiens isthmin 2 homolog (zebrafish) (ISM2), transcript variant 1, mRNA [NM_199296 NM_199296 3.46 down 0.0497 PAOX polyamine oxidase (exo‐N4‐amino) Homo sapiens polyamine oxidase (exo‐N4‐amino) (PAOX), transcript variant 1, mRNA [NM_152NM_152911 3.46 down 0.0253 TTC39B tetratricopeptide repeat domain 39B Homo sapiens tetratricopeptide repeat domain 39B (TTC39B), mRNA [NM_152574] NM_152574 3.44 up 0.0317 CCDC65 coiled‐coil domain containing 65 Homo sapiens coiled‐coil domain containing 65 (CCDC65), mRNA [NM_033124 NM_033124 3.43 up 0.0235 PHEX phosphate regulating endopeptidase homolog, X‐linked Homo sapiens phosphate regulating endopeptidase homolog, X‐linked (PHEX), mRNA [NM_000NM_000444 3.43 up 0.0431 LOC100130111 hypothetical protein LOC100130111 PREDICTED: Homo sapiens hypothetical protein LOC100130111 (LOC100130111), miscRNA [XR XR_078492 3.42 down 0.0224 SLC2A10 solute carrier family 2 (facilitated glucose transporter), member 10 Homo sapiens solute carrier family 2 (facilitated glucose transporter), member 10 (SLC2A10), mNM_030777 3.42 up 0.0435 CYP3A7 cytochrome P450, family 3, subfamily A, polypeptide 7 Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 7 (CYP3A7), mRNA [NM_00NM_000765 3.40 up 0.0101 PSMB9 proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2 Homo sapiens proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional pNM_002800 3.39 up 0.0235 NOTCH2 Notch homolog 2 (Drosophila) Homo sapiens Notch homolog 2 (Drosophila) (NOTCH2), mRNA [NM_024408] NM_024408 3.39 up 0.0101 ISX intestine‐specific homeobox Homo sapiens intestine‐specific homeobox (ISX), mRNA [NM_001008494] NM_001008494 3.38 up 0.0494 ZC4H2 zinc finger, C4H2 domain containing Homo sapiens zinc finger, C4H2 domain containing (ZC4H2), mRNA [NM_018684 NM_018684 3.37 down 0.0077 SCARA3 scavenger receptor class A, member 3 Homo sapiens scavenger receptor class A, member 3 (SCARA3), transcript variant 2, mRNA [NMNM_182826 3.35 up 0.0306 ZIC2 Zic family member 2 (odd‐paired homolog, Drosophila) Homo sapiens Zic family member 2 (odd‐paired homolog, Drosophila) (ZIC2), mRNA [NM_0071NM_007129 3.35 up 0.0498 AKR1D1 aldo‐keto reductase family 1, member D1 (delta 4‐3‐ketosteroid‐5‐beta‐reductase) Homo sapiens aldo‐keto reductase family 1, member D1 (delta 4‐3‐ketosteroid‐5‐beta‐reducta NM_005989 3.32 up 0.0120 TBX10 T‐box 10 Homo sapiens T‐box 10 (TBX10), mRNA [NM_005995] NM_005995 3.32 down 0.0440 PRKAG2 protein kinase, AMP‐activated, gamma 2 non‐catalytic subunit Homo sapiens protein kinase, AMP‐activated, gamma 2 non‐catalytic subunit (PRKAG2), transc NM_016203 3.31 up 0.0183 LEPR leptin receptor Homo sapiens leptin receptor (LEPR), transcript variant 3, mRNA [NM_001003679 NM_001003679 3.30 down 0.0429 GNMT glycine N‐methyltransferase Homo sapiens glycine N‐methyltransferase (GNMT), mRNA [NM_018960] NM_018960 3.29 up 0.0183 ACE2 angiotensin I converting enzyme (peptidyl‐dipeptidase A) 2 Homo sapiens angiotensin I converting enzyme (peptidyl‐dipeptidase A) 2 (ACE2), mRNA [NM_ NM_021804 3.27 up 0.0386 CYP3A7 cytochrome P450, family 3, subfamily A, polypeptide 7 Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 7 (CYP3A7), mRNA [NM_00NM_000765 3.27 up 0.0130 SLC15A3 solute carrier family 15, member 3 Homo sapiens solute carrier family 15, member 3 (SLC15A3), transcript variant 1, mRNA [NM_0NM_016582 3.26 up 0.0235 SLC37A2 solute carrier family 37 (glycerol‐3‐phosphate transporter), member 2 Homo sapiens solute carrier family 37 (glycerol‐3‐phosphate transporter), member 2 (SLC37A2 NM_198277 3.26 up 0.0182 PLA2G2A phospholipase A2, group IIA (platelets, synovial fluid) Homo sapiens phospholipase A2, group IIA (platelets, synovial fluid) (PLA2G2A), transcript variaNM_000300 3.23 down 0.0149 ACOT11 acyl‐CoA thioesterase 11 Homo sapiens acyl‐CoA thioesterase 11 (ACOT11), transcript variant 2, mRNA [NM_147161] NM_147161 3.22 down 0.0176 ACSM2B acyl‐CoA synthetase medium‐chain family member 2B Homo sapiens acyl‐CoA synthetase medium‐chain family member 2B (ACSM2B), nuclear gene eNM_182617 3.21 up 0.0219 FAM46A family with sequence similarity 46, member A Homo sapiens family with sequence similarity 46, member A (FAM46A), mRNA [NM_017633] NM_017633 3.20 up 0.0267 NIN ninein (GSK3B interacting protein) Homo sapiens ninein (GSK3B interacting protein) (NIN), transcript variant 4, mRNA [NM_01635NM_016350 3.20 up 0.0153 SBK1 SH3‐binding domain kinase 1 Homo sapiens SH3‐binding domain kinase 1 (SBK1), mRNA [NM_001024401] NM_001024401 3.20 up 0.0183 GSTA2 glutathione S‐transferase alpha 2 Homo sapiens glutathione S‐transferase alpha 2 (GSTA2), mRNA [NM_000846] NM_000846 3.19 up 0.0343 NDRG4 NDRG family member 4 Homo sapiens NDRG family member 4 (NDRG4), transcript variant 3, mRNA [NM_022910] NM_022910 3.16 up 0.0127 C3P1 complement component 3 precursor pseudogene Homo sapiens complement component 3 precursor pseudogene (C3P1), non‐coding RNA [NR_0NR_027300 3.16 up 0.0463 CD97 CD97 molecule Homo sapiens CD97 molecule (CD97), transcript variant 1, mRNA [NM_078481] NM_078481 3.15 down 0.0101 LRRC27 leucine rich repeat containing 27 Homo sapiens leucine rich repeat containing 27 (LRRC27), transcript variant 4, mRNA [NM_001NM_001143759 3.13 down 0.0187 HECTD2 HECT domain containing 2 Homo sapiens HECT domain containing 2 (HECTD2), transcript variant 2, mRNA [NM_173497 NM_173497 3.11 down 0.0320 TRIM2 tripartite motif‐containing 2 Homo sapiens tripartite motif‐containing 2 (TRIM2), transcript variant 1, mRNA [NM_015271 NM_015271 3.11 down 0.0283 ANKRD29 ankyrin repeat domain 29 Homo sapiens ankyrin repeat domain 29 (ANKRD29), mRNA [NM_173505] NM_173505 3.11 up 0.0388 CYP3A5 cytochrome P450, family 3, subfamily A, polypeptide 5 Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5), mRNA [NM_00NM_000777 3.09 up 0.0205 Putative ‐XVB (Unconventional myosin‐15B)(Myosin XVBP) [Source:UniProtKB/Swiss‐Prot;Acc:Q96JP2] [ENST00 3.07 down 0.0265 LAYN layilin Homo sapiens layilin (LAYN), mRNA [NM_178834] NM_178834 3.07 up 0.0209 LOC115110 hypothetical protein LOC115110 Homo sapiens hypothetical protein LOC115110 (LOC115110), non‐coding RNA [NR_026927 NR_026927 3.07 up 0.0217 RUNX3 runt‐related transcription factor 3 Homo sapiens runt‐related transcription factor 3 (RUNX3), transcript variant 1, mRNA [NM_001NM_001031680 3.06 down 0.0160 TEX19 testis expressed 19 Homo sapiens testis expressed 19 (TEX19), mRNA [NM_207459] NM_207459 3.05 down 0.0270 NOSTRIN nitric oxide synthase trafficker Homo sapiens nitric oxide synthase trafficker (NOSTRIN), transcript variant 1, mRNA [NM_0529NM_052946 3.04 up 0.0298 SFI1 Sfi1 homolog, spindle assembly associated (yeast) Homo sapiens Sfi1 homolog, spindle assembly associated (yeast) (SFI1), transcript variant 1, mRNM_001007467 3.04 down 0.0181 PNLIPRP2 pancreatic lipase‐related protein 2 Homo sapiens pancreatic lipase‐related protein 2 (PNLIPRP2), mRNA [NM_005396] NM_005396 3.03 up 0.0452 PPARGC1B peroxisome proliferator‐activated receptor gamma, coactivator 1 beta Peroxisome proliferator‐activated receptor gamma coactivator 1‐beta (PPAR‐gamma coactivat BX648997 3.02 up 0.0313 CARD6 caspase recruitment domain family, member 6 Homo sapiens caspase recruitment domain family, member 6 (CARD6), mRNA [NM_032587] NM_032587 3.02 up 0.0343 IL21R interleukin 21 receptor Homo sapiens interleukin 21 receptor (IL21R), transcript variant 2, mRNA [NM_181078 NM_181078 3.01 up 0.0139 HSD17B2 hydroxysteroid (17‐beta) dehydrogenase 2 Homo sapiens hydroxysteroid (17‐beta) dehydrogenase 2 (HSD17B2), mRNA [NM_002153] NM_002153 3.01 up 0.0160 Alpha‐(1,3)‐fucosyltransferase 11 (EC 2.4.1.‐)(Galactoside 3‐L‐fucosyltransferase 11)(FucosyltraBC100994 3.00 down 0.0101 FYN FYN oncogene related to SRC, FGR, YES Homo sapiens FYN oncogene related to SRC, FGR, YES (FYN), transcript variant 1, mRNA [NM_0NM_002037 3.00 up 0.0387 FXYD2 FXYD domain containing ion transport regulator 2 Homo sapiens FXYD domain containing ion transport regulator 2 (FXYD2), transcript variant b, mNM_021603 2.99 up 0.0149 CYP2S1 cytochrome P450, family 2, subfamily S, polypeptide 1 Homo sapiens cytochrome P450, family 2, subfamily S, polypeptide 1 (CYP2S1), mRNA [NM_03 NM_030622 2.98 down 0.0156 FAM46A family with sequence similarity 46, member A Homo sapiens family with sequence similarity 46, member A (FAM46A), mRNA [NM_017633] NM_017633 2.97 up 0.0256 LOC730085 hypothetical LOC730085 PREDICTED: Homo sapiens hypothetical LOC730085 (LOC730085), mRNA [XM_001127205] XM_001127205 2.96 up 0.0235 ENPP2 ectonucleotide /phosphodiesterase 2 Homo sapiens ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), transcript varia NM_006209 2.96 up 0.0417 AQP3 aquaporin 3 (Gill blood group) Homo sapiens aquaporin 3 (Gill blood group) (AQP3), mRNA [NM_004925 NM_004925 2.95 up 0.0130 LOC285758 hypothetical protein LOC285758 Homo sapiens cDNA clone IMAGE:5264351 [BC037331] BC037331 2.95 up 0.0308 PPP1R3G protein phosphatase 1, regulatory (inhibitor) subunit 3G Homo sapiens protein phosphatase 1, regulatory (inhibitor) subunit 3G (PPP1R3G), mRNA [NM NM_001145115 2.94 down 0.0496 MYBPHL myosin binding protein H‐like Homo sapiens myosin binding protein H‐like (MYBPHL), mRNA [NM_001010985] NM_001010985 2.94 down 0.0171 ADH1A alcohol dehydrogenase 1A (class I), alpha polypeptide Homo sapiens alcohol dehydrogenase 1A (class I), alpha polypeptide (ADH1A), mRNA [NM_000NM_000667 2.94 up 0.0316 SYPL2 synaptophysin‐like 2 Homo sapiens synaptophysin‐like 2 (SYPL2), mRNA [NM_001040709] NM_001040709 2.94 up 0.0323 IL15 interleukin 15 Homo sapiens interleukin 15 (IL15), transcript variant 1, mRNA [NM_172174] NM_172174 2.94 up 0.0264 SERPINE2 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 Homo sapiens serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1NM_006216 2.93 up 0.0120 GPNMB glycoprotein (transmembrane) nmb Homo sapiens glycoprotein (transmembrane) nmb (GPNMB), transcript variant 1, mRNA [NM_ NM_001005340 2.92 down 0.0193 PAK1 p21 protein (Cdc42/Rac)‐activated kinase 1 Homo sapiens p21 protein (Cdc42/Rac)‐activated kinase 1 (PAK1), transcript variant 1, mRNA [NNM_001128620 2.92 up 0.0081 UCA1 urothelial cancer associated 1 (non‐protein coding) Homo sapiens urothelial cancer associated 1 (non‐protein coding) (UCA1), non‐coding RNA [NRNR_015379 2.92 up 0.0209 Putative uncharacterized protein ENSP00000382848 [Source:UniProtKB/TrEMBL;Acc:A8MY96]XM_001714753 2.89 up 0.0374 C6orf114 chromosome 6 open reading frame 114 Homo sapiens chromosome 6 open reading frame 114 (C6orf114), mRNA [NM_033069 NM_033069 2.87 up 0.0224 SMPDL3B sphingomyelin phosphodiesterase, acid‐like 3B Homo sapiens sphingomyelin phosphodiesterase, acid‐like 3B (SMPDL3B), transcript variant 2, NM_001009568 2.86 up 0.0290 DUSP13 dual specificity phosphatase 13 Homo sapiens dual specificity phosphatase 13 (DUSP13), transcript variant 1, mRNA [NM_0010NM_001007271 2.86 down 0.0246 ANTXR2 anthrax toxin receptor 2 Homo sapiens anthrax toxin receptor 2 (ANTXR2), transcript variant 1, mRNA [NM_058172 NM_058172 2.84 up 0.0442 ZDHHC14 zinc finger, DHHC‐type containing 14 Homo sapiens zinc finger, DHHC‐type containing 14 (ZDHHC14), transcript variant 2, mRNA [NMNM_153746 2.84 up 0.0291 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 Homo sapiens BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), nuclear gene encodinNM_004052 2.83 down 0.0301 LOC642406 similar to contactin associated protein‐like 3B Contactin‐associated protein‐like 3B Precursor (Cell recognition molecule Caspr3b) [Source:UniAK024257 2.83 down 0.0458 AQP3 aquaporin 3 (Gill blood group) Homo sapiens aquaporin 3 (Gill blood group) (AQP3), mRNA [NM_004925 NM_004925 2.83 up 0.0455 RORC RAR‐related orphan receptor C Homo sapiens RAR‐related orphan receptor C (RORC), transcript variant 1, mRNA [NM_005060 NM_005060 2.82 down 0.0304 SLC22A11 solute carrier family 22 (organic anion/urate transporter), member 11 Homo sapiens solute carrier family 22 (organic anion/urate transporter), member 11 (SLC22A1 NM_018484 2.82 up 0.0081 IL17D interleukin 17D Homo sapiens interleukin 17D (IL17D), mRNA [NM_138284] NM_138284 2.82 up 0.0139 DMRTA1 DMRT‐like family A1 Homo sapiens DMRT‐like family A1 (DMRTA1), mRNA [NM_022160] NM_022160 2.81 up 0.0370 TSPO translocator protein (18kDa) Homo sapiens translocator protein (18kDa) (TSPO), transcript variant PBR, mRNA [NM_000714 NM_000714 2.80 down 0.0171 HSD17B6 hydroxysteroid (17‐beta) dehydrogenase 6 homolog (mouse) Homo sapiens hydroxysteroid (17‐beta) dehydrogenase 6 homolog (mouse) (HSD17B6), mRNA NM_003725 2.80 down 0.0214 ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) Homo sapiens integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) (ITGA3), tra NM_002204 2.78 up 0.0494 FAM113B family with sequence similarity 113, member B Homo sapiens family with sequence similarity 113, member B (FAM113B), mRNA [NM_138371 NM_138371 2.76 up 0.0260 CCDC69 coiled‐coil domain containing 69 Homo sapiens coiled‐coil domain containing 69 (CCDC69), mRNA [NM_015621 NM_015621 2.76 up 0.0127 PFKP phosphofructokinase, platelet Homo sapiens phosphofructokinase, platelet (PFKP), mRNA [NM_002627] NM_002627 2.76 down 0.0321 TUBB6 tubulin, beta 6 Homo sapiens tubulin, beta 6 (TUBB6), mRNA [NM_032525] NM_032525 2.76 down 0.0261 MATN3 matrilin 3 Homo sapiens matrilin 3 (MATN3), mRNA [NM_002381] NM_002381 2.75 down 0.0246 UI‐E‐EJ1‐ajb‐m‐22‐0‐UI.r1 UI‐E‐EJ1 Homo sapiens cDNA clone UI‐E‐EJ1‐ajb‐m‐22‐0‐UI 5', mRNA BM930849 2.75 up 0.0205 SLC6A4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 Homo sapiens solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLCNM_001045 2.75 down 0.0445 IFIT1 interferon‐induced protein with tetratricopeptide repeats 1 Homo sapiens interferon‐induced protein with tetratricopeptide repeats 1 (IFIT1), transcript vaNM_001548 2.73 up 0.0247 DBNDD2 dysbindin (dystrobrevin binding protein 1) domain containing 2 Homo sapiens dysbindin (dystrobrevin binding protein 1) domain containing 2 (DBNDD2), transNM_001048226 2.72 down 0.0232 MICALL2 MICAL‐like 2 Homo sapiens MICAL‐like 2 (MICALL2), transcript variant 1, mRNA [NM_182924] NM_182924 2.72 down 0.0170 EDN1 endothelin 1 Homo sapiens endothelin 1 (EDN1), mRNA [NM_001955] NM_001955 2.72 down 0.0494 FAM166B family with sequence similarity 166, member B Homo sapiens family with sequence similarity 166, member B (FAM166B), transcript variant 2, NM_001099951 2.72 down 0.0414 MPP3 membrane protein, palmitoylated 3 (MAGUK p55 subfamily member 3) Homo sapiens membrane protein, palmitoylated 3 (MAGUK p55 subfamily member 3) (MPP3), NM_001932 2.72 up 0.0176 DLX1 distal‐less homeobox 1 Homo sapiens distal‐less homeobox 1 (DLX1), transcript variant 1, mRNA [NM_178120] NM_178120 2.71 up 0.0466 MATN3 matrilin 3 Homo sapiens matrilin 3 (MATN3), mRNA [NM_002381] NM_002381 2.71 down 0.0279 IFNAR1 interferon (alpha, beta and omega) receptor 1 Homo sapiens interferon (alpha, beta and omega) receptor 1 (IFNAR1), mRNA [NM_000629 NM_000629 2.71 down 0.0253 S100A10 S100 calcium binding protein A10 Homo sapiens S100 calcium binding protein A10 (S100A10), mRNA [NM_002966 NM_002966 2.70 down 0.0240 UBE2L6 ubiquitin‐conjugating enzyme E2L 6 Homo sapiens ubiquitin‐conjugating enzyme E2L 6 (UBE2L6), transcript variant 2, mRNA [NM_1NM_198183 2.69 up 0.0346 PRLR prolactin receptor Homo sapiens prolactin receptor (PRLR), mRNA [NM_000949] NM_000949 2.69 up 0.0301 RRAD Ras‐related associated with diabetes Homo sapiens Ras‐related associated with diabetes (RRAD), transcript variant 2, mRNA [NM_00NM_004165 2.68 up 0.0232 FZD1 frizzled homolog 1 (Drosophila) Homo sapiens frizzled homolog 1 (Drosophila) (FZD1), mRNA [NM_003505] NM_003505 2.68 up 0.0368 ABCG2 ATP‐binding cassette, sub‐family G (WHITE), member 2 Homo sapiens ATP‐binding cassette, sub‐family G (WHITE), member 2 (ABCG2), mRNA [NM_00NM_004827 2.67 down 0.0122 APOF apolipoprotein F Homo sapiens apolipoprotein F (APOF), mRNA [NM_001638] NM_001638 2.67 down 0.0257 HPD 4‐hydroxyphenylpyruvate dioxygenase Homo sapiens 4‐hydroxyphenylpyruvate dioxygenase (HPD), mRNA [NM_002150] NM_002150 2.65 down 0.0235 SYNPO synaptopodin Homo sapiens synaptopodin (SYNPO), transcript variant 1, mRNA [NM_007286] NM_007286 2.65 up 0.0413 MUPCDH mucin‐like protocadherin Homo sapiens mucin‐like protocadherin (MUPCDH), transcript variant 1, mRNA [NM_021924 NM_021924 2.65 down 0.0131 PTP4A3 protein tyrosine phosphatase type IVA, member 3 Homo sapiens protein tyrosine phosphatase type IVA, member 3 (PTP4A3), transcript variant 1 NM_032611 2.65 down 0.0040 CKAP4 ‐associated protein 4 Homo sapiens cytoskeleton‐associated protein 4 (CKAP4), mRNA [NM_006825] NM_006825 2.64 up 0.0243 CCDC114 coiled‐coil domain containing 114 Homo sapiens coiled‐coil domain containing 114 (CCDC114), transcript variant 2, mRNA [NM_1NM_144577 2.64 down 0.0193 SHBG sex hormone‐binding globulin Homo sapiens sex hormone‐binding globulin (SHBG), transcript variant 1, mRNA [NM_001040 NM_001040 2.64 up 0.0224 HMSD histocompatibility (minor) serpin domain containing Homo sapiens histocompatibility (minor) serpin domain containing (HMSD), mRNA [NM_00112NM_001123366 2.64 up 0.0446 NLRP12 NLR family, pyrin domain containing 12 Homo sapiens NLR family, pyrin domain containing 12 (NLRP12), transcript variant 1, mRNA [N NM_033297 2.63 down 0.0160 ANKRD56 ankyrin repeat domain 56 Homo sapiens ankyrin repeat domain 56 (ANKRD56), mRNA [NM_001029870] NM_001029870 2.61 up 0.0317 FSTL3 follistatin‐like 3 (secreted glycoprotein) Homo sapiens follistatin‐like 3 (secreted glycoprotein) (FSTL3), mRNA [NM_005860] NM_005860 2.60 up 0.0343 COL6A1 , type VI, alpha 1 Homo sapiens collagen, type VI, alpha 1 (COL6A1), mRNA [NM_001848] NM_001848 2.60 up 0.0316 AKAP12 A kinase (PRKA) anchor protein 12 Homo sapiens A kinase (PRKA) anchor protein 12 (AKAP12), transcript variant 1, mRNA [NM_00NM_005100 2.59 down 0.0265 RASIP1 Ras interacting protein 1 Homo sapiens Ras interacting protein 1 (RASIP1), mRNA [NM_017805] NM_017805 2.59 down 0.0202 COL4A6 collagen, type IV, alpha 6 Homo sapiens collagen, type IV, alpha 6 (COL4A6), transcript variant B, mRNA [NM_033641 NM_033641 2.59 down 0.0296 TECTB tectorin beta Homo sapiens tectorin beta (TECTB), mRNA [NM_058222] NM_058222 2.59 down 0.0183 STK17B serine/threonine kinase 17b Homo sapiens serine/threonine kinase 17b (STK17B), mRNA [NM_004226] NM_004226 2.58 up 0.0232 ARNTL aryl hydrocarbon receptor nuclear translocator‐like Homo sapiens aryl hydrocarbon receptor nuclear translocator‐like (ARNTL), transcript variant 3NM_001030273 2.58 up 0.0341 TRO trophinin Homo sapiens trophinin (TRO), transcript variant 3, mRNA [NM_016157] NM_016157 2.56 down 0.0224 NIN ninein (GSK3B interacting protein) Homo sapiens ninein (GSK3B interacting protein) (NIN), transcript variant 2, mRNA [NM_02092NM_020921 2.56 up 0.0192 MTHFD2L methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2‐like Homo sapiens methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2‐like (MTHFD2NM_001144978 2.55 down 0.0290 ADH4 alcohol dehydrogenase 4 (class II), pi polypeptide Homo sapiens alcohol dehydrogenase 4 (class II), pi polypeptide (ADH4), mRNA [NM_000670] NM_000670 2.55 up 0.0239 ID4 inhibitor of DNA binding 4, dominant negative helix‐loop‐helix protein Homo sapiens inhibitor of DNA binding 4, dominant negative helix‐loop‐helix protein (ID4), mR NM_001546 2.55 up 0.0329 ZNF326 zinc finger protein 326 Homo sapiens zinc finger protein 326 (ZNF326), transcript variant 3, mRNA [NM_182975 NM_182975 2.54 down 0.0396 HLA class I histocompatibility antigen, B‐18 alpha chain Precursor (MHC class I antigen B*18) [SBC013187 2.53 up 0.0293 STAR steroidogenic acute regulatory protein Homo sapiens steroidogenic acute regulatory protein (STAR), nuclear gene encoding mitochon NM_000349 2.53 up 0.0423 ZSCAN4 zinc finger and SCAN domain containing 4 Homo sapiens zinc finger and SCAN domain containing 4 (ZSCAN4), mRNA [NM_152677 NM_152677 2.53 up 0.0403 ZNF223 zinc finger protein 223 Homo sapiens zinc finger protein 223 (ZNF223), mRNA [NM_013361] NM_013361 2.52 up 0.0175 CDH1 cadherin 1, type 1, E‐cadherin (epithelial) Homo sapiens cadherin 1, type 1, E‐cadherin (epithelial) (CDH1), mRNA [NM_004360 NM_004360 2.52 up 0.0235 LEF1 lymphoid enhancer‐binding factor 1 Homo sapiens lymphoid enhancer‐binding factor 1 (LEF1), transcript variant 1, mRNA [NM_016NM_016269 2.52 down 0.0185 IL1RN interleukin 1 receptor antagonist Homo sapiens interleukin 1 receptor antagonist (IL1RN), transcript variant 1, mRNA [NM_1738 NM_173842 2.52 up 0.0133 CYP3A5 cytochrome P450, family 3, subfamily A, polypeptide 5 Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5), mRNA [NM_00NM_000777 2.51 up 0.0291 LOC285505 hypothetical protein LOC285505 Homo sapiens cDNA FLJ39473 fis, clone PROST2012890 [AK096792] AK096792 2.51 up 0.0258 HKDC1 hexokinase domain containing 1 Homo sapiens hexokinase domain containing 1 (HKDC1), mRNA [NM_025130] NM_025130 2.51 down 0.0183 PIWIL2 piwi‐like 2 (Drosophila) Homo sapiens piwi‐like 2 (Drosophila) (PIWIL2), transcript variant 2, mRNA [NM_018068 NM_018068 2.51 down 0.0313 LY6D lymphocyte antigen 6 complex, locus D Homo sapiens lymphocyte antigen 6 complex, locus D (LY6D), mRNA [NM_003695] NM_003695 2.51 down 0.0343 S100A4 S100 calcium binding protein A4 Homo sapiens S100 calcium binding protein A4 (S100A4), transcript variant 1, mRNA [NM_0029NM_002961 2.50 down 0.0333 MYL9 myosin, light chain 9, regulatory Homo sapiens myosin, light chain 9, regulatory (MYL9), transcript variant 2, mRNA [NM_18152 NM_181526 2.50 down 0.0267 AKAP12 A kinase (PRKA) anchor protein 12 Homo sapiens A kinase (PRKA) anchor protein 12 (AKAP12), transcript variant 2, mRNA [NM_14NM_144497 2.50 down 0.0245 KIAA0922 KIAA0922 Homo sapiens KIAA0922 (KIAA0922), transcript variant 2, mRNA [NM_015196] NM_015196 2.49 up 0.0461 IGSF1 immunoglobulin superfamily, member 1 Homo sapiens immunoglobulin superfamily, member 1 (IGSF1), transcript variant 2, mRNA [NMNM_205833 2.49 up 0.0451 Transmembrane protease, serine 6 (EC 3.4.21.‐)(Matriptase‐2) [Source:UniProtKB/Swiss‐Prot;Acc:Q8IU80] [ENST00000 2.48 up 0.0317 ZFP2 zinc finger protein 2 homolog (mouse) Homo sapiens zinc finger protein 2 homolog (mouse) (ZFP2), mRNA [NM_030613] NM_030613 2.48 down 0.0423 CACNG4 calcium channel, voltage‐dependent, gamma subunit 4 Homo sapiens calcium channel, voltage‐dependent, gamma subunit 4 (CACNG4), mRNA [NM_0NM_014405 2.48 down 0.0494 Putative uncharacterized protein [Source:UniProtKB/TrEMBL;Acc:Q86VG7] [ENST00000335616XR_042132 2.48 up 0.0183 LOC201651 similar to arylacetamide deacetylase (AADAC) Homo sapiens similar to arylacetamide deacetylase (AADAC) (LOC201651), non‐coding RNA [NRNR_026915 2.47 down 0.0274 SLC2A2 solute carrier family 2 (facilitated glucose transporter), member 2 Homo sapiens solute carrier family 2 (facilitated glucose transporter), member 2 (SLC2A2), mRNNM_000340 2.47 up 0.0248 SLC17A2 solute carrier family 17 (sodium phosphate), member 2 Homo sapiens solute carrier family 17 (sodium phosphate), member 2 (SLC17A2), mRNA [NM_0NM_005835 2.47 up 0.0260 Homo sapiens mRNA; cDNA DKFZp686A06106 (from clone DKFZp686A06106) [CR933618 CR933618 2.47 up 0.0498 CXCL2 chemokine (C‐X‐C motif) ligand 2 Homo sapiens chemokine (C‐X‐C motif) ligand 2 (CXCL2), mRNA [NM_002089] NM_002089 2.47 up 0.0181 MOSC2 MOCO sulphurase C‐terminal domain containing 2 Homo sapiens MOCO sulphurase C‐terminal domain containing 2 (MOSC2), mRNA [NM_01789 NM_017898 2.46 up 0.0215 WBP5 WW domain binding protein 5 Homo sapiens WW domain binding protein 5 (WBP5), transcript variant 1, mRNA [NM_016303 NM_016303 2.46 up 0.0270 MOSC2 MOCO sulphurase C‐terminal domain containing 2 Homo sapiens MOCO sulphurase C‐terminal domain containing 2 (MOSC2), mRNA [NM_01789 NM_017898 2.45 up 0.0173 PDIA2 protein disulfide family A, member 2 Homo sapiens protein disulfide isomerase family A, member 2 (PDIA2), mRNA [NM_006849] NM_006849 2.45 down 0.0127 ALDH1A1 aldehyde dehydrogenase 1 family, member A1 Homo sapiens aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), mRNA [NM_000689]NM_000689 2.45 up 0.0282 LOC729680 hypothetical protein LOC729680 PREDICTED: Homo sapiens hypothetical protein LOC729680 (LOC729680), miscRNA [XR_04050 XR_040503 2.44 up 0.0139 SLC25A12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 Homo sapiens solute carrier family 25 (mitochondrial carrier, Aralar), member 12 (SLC25A12), nNM_003705 2.44 down 0.0174 FAM111A family with sequence similarity 111, member A Homo sapiens family with sequence similarity 111, member A (FAM111A), transcript variant 1, NM_022074 2.43 up 0.0204 RAB36 RAB36, member RAS oncogene family Homo sapiens RAB36, member RAS oncogene family (RAB36), mRNA [NM_004914] NM_004914 2.43 down 0.0343 CXCL2 chemokine (C‐X‐C motif) ligand 2 Homo sapiens chemokine (C‐X‐C motif) ligand 2 (CXCL2), mRNA [NM_002089] NM_002089 2.43 up 0.0174 GBP2 guanylate binding protein 2, interferon‐inducible Homo sapiens guanylate binding protein 2, interferon‐inducible (GBP2), mRNA [NM_004120 NM_004120 2.41 up 0.0297 CPN1 carboxypeptidase N, polypeptide 1 Homo sapiens carboxypeptidase N, polypeptide 1 (CPN1), mRNA [NM_001308] NM_001308 2.41 down 0.0300 GLT8D4 8 domain containing 4 Homo sapiens glycosyltransferase 8 domain containing 4 (GLT8D4), mRNA [NM_001080393] NM_001080393 2.41 up 0.0081 MC1R melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) Homo sapiens melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC NM_002386 2.41 down 0.0131 Putative uncharacterized protein FLJ38767 [Source:UniProtKB/Swiss‐Prot;Acc:A8MZ25] [ENST00000399413 2.40 down 0.0191 PIK3R1 phosphoinositide‐3‐kinase, regulatory subunit 1 (alpha) Homo sapiens phosphoinositide‐3‐kinase, regulatory subunit 1 (alpha) (PIK3R1), transcript variaNM_181523 2.40 up 0.0323 CD14 CD14 molecule Homo sapiens CD14 molecule (CD14), transcript variant 1, mRNA [NM_000591] NM_000591 2.40 down 0.0174 LTB lymphotoxin beta (TNF superfamily, member 3) Homo sapiens lymphotoxin beta (TNF superfamily, member 3) (LTB), transcript variant 1, mRNANM_002341 2.39 up 0.0101 NR3C1 nuclear receptor subfamily 3, group C, member 1 ( Homo sapiens nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3NM_001018077 2.39 up 0.0276 HKDC1 hexokinase domain containing 1 Homo sapiens hexokinase domain containing 1 (HKDC1), mRNA [NM_025130] NM_025130 2.39 down 0.0193 MIXL1 Mix1 homeobox‐like 1 (Xenopus laevis) Homo sapiens Mix1 homeobox‐like 1 (Xenopus laevis) (MIXL1), mRNA [NM_031944] NM_031944 2.39 down 0.0367 DNAJB5 DnaJ (Hsp40) homolog, subfamily B, member 5 Homo sapiens DnaJ (Hsp40) homolog, subfamily B, member 5 (DNAJB5), transcript variant 3, mNM_012266 2.38 up 0.0235 RAP1GDS1 , GTP‐GDP dissociation stimulator 1 Homo sapiens RAP1, GTP‐GDP dissociation stimulator 1 (RAP1GDS1), transcript variant 1, mRN NM_001100426 2.38 down 0.0101 TBC1D10C TBC1 domain family, member 10C Homo sapiens TBC1 domain family, member 10C (TBC1D10C), mRNA [NM_198517] NM_198517 2.38 down 0.0259 DLX2 distal‐less homeobox 2 Homo sapiens distal‐less homeobox 2 (DLX2), mRNA [NM_004405] NM_004405 2.38 up 0.0261 NCEH1 neutral cholesterol ester 1 Homo sapiens neutral cholesterol ester hydrolase 1 (NCEH1), transcript variant 2, mRNA [NM_0NM_020792 2.38 down 0.0081 PARP9 poly (ADP‐ribose) polymerase family, member 9 Homo sapiens poly (ADP‐ribose) polymerase family, member 9 (PARP9), transcript variant 1, m NM_031458 2.37 up 0.0120 TLE4 ‐like enhancer of split 4 (E(sp1) homolog, Drosophila) Homo sapiens transducin‐like enhancer of split 4 (E(sp1) homolog, Drosophila) (TLE4), mRNA [NNM_007005 2.37 down 0.0318 MAGI2 membrane associated guanylate kinase, WW and PDZ domain containing 2 Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGNM_012301 2.37 down 0.0343 LEPR leptin receptor Homo sapiens leptin receptor (LEPR), transcript variant 2, mRNA [NM_001003680 NM_001003680 2.37 down 0.0460 PEG10 paternally expressed 10 Homo sapiens paternally expressed 10 (PEG10), transcript variant 1, mRNA [NM_001040152] NM_001040152 2.36 up 0.0279 ANXA2P3 pseudogene 3 Homo sapiens annexin A2 pseudogene 3 (ANXA2P3), non‐coding RNA [NR_001446] NR_001446 2.36 down 0.0467 INPP5F inositol polyphosphate‐5‐phosphatase F Homo sapiens inositol polyphosphate‐5‐phosphatase F (INPP5F), transcript variant 1, mRNA [N NM_014937 2.36 down 0.0133 GPR146 G protein‐coupled receptor 146 Homo sapiens G protein‐coupled receptor 146 (GPR146), mRNA [NM_138445] NM_138445 2.35 down 0.0193 C2orf39 chromosome 2 open reading frame 39 Homo sapiens chromosome 2 open reading frame 39 (C2orf39), mRNA [NM_145038 NM_145038 2.35 down 0.0293 LRRTM4 leucine rich repeat transmembrane neuronal 4 Homo sapiens leucine rich repeat transmembrane neuronal 4 (LRRTM4), transcript variant 2, mNM_024993 2.35 down 0.0447 IGF2AS insulin‐like growth factor 2 antisense Homo sapiens insulin‐like growth factor 2 antisense (IGF2AS), transcript variant 1, non‐coding RNR_028044 2.35 up 0.0385 TRDMT1 tRNA aspartic acid methyltransferase 1 Homo sapiens tRNA aspartic acid methyltransferase 1 (TRDMT1), transcript variant c, mRNA [NNM_176081 2.34 down 0.0253 KIF7 family member 7 Homo sapiens kinesin family member 7 (KIF7), mRNA [NM_198525] NM_198525 2.34 up 0.0272 UPK3A uroplakin 3A Homo sapiens uroplakin 3A (UPK3A), mRNA [NM_006953] NM_006953 2.34 down 0.0319 ACVR1C activin A receptor, type IC Homo sapiens activin A receptor, type IC (ACVR1C), transcript variant 1, mRNA [NM_145259] NM_145259 2.33 up 0.0211 CD8A CD8a molecule Homo sapiens CD8a molecule (CD8A), transcript variant 1, mRNA [NM_001768] NM_001768 2.33 down 0.0242 PLEK2 pleckstrin 2 Homo sapiens pleckstrin 2 (PLEK2), mRNA [NM_016445] NM_016445 2.33 up 0.0322 MVP major vault protein Homo sapiens major vault protein (MVP), transcript variant 1, mRNA [NM_017458] NM_017458 2.33 down 0.0185 UNC13D unc‐13 homolog D (C. elegans) Homo sapiens unc‐13 homolog D (C. elegans) (UNC13D), mRNA [NM_199242] NM_199242 2.33 down 0.0321 LOC375190 hypothetical protein LOC375190 Homo sapiens hypothetical protein LOC375190 (LOC375190), mRNA [NM_001145710 NM_001145710 2.33 down 0.0378 NTN4 netrin 4 Homo sapiens netrin 4 (NTN4), mRNA [NM_021229] NM_021229 2.32 up 0.0199 C10orf79 chromosome 10 open reading frame 79 Homo sapiens chromosome 10 open reading frame 79 (C10orf79), mRNA [NM_025145 NM_025145 2.31 up 0.0413 GAS2L3 growth arrest‐specific 2 like 3 Homo sapiens growth arrest‐specific 2 like 3 (GAS2L3), mRNA [NM_174942] NM_174942 2.31 down 0.0286 LOC388242 coiled‐coil domain containing 101 pseudogene Homo sapiens coiled‐coil domain containing 101 pseudogene (LOC388242), non‐coding RNA [NNR_002556 2.30 down 0.0444 KIAA1257 KIAA1257 Homo sapiens KIAA1257 (KIAA1257), mRNA [NM_020741] NM_020741 2.28 up 0.0355 PLA2G1B phospholipase A2, group IB () Homo sapiens phospholipase A2, group IB (pancreas) (PLA2G1B), mRNA [NM_000928] NM_000928 2.28 up 0.0368 RBM24 RNA binding motif protein 24 Homo sapiens RNA binding motif protein 24 (RBM24), transcript variant 2, mRNA [NM_153020NM_153020 2.28 down 0.0077 ENDOD1 endonuclease domain containing 1 Homo sapiens endonuclease domain containing 1 (ENDOD1), mRNA [NM_015036] NM_015036 2.28 down 0.0224 AKAP14 A kinase (PRKA) anchor protein 14 Homo sapiens A kinase (PRKA) anchor protein 14 (AKAP14), transcript variant 1, mRNA [NM_17NM_178813 2.27 up 0.0278 KLRC3 killer cell lectin‐like receptor subfamily C, member 3 Homo sapiens killer cell lectin‐like receptor subfamily C, member 3 (KLRC3), transcript variant 2NM_007333 2.27 up 0.0338 CARD17 caspase recruitment domain family, member 17 Homo sapiens caspase recruitment domain family, member 17 (CARD17), mRNA [NM_0010072NM_001007232 2.26 up 0.0165 ANXA2P1 annexin A2 pseudogene 1 Homo sapiens annexin A2 pseudogene 1 (ANXA2P1), non‐coding RNA [NR_001562] NR_001562 2.25 down 0.0411 SYNE1 spectrin repeat containing, nuclear envelope 1 Homo sapiens spectrin repeat containing, nuclear envelope 1 (SYNE1), transcript variant 1, mR NM_182961 2.25 up 0.0197 RECK reversion‐inducing‐cysteine‐rich protein with kazal motifs Homo sapiens reversion‐inducing‐cysteine‐rich protein with kazal motifs (RECK), mRNA [NM_0 NM_021111 2.25 up 0.0468 TRIM22 tripartite motif‐containing 22 Homo sapiens tripartite motif‐containing 22 (TRIM22), mRNA [NM_006074] NM_006074 2.25 up 0.0401 ASAH2 N‐acylsphingosine amidohydrolase (non‐lysosomal ceramidase) 2 Homo sapiens N‐acylsphingosine amidohydrolase (non‐lysosomal ceramidase) 2 (ASAH2), transNM_019893 2.24 down 0.0205 PLEKHB1 pleckstrin homology domain containing, family B (evectins) member 1 Homo sapiens pleckstrin homology domain containing, family B (evectins) member 1 (PLEKHB1NM_021200 2.24 down 0.0275 TRMT12 tRNA methyltransferase 12 homolog (S. cerevisiae) Homo sapiens tRNA methyltransferase 12 homolog (S. cerevisiae) (TRMT12), mRNA [NM_0179 NM_017956 2.24 up 0.0499 CD8B CD8b molecule Homo sapiens CD8b molecule (CD8B), transcript variant 4, mRNA [NM_172102] NM_172102 2.24 down 0.0193 ALDH1A1 aldehyde dehydrogenase 1 family, member A1 Homo sapiens aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), mRNA [NM_000689]NM_000689 2.24 up 0.0277 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha polypeptide) Homo sapiens integrin, alpha 5 (fibronectin receptor, alpha polypeptide) (ITGA5), mRNA [NM_0NM_002205 2.23 up 0.0435 TMEM59L transmembrane protein 59‐like Homo sapiens transmembrane protein 59‐like (TMEM59L), mRNA [NM_012109] NM_012109 2.23 up 0.0138 HERC5 hect domain and RLD 5 Homo sapiens hect domain and RLD 5 (HERC5), mRNA [NM_016323] NM_016323 2.23 up 0.0336 HMOX1 heme oxygenase (decycling) 1 Homo sapiens heme oxygenase (decycling) 1 (HMOX1), mRNA [NM_002133] NM_002133 2.23 up 0.0221 ANXA2 annexin A2 Homo sapiens annexin A2 (ANXA2), transcript variant 2, mRNA [NM_001002857] NM_001002857 2.22 down 0.0352 RICH2 Rho‐type GTPase‐activating protein RICH2 Homo sapiens Rho‐type GTPase‐activating protein RICH2 (RICH2), mRNA [NM_014859] NM_014859 2.22 up 0.0423 CYP7A1 cytochrome P450, family 7, subfamily A, polypeptide 1 Homo sapiens cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), mRNA [NM_00NM_000780 2.22 up 0.0484 FOXD1 Homo sapiens forkhead box D1 (FOXD1), mRNA [NM_004472] NM_004472 2.22 up 0.0219 ELOVL7 ELOVL family member 7, elongation of long chain fatty acids (yeast) Homo sapiens ELOVL family member 7, elongation of long chain fatty acids (yeast) (ELOVL7), tr NM_024930 2.22 up 0.0253 SGPL1 sphingosine‐1‐phosphate 1 Homo sapiens sphingosine‐1‐phosphate lyase 1 (SGPL1), mRNA [NM_003901] NM_003901 2.22 down 0.0102 CACNA2D4 calcium channel, voltage‐dependent, alpha 2/delta subunit 4 Homo sapiens calcium channel, voltage‐dependent, alpha 2/delta subunit 4 (CACNA2D4), mRN NM_172364 2.22 down 0.0490 SMPD1 sphingomyelin phosphodiesterase 1, acid lysosomal Homo sapiens sphingomyelin phosphodiesterase 1, acid lysosomal (SMPD1), transcript variant NM_000543 2.22 up 0.0154 RHOU ras homolog gene family, member U Homo sapiens ras homolog gene family, member U (RHOU), mRNA [NM_021205] NM_021205 2.22 up 0.0455 TRDMT1 tRNA aspartic acid methyltransferase 1 Homo sapiens tRNA aspartic acid methyltransferase 1 (TRDMT1), transcript variant a, mRNA [NNM_004412 2.22 down 0.0252 P2RX5 purinergic receptor P2X, ligand‐gated ion channel, 5 Homo sapiens purinergic receptor P2X, ligand‐gated ion channel, 5 (P2RX5), transcript variant 1NM_002561 2.21 up 0.0254 TGM2 transglutaminase 2 (C polypeptide, protein‐glutamine‐gamma‐glutamyltransferase) Homo sapiens transglutaminase 2 (C polypeptide, protein‐glutamine‐gamma‐glutamyltransferaNM_004613 2.21 down 0.0167 TSPAN14 tetraspanin 14 Homo sapiens tetraspanin 14 (TSPAN14), transcript variant 1, mRNA [NM_030927] NM_030927 2.21 down 0.0205 Sodium‐dependent phosphate transport protein 3 (Sodium/phosphate cotransporter 3)(Na(+)/PI cotransporter 3)(Solu 2.20 up 0.0362 RICH2 Rho‐type GTPase‐activating protein RICH2 Homo sapiens Rho‐type GTPase‐activating protein RICH2 (RICH2), mRNA [NM_014859] NM_014859 2.20 up 0.0246 ADAM11 ADAM metallopeptidase domain 11 Homo sapiens ADAM metallopeptidase domain 11 (ADAM11), mRNA [NM_002390] NM_002390 2.20 up 0.0232 GFI1 growth factor independent 1 transcription repressor Homo sapiens growth factor independent 1 transcription repressor (GFI1), transcript variant 1, NM_005263 2.20 down 0.0202 DBN1 drebrin 1 Homo sapiens drebrin 1 (DBN1), transcript variant 2, mRNA [NM_080881] NM_080881 2.20 up 0.0254 EPB41L4B erythrocyte membrane protein band 4.1 like 4B Homo sapiens erythrocyte membrane protein band 4.1 like 4B (EPB41L4B), transcript variant 2 NM_019114 2.18 up 0.0492 INPP5F inositol polyphosphate‐5‐phosphatase F Homo sapiens inositol polyphosphate‐5‐phosphatase F (INPP5F), transcript variant 1, mRNA [N NM_014937 2.18 down 0.0101 LOC100131208 similar to hCG2045825 Homo sapiens cDNA FLJ42971 fis, clone BRSTN2018083 [AK124961] AK124961 2.18 up 0.0414 Collagen alpha‐6(IV) chain Precursor [Source:UniProtKB/Swiss‐Prot;Acc:Q14031] [ENST000003BC005305 2.17 down 0.0370 SLCO4A1 solute carrier organic anion transporter family, member 4A1 Homo sapiens solute carrier organic anion transporter family, member 4A1 (SLCO4A1), mRNA [NM_016354 2.17 up 0.0261 SH2D2A SH2 domain protein 2A Homo sapiens SH2 domain protein 2A (SH2D2A), transcript variant 2, mRNA [NM_003975 NM_003975 2.17 up 0.0414 TLE4 transducin‐like enhancer of split 4 (E(sp1) homolog, Drosophila) Homo sapiens transducin‐like enhancer of split 4 (E(sp1) homolog, Drosophila) (TLE4), mRNA [NNM_007005 2.17 down 0.0331 CD40 CD40 molecule, TNF receptor superfamily member 5 Homo sapiens CD40 molecule, TNF receptor superfamily member 5 (CD40), transcript variant 1NM_001250 2.17 up 0.0196 GFOD1 glucose‐fructose domain containing 1 Homo sapiens glucose‐fructose oxidoreductase domain containing 1 (GFOD1), mRNA [NM_018NM_018988 2.17 up 0.0346 TET2 tet oncogene family member 2 Homo sapiens tet oncogene family member 2 (TET2), transcript variant 1, mRNA [NM_0011272NM_001127208 2.17 up 0.0204 LECT2 leukocyte cell‐derived chemotaxin 2 Homo sapiens leukocyte cell‐derived chemotaxin 2 (LECT2), mRNA [NM_002302] NM_002302 2.16 up 0.0171 NR3C1 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor Homo sapiens nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3NM_001018077 2.16 up 0.0353 SAA4 serum amyloid A4, constitutive Homo sapiens serum amyloid A4, constitutive (SAA4), mRNA [NM_006512] NM_006512 2.16 up 0.0103 NETO2 neuropilin (NRP) and tolloid (TLL)‐like 2 Homo sapiens neuropilin (NRP) and tolloid (TLL)‐like 2 (NETO2), mRNA [NM_018092 NM_018092 2.16 down 0.0149 INPP5F inositol polyphosphate‐5‐phosphatase F Homo sapiens inositol polyphosphate‐5‐phosphatase F (INPP5F), transcript variant 3, transcribeNR_003251 2.16 down 0.0091 STK17B serine/threonine kinase 17b Homo sapiens serine/threonine kinase 17b (STK17B), mRNA [NM_004226] NM_004226 2.15 up 0.0181 ALDH3A1 aldehyde dehydrogenase 3 family, memberA1 Homo sapiens aldehyde dehydrogenase 3 family, memberA1 (ALDH3A1), transcript variant 1, mNM_001135168 2.15 down 0.0187 TMEM8B transmembrane protein 8B Homo sapiens transmembrane protein 8B (TMEM8B), transcript variant 1, mRNA [NM_001042 NM_001042590 2.15 up 0.0107 LOC100128288 hypothetical protein LOC100128288 Homo sapiens hypothetical protein LOC100128288 (LOC100128288), non‐coding RNA [NR_024NR_024447 2.15 up 0.0232 ZNF284 zinc finger protein 284 Zinc finger protein 284 [Source:UniProtKB/Swiss‐Prot;Acc:Q2VY69] [ENST00000328297 CR936662 2.14 up 0.0154 MSRB2 methionine sulfoxide reductase B2 Homo sapiens methionine sulfoxide reductase B2 (MSRB2), mRNA [NM_012228] NM_012228 2.14 up 0.0196 GOLM1 golgi membrane protein 1 Homo sapiens golgi membrane protein 1 (GOLM1), transcript variant 1, mRNA [NM_016548] NM_016548 2.14 up 0.0372 C20orf46 chromosome 20 open reading frame 46 Homo sapiens chromosome 20 open reading frame 46 (C20orf46), mRNA [NM_018354 NM_018354 2.13 down 0.0253 MSRB2 methionine sulfoxide reductase B2 Homo sapiens methionine sulfoxide reductase B2 (MSRB2), mRNA [NM_012228] NM_012228 2.12 up 0.0209 MCF2L MCF.2 cell line derived transforming sequence‐like Homo sapiens MCF.2 cell line derived transforming sequence‐like (MCF2L), transcript variant 2 NM_024979 2.11 up 0.0246 CCDC88B coiled‐coil domain containing 88B Homo sapiens coiled‐coil domain containing 88B (CCDC88B), mRNA [NM_032251 NM_032251 2.11 down 0.0214 FAM111B family with sequence similarity 111, member B Homo sapiens family with sequence similarity 111, member B (FAM111B), transcript variant 1, NM_198947 2.10 up 0.0107 FLJ37644 hypothetical gene supported by AK094963 Homo sapiens cDNA FLJ37644 fis, clone BRHIP2000239 [AK094963] AK094963 2.10 up 0.0423 ACSL1 acyl‐CoA synthetase long‐chain family member 1 Homo sapiens acyl‐CoA synthetase long‐chain family member 1 (ACSL1), mRNA [NM_001995] NM_001995 2.10 up 0.0149 LOC727916 hypothetical protein LOC727916 PREDICTED: Homo sapiens hypothetical protein LOC727916 (LOC727916), miscRNA [XR_03995 XR_039953 2.10 up 0.0244 MYH7B myosin, heavy chain 7B, cardiac muscle, beta Homo sapiens myosin, heavy chain 7B, cardiac muscle, beta (MYH7B), mRNA [NM_020884] NM_020884 2.10 down 0.0386 MCF2L MCF.2 cell line derived transforming sequence‐like Homo sapiens MCF.2 cell line derived transforming sequence‐like (MCF2L), transcript variant 2 NM_024979 2.10 up 0.0213 BTG2 BTG family, member 2 Homo sapiens BTG family, member 2 (BTG2), mRNA [NM_006763] NM_006763 2.10 up 0.0176 IFIT3 interferon‐induced protein with tetratricopeptide repeats 3 Homo sapiens interferon‐induced protein with tetratricopeptide repeats 3 (IFIT3), transcript vaNM_001549 2.09 up 0.0133 NUDT19 nudix (nucleoside diphosphate linked moiety X)‐type motif 19 Homo sapiens nudix (nucleoside diphosphate linked moiety X)‐type motif 19 (NUDT19), mRNA NM_001105570 2.09 down 0.0171 STXBP1 syntaxin binding protein 1 Homo sapiens syntaxin binding protein 1 (STXBP1), transcript variant 1, mRNA [NM_003165 NM_003165 2.09 up 0.0368 SDPR serum deprivation response Homo sapiens serum deprivation response (SDPR), mRNA [NM_004657] NM_004657 2.09 down 0.0186 CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1 Homo sapiens cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), mRNA [NM_00NM_000499 2.09 up 0.0252 ANXA11 annexin A11 Annexin A11 (Annexin‐11)(Annexin XI)(Calcyclin‐associated annexin 50)(CAP‐50)(56 kDa autoantigen) [Source:UniProtK 2.09 down 0.0399 SALL2 sal‐like 2 (Drosophila) Homo sapiens sal‐like 2 (Drosophila) (SALL2), mRNA [NM_005407] NM_005407 2.09 down 0.0239 ANUBL1 AN1, ubiquitin‐like, homolog (Xenopus laevis) Homo sapiens AN1, ubiquitin‐like, homolog (Xenopus laevis) (ANUBL1), transcript variant 1, mRNM_174890 2.08 down 0.0101 IFITM1 interferon induced transmembrane protein 1 (9‐27) Homo sapiens interferon induced transmembrane protein 1 (9‐27) (IFITM1), mRNA [NM_00364NM_003641 2.07 down 0.0450 ULK4 unc‐51‐like kinase 4 (C. elegans) Homo sapiens unc‐51‐like kinase 4 (C. elegans) (ULK4), mRNA [NM_017886] NM_017886 2.07 down 0.0261 TMIGD2 transmembrane and immunoglobulin domain containing 2 Homo sapiens transmembrane and immunoglobulin domain containing 2 (TMIGD2), mRNA [NMNM_144615 2.07 down 0.0321 KANK3 KN motif and ankyrin repeat domains 3 Homo sapiens KN motif and ankyrin repeat domains 3 (KANK3), mRNA [NM_198471] NM_198471 2.07 up 0.0410 RP11‐529I10.4 deleted in a mouse model of primary ciliary dyskinesia Homo sapiens deleted in a mouse model of primary ciliary dyskinesia (RP11‐529I10.4), mRNA [ NM_015448 2.06 down 0.0160 TLE3 transducin‐like enhancer of split 3 (E(sp1) homolog, Drosophila) Homo sapiens transducin‐like enhancer of split 3 (E(sp1) homolog, Drosophila) (TLE3), transcripNM_005078 2.06 down 0.0492 ZNF226 zinc finger protein 226 Homo sapiens zinc finger protein 226 (ZNF226), transcript variant 3, mRNA [NM_001032374 NM_001032374 2.06 up 0.0246 KIAA0513 KIAA0513 Homo sapiens KIAA0513 (KIAA0513), mRNA [NM_014732] NM_014732 2.05 up 0.0488 hCG_1811732 hypothetical LOC151534 Homo sapiens hypothetical LOC151534 (LOC151534), non‐coding RNA [NR_024606 NR_024606 2.05 up 0.0428 LOC222699 transducer of ERBB2, 2 pseudogene Homo sapiens transducer of ERBB2, 2 pseudogene (LOC222699), non‐coding RNA [NR_002936 NR_002936 2.05 up 0.0460 SYNJ2BP synaptojanin 2 binding protein Homo sapiens synaptojanin 2 binding protein (SYNJ2BP), mRNA [NM_018373] NM_018373 2.05 up 0.0362 DNMT3A DNA (cytosine‐5‐)‐methyltransferase 3 alpha Homo sapiens DNA (cytosine‐5‐)‐methyltransferase 3 alpha (DNMT3A), transcript variant 4, mRNM_175630 2.05 down 0.0202 PPL periplakin Homo sapiens periplakin (PPL), mRNA [NM_002705] NM_002705 2.04 up 0.0355 ASB2 ankyrin repeat and SOCS box‐containing 2 Homo sapiens ankyrin repeat and SOCS box‐containing 2 (ASB2), mRNA [NM_016150] NM_016150 2.04 up 0.0497 PON3 paraoxonase 3 Homo sapiens paraoxonase 3 (PON3), mRNA [NM_000940] NM_000940 2.04 up 0.0333 AHRR aryl‐hydrocarbon receptor repressor Homo sapiens aryl‐hydrocarbon receptor repressor (AHRR), mRNA [NM_020731] NM_020731 2.04 up 0.0232 PRKD3 protein kinase D3 Homo sapiens protein kinase D3 (PRKD3), mRNA [NM_005813] NM_005813 2.04 up 0.0133 RADIL Ras association and DIL domains Homo sapiens Ras association and DIL domains (RADIL), mRNA [NM_018059] NM_018059 2.04 down 0.0463 CCDC46 coiled‐coil domain containing 46 Homo sapiens coiled‐coil domain containing 46 (CCDC46), transcript variant 1, mRNA [NM_145NM_145036 2.04 down 0.0232 BAG3 BCL2‐associated athanogene 3 Homo sapiens BCL2‐associated athanogene 3 (BAG3), mRNA [NM_004281] NM_004281 2.03 down 0.0232 LOC338620 hypothetical protein LOC338620 Homo sapiens hypothetical protein LOC338620, mRNA (cDNA clone IMAGE:6023208), partial cdBC043009 2.03 down 0.0343 KANK4 KN motif and ankyrin repeat domains 4 Homo sapiens KN motif and ankyrin repeat domains 4 (KANK4), mRNA [NM_181712] NM_181712 2.03 up 0.0213 ID2 inhibitor of DNA binding 2, dominant negative helix‐loop‐helix protein Homo sapiens inhibitor of DNA binding 2, dominant negative helix‐loop‐helix protein (ID2), mR NM_002166 2.03 up 0.0286 Ras association domain‐containing protein 4 [Source:UniProtKB/Swiss‐Prot;Acc:Q9H2L5] [ENST00000374414 2.03 down 0.0254 NAT8L N‐acetyltransferase 8‐like (GCN5‐related, putative) Homo sapiens N‐acetyltransferase 8‐like (GCN5‐related, putative) (NAT8L), mRNA [NM_178557NM_178557 2.02 up 0.0432 LOC344887 similar to hCG2041270 PREDICTED: Homo sapiens similar to hCG2041270 (LOC344887), miscRNA [XR_017497] XR_017497 2.02 down 0.0408 RUFY2 RUN and FYVE domain containing 2 Homo sapiens RUN and FYVE domain containing 2 (RUFY2), transcript variant 1, mRNA [NM_01NM_017987 2.02 down 0.0100 PRKCH protein kinase C, eta Homo sapiens protein kinase C, eta (PRKCH), mRNA [NM_006255] NM_006255 2.02 up 0.0492 RBP1 retinol binding protein 1, cellular Homo sapiens retinol binding protein 1, cellular (RBP1), transcript variant 1, mRNA [NM_00289NM_002899 2.02 down 0.0293 FER fer (fps/fes related) tyrosine kinase Homo sapiens fer (fps/fes related) tyrosine kinase (FER), mRNA [NM_005246] NM_005246 2.01 up 0.0156 PHIP pleckstrin homology domain interacting protein Homo sapiens pleckstrin homology domain interacting protein (PHIP), mRNA [NM_017934] NM_017934 2.01 up 0.0431 ZNF155 zinc finger protein 155 Homo sapiens zinc finger protein 155 (ZNF155), transcript variant 1, mRNA [NM_003445 NM_003445 2.01 up 0.0368 TREM2 triggering receptor expressed on myeloid cells 2 Homo sapiens triggering receptor expressed on myeloid cells 2 (TREM2), mRNA [NM_018965] NM_018965 2.01 up 0.0499 RP11‐529I10.4 deleted in a mouse model of primary ciliary dyskinesia Homo sapiens deleted in a mouse model of primary ciliary dyskinesia (RP11‐529I10.4), mRNA [ NM_015448 2.01 down 0.0118 RNF148 ring finger protein 148 Homo sapiens ring finger protein 148 (RNF148), mRNA [NM_198085] NM_198085 2.01 up 0.0432 ZDHHC14 zinc finger, DHHC‐type containing 14 Homo sapiens zinc finger, DHHC‐type containing 14 (ZDHHC14), transcript variant 2, mRNA [NMNM_153746 2.00 up 0.0460 LOC730101 hypothetical LOC730101 Homo sapiens hypothetical LOC730101 (LOC730101), transcript variant 1, non‐coding RNA [NR NR_024403 2.00 up 0.0470 Supplemental Table 3: Putative transcription factor binding sites identified between nt -10090 to -10037 in the 5’-flanking region of the UGT2B4 gene

CoreSimilarity/ Sequence Core Location in Matrix Description Strand Matrix Similarity (Core nucleotides capitalized) LS Mutants V$BRN5.03 Brn-5, POU-VI protein class (also known as emb and CNS-1) (-) 1.00/0.774 aTAATgaaattaagacagaaatc LS2, LS3 V$DLX3.01 Distal-less 3 homeodomain transcription factor (+) 1.00/0.922 tctTAATttcatt LS1, LS2 V$HOXB8.01 Homeobox B8 / Hox-2delta (-) 1.00/0.894 taatgaaATTAagacagaa LS1, LS2 V$BRN2.03 Brn-2, POU-III protein class (+) 1.00/0.968 tctgtcttaATTTcattat LS1, LS2 V$NKX61.01 NK6 homeobox 1 (+) 1.00/0.961 tgtcTTAAtttcatt LS1 V$MEOX1.01 Mesenchyme homeobox 1 (+) 1.00/0.854 ctgtcttAATTtcattatt LS1, LS2 Homeo domain factor Nkx-2.5/Csx, tinman homolog low V$NKX25.02 affinity sites (+) 1.00/0.957 tgtctTAATttcattattt LS1, LS2 V$CART1.01 Cart-1 (cartilage homeoprotein 1) (-) 1.00/0.945 gcaaaTAATgaaattaagaca LS2, LS3 V$CART1.01 Cart-1 (cartilage homeoprotein 1) (+) 1.00/0.975 tgtctTAATttcattatttgc LS1, LS2 Octamer-binding transcription factor-1, POU class 2 V$OCT1.03 homeobox 1 (POU2F1) (+) 1.00/0.876 ttaatttcATTAttt LS2, LS3 V$BRN5.04 POU class 6 homeobox 1 (POU6F1) (+) 1.00/0.854 gtcttaatttcATTAtttgccca LS2, LS3 Xenopus homeodomain factor Xvent-2; early BMP signaling V$XVENT2.01 response (-) 0.750/0.842 tgggcAAATaatgaaattaag LS3 V$ISL1.01 Pancreatic and intestinal lim-homeodomain factor (-) 1.00/0.835 ttgggcaaaTAATgaaattaaga LS2, LS3 V$NKX63.01 NK6 homeobox 3 (-) 1.00/0.859 gcaaaTAATgaaatt LS2, LS3 V$HOXB3.01 Homeobox B3 / Hox 2-gamma (+) 0.868/0.876 taattTCATtatttgccca LS2 V$RHOX6.01 Reproductive homeobox 6, placenta specific homeobox 1 (+) 0.780/0840 taattTCATtatttgcccaaa LS2 V$GSH1.01 Homeobox transcription factor Gsh-1 (-) 1.00/0.897 tttgggcaaaTAATgaaat LS2, LS3 V$HNF3.01 Hepatocyte nuclear factor 3 (alpha, beta) (FOXA1, FOXA2) (-) 0.971/0.990 tttgggcAAATaatgaa LS3 V$CEBPA.01 CCAAT/enhancer binding protein alpha (-) 1.00/0.948 acttttggGCAAata LS3, LS4 Hypermethylated in cancer 1 (secondary DNA binding V$HIC1.02 preference) (+) 1.00/0.953 attTGCCcaaaag LS3, LS4 V$PPARG.03 Peroxisome proliferator-activated receptor gamma, DR1 sites (+) 0.764/0.845 cattatttgcccAAAAgtcattc LS4, LS5 ZNF143 is the human ortholog of Xenopus Staf, ZNF76 is a V$ZNF76 143.01 DNA binding protein related to ZNF143 and Staf (+) 1.00/0.778 atttcattatttgCCCAaaagtcattcagga LS4 V$HBP1.02 HMG box-containing protein 1 (-) 1.00/0.886 actcctgAATGacttttgggcaaat LS5, LS6 V$PBX1_MEIS1.01 for a Pbx1/Meis1 heterodimer (-) 0.758/0.755 ctccTGAAtgacttttg LS5, LS6 V$PAX6.03 Pax-6 paired domain binding site (-) 0.839/0.771 tgactACTCctgaatgact LS6, LS7 V$HOXC8.01 Homeobox C8 / Hox-3alpha (-) 0.770/0.854 gcaaataATGActactcct LS7, LS8 Barx2, homeobox transcription factor that preferentially binds V$BARX2.01 to paired TAAT motifs (-) 1.00/0.951 gggcaaaTAATgactactc LS8 V$NKX63.01 NK6 homeobox 3 (-) 1.00/0.884 gcaaaTAATgactac LS8 V$HOXB3.01 Homeobox B3 / Hox 2-gamma (+) 0.868/0.861 agtagTCATtatttgccca LS7, LS8

Supplemental Table 4. Prediction of miRNA binding sites in the human UGT2B4 3'-UTR by 10 prediction programs. miRNAs that were predicted by at least 5 of the 10 programs are shown. Gray (1)=predicted; White (0)=not predicted miRNA DIANAmT miRanda miRDB miRWalk RNAhybrid PICTAR4 PICTAR5 PITA RNA22 Targetscan Sum hsa-miR-135b 11110011017 hsa-miR-135a 11110011017 hsa-miR-649 11110010016 hsa-miR-578 11110010016 hsa-miR-944 11110010016 hsa-miR-629 11010010015 hsa-miR-101 11010010015 hsa-miR-496 11010010015 hsa-miR-576-5p 11010010015 hsa-miR-643 11010010015 hsa-miR-203 11010010015 hsa-miR-579 11010010015 hsa-miR-542-3p 11010010015 hsa-miR-223 11010010015 hsa-miR-410 11010010015 hsa-miR-516a-3p 11010010015 hsa-miR-587 11010010015 hsa-miR-450b-5p 11010010015 hsa-miR-488 11010010015 hsa-miR-624 11010010015 hsa-miR-216b 11010010015 hsa-miR-489 11010010015 hsa-miR-513a-3p 01110010015 -10503 -10090 -10037

-112 0 +13

-54 -48 -42 -36 -30 -24 -18 -12 -6 -1

-10090 TCTTAA TTTCAT TATTTG CCCAAA AGTCAT TCAGGA GTAGTC ATTATT TGCCCA -10037 LS1 LS2 LS3 LS4 LS5 LS6 LS7 LS8 LS9

LS1 LS2 LS3 LS4 LS5 LS6 LS7 LS8 LS9 BamHI site GGATCC

Supplemental Fig. 1. Schematic representation of linker scanning mutagenesis of nucleotides - 10090 to -10037 within the (-10503:-10037) fragment. b -12072 -10037 B

b -11524 -10037 B

b -11114 -10037 B

b -10767 -10037 B

b -10503 -10037 B

a shPAPSS1/2 shNT -112 +13 A

0 1 2 3 4 5 6 7 8 9 10

Relative Luciferase Activity

Supplemental Fig. 2. Transient transfection analysis of a deletion series of reporter plasmids constructed from the (-12072:-10037)(-112:+13)-UGT2B4-Luc reporter. shPAPSS1/2 cells and shNT cells were transfected with the indicated reporters and 48 hours later were harvested for measurement of luciferase activities. Each bar represents mean ± S.D. of normalized

(firefly/Renilla) luciferase measurements relative to the activity measured in cells transfected with (-112:+13)-UGT2B4-Luc within each cell line (n=9 wells per group, derived from combining data from three independent experiments with triplicate transfection). For shNT cells, groups not sharing an upper case letter are significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by one-way ANOVA and Neuman-Keuls test).

c -10503 -10037 C

b -10391 -10037 BC

b -10276 -10037 BC

b -10159 -10037 B

a shPAPSS1/2 -112 +13 A shNT

0 1 2 3 4 5 6

Relative Luciferase Activity

Supplemental Fig. 3. Transient transfection analysis of a deletion series of reporter plasmids constructed from the (-10503:-10037)(-112:+13)-UGT2B4-Luc reporter. shPAPSS1/2 cells and shNT cells were transfected with the indicated reporters and 48 hours later were harvested for measurement of luciferase activities. Each bar represents mean ± S.D. of normalized

(firefly/Renilla) luciferase measurements relative to the activity measured in cells transfected with (-112:+13)-UGT2B4-Luc within each cell line (n=9 wells per group, derived from combining data from three independent experiments with triplicate transfection). For shNT cells, groups not sharing an upper case letter are significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by one-way ANOVA and Neuman-Keuls test). c -10159 -10037 C

c -10136 -10037 C

c -10111 -10037 C

b -10080 -10037 B

a shPAPSS1/2 -112 +13 A shNT

0 1 2 3 4 5 6 7 8 9 10

Relative Luciferase Activity

Supplemental Fig. 4. Transient transfection analysis of a deletion series of reporter plasmids constructed from the (-10159:-10037)(-112:+13)-UGT2B4-Luc reporter. shPAPSS1/2 cells and shNT cells were transfected with the indicated reporters and 48 hours later were harvested for measurement of luciferase activities. Each bar represents mean ± S.D. of normalized

(firefly/Renilla) luciferase measurements relative to the activity measured in cells transfected with (-112:+13)-UGT2B4-Luc within each cell line (n=9 wells per group, derived from combining data from three independent experiments with triplicate transfection). For shNT cells, groups not sharing an upper case letter are significantly different from each other, p<0.05. For shPAPSS1/2 cells, groups not sharing a lower case letter are significantly different from each other, p<0.05 (by one-way ANOVA and Neuman-Keuls test).