the role of nuclear receptors in the regulation of...

1

The Role of Nuclear Receptors in the Regulation of Bilirubin

Conjugation by UDP-glucuronosyltransferase 1A1

Lori W.E. van der Schoor (1957457)

Supervision: Prof. dr. Johan W. Jonker and Weilin Liu (MSc)

Department of Pediatrics

Center for Liver, Digestive and Metabolic Diseases

University Medical Center Groningen

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TABLE OF CONTENTS

Abstract page 3

Samenvatting page 4

Background page 5-6

Introduction page 6-8

Materials and Methods page 8-11

Results page 11-18

Discussion page 18-22

References page 23-27

Appendices page 28-37

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ABSTRACT

Background: Severe unconjugated hyperbilirubinemia is associated with the development of

kernicterus, resulting in permanent neurological damage or even death. UGT1A1 is the rate

limiting enzyme in bilirubin secretion and plays a pivotal role in the genesis of unconjugated

hyperbilirubinemia, as occurs in Crigler Najjar syndrome or neonatal hyperbilirubinemia. The

expression level of UGT1A1 is partially determined by the transcriptional activity of the

UGT1A1 gene. The transcription of many metabolic genes has shown to be partially modulated

by nuclear receptors (NR), which are ligand-activated transcription factors. We aimed to identify

NRs that play a role in the regulation of UGT1A1 expression.

Methods: A dual luciferase-based promoter activity screen was performed on 2.3kb rat UGT1A1

promoter (rUGT1A1) together with all known NRs (49 members) to identify NRs-rUGT1A1

interactions. In silico analysis was performed to identify the response element of the identified

NRs in the rUGT1A1 promoter. The identified response element was mutated by overlap

extension PCR followed by dual-luciferase assay to assess the change on NRs-rUGT1A1

interactions.

Results: The Glucocorticoid Receptor (GR) and the Progesterone Receptor (PR) were shown to

induce rUGT1A1 expression when stimulated with their ligands, dexamethasone (DEX) and

progesterone (PRG), respectively. In subsequent experiments, GR with DEX significantly

induced rUGT1A1 10-17-fold in CV1 cells, and 26-fold in HepG2 cells. PR with PRG

significantly induced rUGT1A1 10-13 fold in CV-1 cells and 10-fold in HepG2 cells.

We identified a GR response element at -77/-95 bp from the rUGT1A1 gene. Mutation of this

binding site completely abolished both the GR- and PR-mediated rUGT1A1 induction.

Conclusion: Our data clearly demonstrate that GR and PR strongly induce rUGT1A1 expression

in hepatoctyes by direct binding to the proximal UGT1A1 promoter at -77/-95 bp upstream the

transcription starting site. Thus, regulation of GR and PR by their ligands could serve as a

potential therapy for hyperbilirubinemia by inducing UGT1A1 expression.

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SAMENVATTING

Achtergrond: Ernstige ongeconjugeerde hyperbilirubinemie is geassocieerd met het ontstaan van

kernicterus. Kernicterus kan resulteren in permanente neurologische schade met mogelijk de

dood tot gevolg. UGT1A1 is het snelheidsbeperkende enzym in bilirubine uitscheiding en speelt

een essentiële rol in het ontstaan van ongeconjugeerde hyperbilirubinemie. Ongeconjugeerde

hyperbilirubinemie is het kenmerk van Crigler Najjar syndroom en neonatale

hyperbilirubinemie. UGT1A1 expressie is gedeeltelijk afhankelijk van de transcriptionele

activiteit van het UGT1A1 gen. De transcriptie van veel metabole gene wordt beïnvloed door

Nucleaire Receptotoren NR), ligand-geactiveerde transcriptie factoren. Ons doel was om NRs te

identificeren die een rol spelen in de transcriptionele regulatie van UGT1A1.

Materiaal en Methode: Om NRs te identificeren die mogelijk interacteren met UGT1A1, hebben

we een promoter activiteits screen uitgevoerd op een 2.3 kb UGT1A1 rat promoter (rUGT1A1),

samen met alle bekende NRs. De screen was gebaseerd op een duale luciferase assay. Om het

bindingselement van de NRs aan de UGT1A1 promoter te identificeren, zijn de

bindingselementen voorspeld door middel van in silico analyse. Het geïdentificeerde

bindingselement werd gemuteerd met overlap extension PCR. Vervolgens werd de duale

luciferase assay weer uitgevoerd om de invloed van de mutatie op de NR-promoter interacties te

beoordelen.

Resultaten: Wij hebben aangetoond dat de Glucocorticoid Receptor (GR) en de Progesteron

Receptor (PR) rUGT1A1 expressie kunnen induceren als ze gestimuleerd worden door hun

liganden, dexamethasone (DEX) en progesteron (PRG). In de daarop volgende experimenten,

verooraakte GR met DEX een significante 10-17x inductie van rUGT1A1 in CV-1 cell en 26x in

HepG2 cellen. PR met PRG veroorzaakte een significante 10-13x inductie in CV-1 cellen en 10x

in HepG2 cellen. We hebben een GR-bindingsplaats geïdentificeerd op een afstand van -77/-95

bp van het rUGT1A1 gen. Na mutatie van deze bindingsplaats, waren zowel de GR- als de PR-

gemediëerde inductie verdwenen.

Conclusie: Onze resultaten tonen aan dat GR en PR rUGT1A1 expressie sterk induceren via

directe binding aan de bindingsplaats in de proximale UGT1A1 promoter op een afstand van -

77/-95 bp van het UGT1A1 gen. Dit geeft aan de regulatie van GR en PR door hun liganden zou

kunnen dienen als een potentiële therapie voor hyperbilirubinemie, door het induceren van

UGT1A1 expressie.

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BACKGROUND

Bilirubin, a yellow coloured bile pigment, is produced by the degradation of heme in the

reticuloendothelial system (RES) (1). Bilirubin mainly originates from hemoglobin in

erythrocytes (1), and additionally from myoglobin (2) or mitochondrial components (3). Several

bilirubin associated diseases are identified. Generally, we distinguish unconjugated

hyperbilirubinemia diseases such as neonatal unconjugated hyperbilirubinemia (4), Crigler-

Najjar syndrome (5,6) and Gilbert syndrome (6); conjugated hyperbilirubinemia, as in Dubin

Johnson syndrome (7); and mixed unconjugated and conjugated hyperbilirubinemia, as in Rotor

syndrome (8). Each of these diseases affects a different step in the process of hepatic bilirubin

detoxification.

Hepatic bilirubin detoxification

Detoxification of bilirubin is a multistep process (Fig. 1). At the basolateral membrane,

hepatocytes take up unconjugated bilirubin from the portal blood. This uptake is mediated by

members of the organic anion-transporting polypeptide (OATP)- family (9). These proteins

mediate sodium-independent membrane transport of numerous endogenous and xenobiotic

amphipathic compounds (10). OATP1B1 and OATP1B3, which are localized in the basolateral

hepatocyte membrane, play a major role in bilirubin uptake (9,11,12). Although these receptors

share 80% identical amino acids (13) and an overlapping substrate specificity, OATP1B1 mainly

mediates unconjugated bilirubin transport (9), whereas OATP1B3 seems to play a role in re-

uptake of conjugated bilirubin (12). Failure of these transporters results in Rotor syndrome, an

autosomal recessive disease that presents as benign mixed conjugated and unconjugated

hyperbilirubinemia (8).

After uptake in the hepatocyte, unconjugated bilirubin is glucuronidated (conjugated) by the

enzyme UDP-glucuronosyltransferase (UGT) 1A1 (14). UGT1A1 is an UGT, an enzyme of the

glucuronidation pathway that transforms small lipophilic molecules, such as hormones, steroids,

drugs and bilirubin, into water soluble, extractable metabolites (15). UGT1A1 is mainly

expressed in the liver, but also present in the small intestine and colon (16). Failure or absence of

this enzyme, as seen in Crigler-Najjer syndrome, Gilbert syndrome and neonatal

hyperbilirubinemia, leads to accumulation of unconjugated bilirubin (17).

After conjugation, bilirubin is exported from the hepatocytes to the bile canaliculi, which drain

into the small intestine. Alternatively, conjugated bilirubin is transported back into the blood at

the basolateral hepatocyte membrane, after which it can be excreted in urine (18). Conjugated

bilirubin transport across the hepatocyte membranes is mediated by specific ATP-binding

cassette (ABC) transporters, ABCC2 (19,20), and ABCC3 . ABC-transporters bind and

hydrolyse ATP to enable active transport of various substrates across membranes of various cell

types. Absence of ABCC2 results in Dubin-Johnson syndrome, which is characterized by

jaundice by conjugated hyperbilirubinemia and liver pigmentation.

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INTRODUCTION Severe unconjugated hyperbilirubinemia is associated with the development of kernicterus,

which results in permanent brain damage and potentially death. Bilirubin induced neurotoxicity

is characterized by sensorineural deafness, gaze abnormalities and cerebral palsy. UGT1A1 is the

rate limiting enzyme in bilirubin secretion and therefore plays a pivotal role in the development

of unconjugated hyperbilirubinemia. The most well-known UGT1A1 related disease is Crigler-

Najjar (CN), an autosomal recessive diseases in which UGT1A1 is completely (type I) or

partially (type II) absent, resulting in severely elevated UCB levels (5,21). Especially CN type I

patients present with high UCB levels and kernicterus early in life (5).

Apart from inheritable UGT1A1 deficiencies, almost all premature neonates present with

decreased UGT1A1 activity and subsequent hyperbilirubinemia. UGT1A1 activity is only 0.1%

of adult levels at 17-30 weeks of gestation, increasing to 1% of adult level between 30 and 40

weeks. Adult levels are reached at 6-12 weeks after gestation (22). Insufficient UGT1A1 activity,

often in combination with elevated erythrocyte turnover and/or delayted intestinal bilirubin

transit, leads to neonatal hyperbilirubinemia, affecting almost all premature neonates (4).

In current practice, no preventative therapy is available for hyperbilirubinemia risk populations.

After diagnosis, the UV-light phototherapy is commonly applied to convert bilirubin in the blood

into water soluble isomers, which are not neurotoxic and can be easily excreted. Although this

therapy has been proven to be effective, it is not sufficiently adequate for CN patients, who may

need up to 16 hours a day of phototherapy in order to prevent permanent brain damage and liver

failure (23). In neonates, phototherapy has not been able to make kernicterus disappear, and

especially in preterm neonates, both phototherapy and exchange transfusions are associated with

significant morbidity and mortality. Alternatively, enhancement of bilirubin conjugation by the

Figure 1: Hepatic bilirubin detoxification pathway

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liver might reduce unconjugated bilirubin levels and thereby reduces the severity and

consequences of this disease.

Conjugation activity is determined by the expression level of UGT1A1, which is partially

determined by the transcriptional activity of the UGT1A1 gene, encoding the enzyme. The

transcription of many metabolic enzymes and transporter genes has shown to be partially

modulated by stimulation of a nuclear receptor (NR).

NRs are a large family of 49 ligand-activated transcription factors that act as transcriptional

switches on large sets of genes in response to lipophilic hormones, vitamins, dietary lipids or

other intracellular signals (Fig. 2) (24). NRs are the primary targets for drug development,

especially in metabolic diseases. Currently, 13% of FDA approved drugs target NRs, including

drugs for treating hyperlipidimia (fibrates), insuline resistance (TZDs), inflammation

(dexamethasone) and cancer (tamoxifen) (25). UGT1A1 expression is already known to be

regulated by the various transcription factors, such as hepatic nuclear factor (HNF) 1α and

several NRs; pregnane X receptor (PXR), constitutive androstane receptor (CAR) and

glucocorticoid receptor (GR) (26).

Phenobarbital, which has been the golden standard therapy of neonatal hyperbilirubinemia before

the introduction of phototherapy, is known to increase UGT1A1 gene expression in the liver (17)

by stimulating CAR, which binds to the phenobarbital response enhancer module (PBREM)

(27,28). PBREM is an enhancer region upstream to the UGT1A1 promoter, composed of various

NR-binding sites and serves as a binding site for CAR, PXR and GR (26,27,29,30).

Transcription of UGT1A1 can be up-regulated by binding of these NRs to PBREM. However,

the role of other NRs in regulating UGT1A1 expression by binding to the proximal promoter of

UGT1A1 is largely unknown. In the current study, we aimed to identify additional NRs that play

Figure 2: Nuclear Receptor-mediated gene transcription. Endogenous or exogenous ligands bind to the

ligand binding domain (LBD) of the receptor. Consequently, the DNA-binding domain (DBD) binds to a

response element in the gene promoter, thereby inducing transcription of the respective gene.

8

a role in the regulation of UGT1A1 expression. Since NRs activities can be specifically

modulated by their ligands, identification of NRs that regulate UGT1A1 expression could lead

to the development of ligand-based new drugs to improve bilirubin conjugation and cure

hyperbilirubinemia in neonates and CN patients, thereby reducing the risk of liver failure,

kernicterus and permanent neurological damage. In comparison to conventional phototherapy,

NR-targeted therapy could potentially be a more effective treatment since it specifically regulates

the expression of UGT1A1, which is the underlying problem in unconjugated

hyperbilirubinemia. In addition, identification of these NRs could potentially help to explain

drug-induced jaundice.

MATERIALS AND METHODS

Concept

The Jonker group has developed a high throughput NR-promoter screen to specifically evaluate

the functional regulation of genes by any NR in a given context (i.e., in the presence or absence

of ligand, in different cell lines etc.). The NR-promoter screen allows to test the roles of all 49

NRs in regulation of gene promoters, using dual luciferase-based reporter assays. Based on the

NR(s) identified in the screen, promoters can be further characterized for response elements of

these respective NRs and their role in protein expression in vitro and eventually in vivo. We used

this screen to determine the transcriptional regulation of the rat UGT1A1 promoter (rUGT1A1)

by NRs. The protocol of the entire screen can be found under ‘Appendices’.

Cell culture

CV1 cells, derived from African Green monkey kidney, were cultured in Dulbecco’s Modified

Eagle Medium (DMEM) (Life Technologies, Carlsbad, CA ) supplemented with high glucose

(4.5g/L), pyruvate, 10% FBS, penicillin and streptomycin at 37°C under 5% CO2 in 75 cm²

culture flasks (Corning Incorporated, Corning , NY). Passage number was 15-35.

HepG2 cells, human hepatocellular liver carcinoma cells, were cultured in DMEM supplemented

with 10% FBS, penicillin and streptomycin at 37 °C under 5% CO2 in 75 cm² culture flasks

(Corning). Passage number was 7-40.

Plasmids and cloning

rUGT1A1 promoter fragments, 2.3 and 1.1 kb respectively, were amplified from rat genomic

DNA using high-fidelity PfuTurbo DNA Polymerase (Agilent Technologies, Santa Clara, CA),

followed by ligation into pGL3-basic luciferase vector (Promega, Madison, WI) digested by

either XhoI and HindIII (1.1 kb) or KpnI and XhoI (2.3 kb). Point mutations were introduced by

2-step overlap-extension PCR using primers carrying mutated nucleotides and rUGT1A1

promoter fragment as a template. PCR products and digested vector were purified by Zymoclean

gel DNA recovery kit. Vectors were isolated and purified by Zyppy plasmid Miniprep

(Zymoresearch, Irvine, CA) or Sigma Midiprep kit (Sigma-Aldrich, St. Louis, MO). All

constructs were sequenced before experiments by Baseclear (Leiden, NL). The primers used for

cloning are listed in the Table I. Primers were from Biolegio (Nijmegen, NL).

PCR (Biorad T100 Thermal Cycler) was performed by using a standard protocol and gene

expression was measured by RT-PCR StepOnePlus (Applied Biosystems, Foster City, CA). The

PCR program is shown in Table II.

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Table I: Primers used for cloning of rUGT1A1 promoter constructs

Primer name Sequence 5 >>> 3

rUGT1A1 F(1.1) XhoI atcCTCGAGgcaaggcacccctctagtgatgctc

rUGT1A1 R(1.1) HindIII gtgaagcttgcgggttacatttgttctgtttgtt

rUGT1A1 F(2.3) KnpI atcGGTACCatgatcatgtcaaatactcattcac

rUGT1A1 F(2.3) XhoI atcCTCGAGgcgggttacatttgttctgtttgtt

rUGT1A1 mF AAAtccacCTCTagaactcagctgcctgatttccac

rUGT1A1 mR gagttctAGAGgtggatttgcatcaaagaactggct

Table II: PCR program used for cloning of rUGT1A1 promoter constructs

PCR program

Temperature Duration

95 °C 5 minutes

94 °C 30 seconds

52 °C 30 seconds

72 °C Based on the length of amplicon (500 ~ 600 bp per 30 seconds)

Return to 94 °C for 2 cycles

94 °C

30 seconds

58 °C

30 seconds

72 °C Based on the length of amplicon (500 ~ 600 bp per 30 seconds)

Return to 94 °C for 32 cycles

72 °C 5 minutes

10 °C

E.coli transformation

Top10 chemically competent E.coli cells (Invitrogen, Carlsbad, CA) were used for

transformation and vector proliferation. Briefly, few μl of ligation mixture was incubated with

cells on ice for 5 minutes, then followed by heat shock at 42 °C for 30 seconds, quickly put back

on ice. Cells were recovered at 37 °C for one hour in the provided S.O.C medium, then spread

on ampicillin (50 mg/l) LB agar plate for selection.

Nuclear receptor-promoter screen

CV1 cells were cultured in 48-well plates (Corning), 30.000 cells/well. After 24 hours, cells were

transfected with 2.3 kb rat UGT1A1 promoter vector linked to a luciferase reporter (100

ng/well). Simultaneously, cells were co-transfected with one of 49 different vectors (pcDNA3.1-

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V5H6 backbone, Invitrogen) each containing the cDNA of a member of the NR-superfamily (50

ng/well) together with a TK-Renilla vector (10 ng/well) as the internal control.

Empty cDNA3.1 vector (50 ng/well) was used as negative control (basic promoter activity). In

case of heterodimeric NRs, cells were co-transfected with RXRα-vector (25 ng/well). FuGene

HD (Fugent LLC, Madison, WI) was used as transfection reagent (0.48 µl/well) with ratio 1:3

(µl FugeneHD : µg vector). As the transfection control, one well of cells was transfected with

160 ng pLenti-eGFP vector only in each experiment. GFP signal was detected using a

DMI6000B microscope (LEICA Microsystems, Wetzlar, DE) to get impression of transfection

efficiency.

24 Hours after transfection, NR ligands were added (where applicable) in DMEM, supplemented

with 10% stripped serum, penicillin and streptomycin. For ethanol-dissolved ligands, ethanol

was used as control. For DMSO-dissolved ligands, DMSO was used as control. All experiments

were performed in triplo. 24 hours after ligand treatment, cells were incubated 10 minutes with

50 µl Passive Lysis Buffer (Promega). Luciferase and Renilla activity were measured using Dual

Luciferase Assay (Promega) and results were read on a luminometer (Promega). Luciferase

signal, corrected for variation in transfection efficiency (Luciferase signal/Renilla signal),

represented UGT1A1 promoter activity.

Response element identification

To determine whether GR and PR exerted their effects via either distal or proximal promoter, we

assessed GR- and PR-mediated transcription in a shorter rat UGT1A1 promoter sequence of 1.1

kb, using an adjusted dual-luciferase protocol. The 1.1 kb UGT1A1 promoter vector linked to a

luciferase transporter, was transfected into CV1 cells (30.000 cells/well; 100 ng vector/well),

together with cDNA3.1 vectors containing GR and PR promoters respectively (50 ng/well), and

with a TK-Renilla vector (10ng/well). FuGene HD was used as transfection reagent (0.48

µl/well). 24 Hours after transfection, 0,3 ul (1mM) dexamethasone (DEX) per well was added to

the GR-transfected wells and 0,3 ul (50µM) progesterone (PRG) to the PR-transfected cells.

DEX and PRG were added in DMEM, supplemented with 10% stripped serum, penicillin and

streptomycin. Ethanol was used as negative control. 24 hours after ligand treatment, cells were

incubated 10 minutes with 50 µl Passive Lysis Buffer. Luciferase and Renilla activity were

measured using Dual Luciferase Assay and results were read on a luminometer.

To identify the response elements of GR and PR in the UGT1A1 promoter, first in silico analysis

was performed on both the rat and human UGT1A1 promoter sequences up to 3.5 kb, using

MatInspector Software. In the rat promoter, a GR-response element (GRE),

aaaGAACActctCTCC, was predicted at -77/-95 bp upstream the UGT1A1 gene. This element

was mutated by overlap extension PCR as described above in the manner of adenine (A) to

cytosine (C) and thymine (T) to guanine (G). Consequently, the mutated UGT1A1 promoter had

9 changed nucleotides (aaaTCCACctctAGAA) in the predicted GRE.

Subsequently, the mutated UGT1A1 1.1kb promoter sequence was tested for GR- and PR

mediated transcriptional regulation in CV1 cells using the Dual Luciferase Assay as described

above.

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UGT1A1 regulation by NRs in liver cellsTranscriptional regulation of both wild-type (1.1kb)

and mutated rUGT1A1 promoter was tested in HepG2 cells, using the same Dual Luciferase

Assay, adjusted for the predicted lower transfection efficiency in this cell line compared to CV1

cells. We used 60.000 cells/well, 150 ng rUGT1A1 promoter vector/well, 75 ng GR or PR

promoter vector/well respectively, and 20 ng Renilla-luciferase vector/well. The same amounts

of DEX and PRG were added.

Additional experiments

rUGT1A1 promoter was co-transfected with cDNA3.1 as the NR control, followed by DEX,

PRG or cortisol treatment. In addition, rUGT1A1 was co-transfected with MR, the most specific

target of cortisol.

To determine whether the GR-DEX complex induced rUGT1A1 transcription in a dose

dependent manner, we tested UGT1A1 expression after co-transfection with different doses of

GR; 0 ng, 5 ng, 25 ng, 50 ng and 100 ng. The DEX-dose was unchanged.

To determine whether the PRG-mediated rUGT1A1 induction was exclusively mediated via PR,

we assessed rUGT1A1 expression after co-transfection with PXR followed by PRG and ligand

pregnenolone-16alpha-carbonitrile (PCN) treatment.

Statistical analysis

Differences in rUGT1A1 expression were analysed using the independent-samples t-test.

Statistical significance was considered reached at p ≤ 0.05. Analyses were performed using IBM

SPSS Statistics 20 for Windows Software (SPSS Inc., IL, USA).

RESULTS

Transcriptional regulation of rUGT1A1 by Nuclear Receptor Family

To identify the potential role of NRs in the transcriptional regulation of UGT1A1, we screened

all 49 members of the NR family for possible interaction with a 2.3kb rUGT1A1 promoter

fragment (Fig. 3). Thereby we identified five NRs that clearly induced rUGT1A1 expression,

namely Estrogen Related Receptor 3 (ERR3), Sterodiogenic Factor 1 (SF-1) and GR, PR and

Androgen Receptor (AR) in presence of their ligands, DEX, PRG, and androstane respectively

(Fig. 3B). The other NRs, which did not result in a clear induction, were therefore not considered

further. Since the strongest induction was observed after co-transfection with GR and PR, further

experiments focused on the GR- and PR- mediated rUGT1A1 expression.

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Transcriptional regulation of rUGT1A1 by GR and PR

GR- and PR- mediated rUGT1A1 transcription was only observed after stimulation with DEX

and PRG respectively. From these data, we could not determine whether the transcription was

mediated by the NR-ligand complex (GR-DEX and PR-PRG), or only by the ligand, independent

of the co-transfected NR. Therefore, in addition to GR, PR, GR-DEX and PR-PRG treatment, a

1.1kb rUGT1A1 promoter fragment was stimulated with DEX and PRG alone (Figure 4A and B

respectively). DEX alone caused a 1.4-fold induction, whereas PRG alone resulted in a 1.7-fold

induction of rUGT1A1. GR-DEX caused a 17-fold and PR-PRG a 13-fold induction. GR without

DEX and PR without PRG did not cause a significant rUGT1A1 induction. These results

Figure 3: A) UGT1A1 induction mediated by heterodimeric NRs. No clear induction of rUGT1A1 can be observed.

B) UGT1A1induction mediated by homodimeric NRs. Note the clear induction of rUGT1A1 by ERR3, SF-1, AR +

androstane and especially GR + dexamethasone and PR + progesterone.

05

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demonstrate that the previously identified DEX and PRG mediated rUGT1A1 induction, is

mediated by NR-ligand complexes and is therefore NR-dependent.

To reaffirm the seemingly pivotal role of GR in GR-DEX-mediated rUGT1A1 induction, a GR-

dose dependent experiment was performed. Figure 5 shows that GR-DEX up-regulates UGT1A1

transcription in a GR-dose dependent manner in a range of 5-100 ng GR per 30.000 cells. Since

the amount of DEX was kept constant during the experiment, these data confirm that the GR-

DEX-mediated rUGT1A1 induction is GR-dependent.

2A 2A

4B

Figure 4: A) DEX causes a significant 1,4-fold induction of UGT1A1 expression (p = 0,005). GR does not

lead to a significant induction of UGT1A1 expression (p=0,122). GR-DEX causes a significant 17-fold

induction (p = 0,006). B) PRG causes a significant 1,7-fold induction of UGT1A1 expression (p = 0,012). PR

co-transfection does not lead to a significant induction of UGT1A1 expression (p = 0,136). PR-PRG causes a

significant 13-fold induction (p = 0,005)

*=significant difference at a p-value <0.05, when compared to cDNA3.1

4A

14

DEX is the most specific ligand of GR, but GR is known to be activated by other ligands that

usually activate homologous NRs. Cortisol is the most specific ligand of the Mineralocorticoid

Receptor, a well-known GR homologue. In addition, cortisol is a less specific ligand of GR. To

test whether GR-mediated rUGT1A1 induction was specifically induced by DEX only,

rUGT1A1 was stimulated with cortisol, after GR co-transfection. GR-cortisol caused a 15-fold

induction, whereas cortisol alone caused a 2,1-fold induction of rUGT1A1. MR co-transfection

in absence of its ligand caused a 1,3-fold induction. MR in combination with cortisol caused a

2,4-fold induction when compared to control (Fig. 6). These results show that GR-mediated

rUGT1A1 regulation can be induced by several GR-ligands.

0

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0 5 25 50 100

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GR dose (ng)

6

5

Figure 5: UGT1A1 expression is induced by GR when stimulated with DEX in a GR-dose

dependent manner.

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60

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80

90

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* * *

*

*

15

GR and PR are known homologues and have similar DNA-binding and ligand-binding domains.

Since both NRs mediate rUGT1A1 induction, the responsible mechanisms were expected to be

similar or potentially the same. Therefore, we tested whether GR-mediated rUGT1A1 induction

could be induced by PRG and whether PR-mediated rUGT1A1 induction could be induced by

DEX. GR-PRG caused a 6,7-fold induction, whereas GR-DEX caused a 9,6-fold induction in

this experiment. PR-DEX caused a 2,3-fold induction and PR-PRG resulted in a 9,7-fold

induction of rUGT1A1 (Fig. 7). These results confirm that GR and PR are, at least in part,

activated by the same ligands. Furthermore, the data indicate that GR and PR can collaborate in

mediating DEX- and PRG- induced rUGT1A1 transcription.

Our data show that PRG can induce rUGT1A1 when interacting with GR, but specifically with PR.

However, PR is only expressed at minimal levels in liver (39), and the UGT1A1-inducing effect

of PRG has been contributed to PXR, a less specific PRG target (38,40), in combination with

RXR (PXR/RXR). In the NR-promoter screen, PXR did not induce rUGT1A1, neither alone,

nor in combination with its ligand, PCN. However, since PXR/RXR had not been tested in

combination with PRG, PRG-mediated rUGT1A1 induction was assessed after either PR or

PXR/RXR co-transfection. PXR/RXR co-transfection without PRG stimulation caused a 1,7-fold

0

2

4

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*

*

*

*

Figure 7: rUGT1A1 expression was significantly induced 6,7-fold (p = 0,003) after co-transfection with GR,

stimulated with PRG. GR-DEX caused a significant 9,6-fold induction (p = 0,008). PR stimulated with DEX

caused a significant 2,3-fold induction of rUGT1A1. PR-PRG caused a significant 9,7-fold induction (p =

0,026).


7

Figure 6: Cortisol caused a significant 2,1-fold induction (p = 0,000) when compared to

cDNA3.1. Co-transfection with MR caused a significant 1,3-fold induction (p = 0,008). MR

stimulated with cortisol caused a significant 2,4-fold induction (p = 0,002). Co-transfection of

GR caused a significant 15-fold rUGT1A1 induction after cortisol stimulation (p = 0,000) and a

17-fold induction after DEX stimulation (p = 0,006).


16

induction. Stimulation with PRG did not induce rUGT1A1 any further. Stimulation of PXR/RXR

co-transfected cells with the specific ligand PCN caused a decrease in UGT1A1 expression. PR-

PRG caused a 13-fold induction in this experiment (Fig. 8).

GR and PR Response element identification

After identification of GR and PR as rUGT1A1 transcription regulators, we aimed to reveal the

mechanism of induction. NRs can induce gene transcription in an either direct or indirect

manner. Upon ligand activation, direct induction involves direct binding of the DNA-binding

domain to the promoter of a gene, thereby inducing transcription of the respective gene. In

indirect induction, ligand activation directly induces the transcription of a gene, which results in

the production of the protein encoded for by this gene. This protein, which can be a transcription

factor or ligand by itself, subsequently induces the transcription of the gene of interest.

To determine if the identified GR- and PR-mediated regulation is due to direct NR-promoter

binding, the rUGT1A1 promoter had to be analyzed for potential GR and PR binding-sites. We

observed GR- and PR-mediated rUGT1A1 induction in the 2,3 kb rUGT1A1 promoter (Fig. 3).

The induction was still present after truncation to a 1 kb promoter sequence (Fig.4), indicating

that a potential binding-site is present in the 1 kb sequence. Within this 1 kb sequence, in silico

analysis predicted a potential GR-response element distanced at -77/-95 bp from the rUGT1A1

gene. No specific PR-response elements were predicted. To determine whether the identified

sequence comprised a direct GR-binding site, the binding site was mutated by point mutations.

8

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60Lu

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Figure 8: PXR/RXR co-transfection caused a significant 1,7-fold induction in UGT1A1 expression (p =

0,047). PXR/RXR stimulated with PRG also caused a significant 1,7-fold induction (p = 0,004). PXR/RXR

stimulated with PCN caused a significant 1,7-fold decrease (p = 0,002). PR-PRG caused a significant 13-

fold induction (p = 0,005).


* *

*

*

17

After mutation, both GR-DEX- and PR-PRG-mediated rUGT1A1 induction was completely

abolished (Fig. 9). These data show that both GR and PR mediate rUGT1A1 transcription by

direct binding to the rUGT1A1 promoter to a binding-site located 77/95 bp upstream the

rUGT1A1 gene.

UGT1A1 regulation in liver cells

All data displayed above were derived from experiments performed using CV-1 cells. CV-1 is a

well-established and commonly used model for testing NR-mediated transcriptional regulation.

However, CV1 is an African Green Monkey derived fibroblast cell line, and may not be

susceptible to the same regulatory pathways as human hepatocytes. In addition, human kidney

cells do not express UGT1A1 . Thereby, these cells potentially limit the physiological relevance

of our results. Therefore, we reproduced our findings from CV1 in HepG2 cells, a human

hepatocellular carcinoma cell line. Figure 10 shows the GR- and PR- mediated regulation of

rUGT1A1 in HepG2 cells. As in CV-1 cells, a relatively small but significant induction of wild-

type UGT1A1 can be observed after DEX treatment. In contrast, PRG did not induce UGT1A1

in both wild-type and mutated promoter. GR-DEX caused a 26-fold induction in the wild-type

UGT1A1 promoter, but did not affect the mutated promoter. PR-PRG caused a 9,7-fold

induction in the wild-type promoter, but no induction in the mutant promoter. These results show

that rUGT1A1 is induced by GR-DEX and PR-PRG in human liver cells via direct binding to the

rUGT1A1 promoter.

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35

cDN

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.1 GR

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+ d

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+ d

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Nuclear receptors (+ligand)

*

* 9

= wild-type UGT1A1

promoter

= mutated UGT1A1 promoter

Figure 9: The GR-DEX mediated rUGT1A1 induction was 50-fold lower in the mutated promoter

compared to the wild-type UGT1A1 promoter (p = 0,013). PR-PRG mediated rUGT1A1 induction was

14-fold lower in the mutated promoter compared to the wild-type promoter (p = 0,002). In the mutated

rUGT1A1 promoter, GR caused a significant 1,7-fold rUGT1A1 induction (p = 0,023). GR-DEX

caused a significant 1,5-fold induction (p=0,013). PR did not cause a significant induction (p = 0,076),

but PR-PRG significantly induced rUGT1A1 1,5-fold (p = 0,038).

18

DISCUSSION

We have shown that rUGT1A1 transcription can be up-regulated in liver cells by DEX, PRG and

cortisol treatment via direct binding of ligand-activated GR and PR to the rat UGT1A1 promoter.

Furthermore, we identified a response element shared for GR and PR (GAACActctCTCC) in

rUGT1A1 promoter at -77/-95 bp upstream of the transcription site. Mutation of this sequence

completely inactivated both GR- and PR-mediated rUGT1A1 induction.

UGT1A1 induction by DEX has been documented in vivo in liver (31) and in vitro in primary rat

hepatocytes (32) and HepG2 cells (33). However, the molecular mechanism of this response has

not been fully elucidated.

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100

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10 = wild-type UGT1A1

promoter

= mutated UGT1A1 promoter

*

*

*

#

Figure 10: In HepG2 cells, using the wild-type UGT1A1 promoter, DEX significantly induced UGT1A1

1,4-fold (p = 0,002) and GR-DEX caused a significant 26-fold induction (p = 0,014). GR without DEX

did not affect UGT1A1 expression (p = 0,069). PRG did not affect UGT1A1 expression (p = 0,848) and

PR alone did not cause an induction (p = 0,096). PR-PRG caused a significant 9,7-fold induction

(p=0,002). Using the mutated UGT1A1 promoter, Dexamethasone, GR and GR-DEX did not induce

UGT1A1 expression (p = 0,092, 0,053 and 0,197, respectively). PR significantly decreased UGT1A1 (p

= 0,011). PRG or PR-PRG did not affect UGT1A1 (p=0,250 and 0,270, respectively), using the mutated

promoter.

*=significant difference at a p-value <0.05, when compared to cDNA3.1, using the wild-type UGT1A1

promoter

#=significant difference at a p-value <0,05, when compared to cDNA3.1, using the mutated UGT1A1

promoter

19

DEX is the most specific ligand of GR, and the GR-DEX complex is known to activate the

transcription of several metabolic genes, including CYP3A4, CYP2B6 and CYP2C8 (34).The

complex can up-regulate gene expression in a direct fashion, by binding to a Glucocorticoid

Response Element (GRE) in the promoter of the respective gene. In addition, the GR-DEX

complex can indirectly regulate transcription, by inducing the transcription of other transcription

factors. DEX has been reported to increase the expression of PXR, CAR and RXRα in cultured

human hepatocytes (34,35). These transcription factors can subsequently enhance transcription

of indirect GR target genes.

Most DEX effects are mediated by the GR-DEX complex, but DEX has also been shown to be a

direct ligand of PXR (36), indicating DEX can also work in an GR-independent manner.

With regards to the human UGT1A1 (hUGT1A1) promoter, Sugatani et al. identified two DEX

responsive GREs, located in the distal enhancer module PBREM, which is located at

−3483/−3194 bp distance of the UGT1A1 gene (28). PBREM contains response elements for

PXR, CAR and 2 GREs. Mutation of these GREs has shown to decrease DEX-induced PBREM

activity around 75%. However, in presence of CAR or PXR, the DEX-induced PBREM activity

remained around 90% in the GRE mutant PBREM. DEX treatment did not increase CAR or PXR

expression (28). These results indicate that GR-DEX can induce its effects on UGT1A1 both

directly and indirectly. Our results are not affected by potential PBREM interactions, since the

rat promoter does not contain a PBREM. Moreover, we used proximal promoters with a maximal

length of 2.3 kb, whereas PBREM is located more distal from the hUGT1A1 gene.

Although GR-DEX induction of hUGT1A1 transcription has shown to work via PBREM, Usui et

al. showed that a shorter hUGT1A1 promoter sequence, up to -3174 bp distance of the UGT1A1

gene, is also activated by DEX in HepG2 cells (33). This promoter does not include PBREM,

indicating the existence of an additional mechanism of DEX-induced hUGT1A1 transcription.

When co-expressed with GR, the induction of this promoter was enhanced seven-fold by DEX

and this response was inhibited by a GR antagonist, RU486 (37). By truncation studies, the

essential sequence through which this response was mediated, was identified at -75/-63 bp from

the hUGT1A1 gene. However, no direct GR-binding to this sequence was shown, indicating no

direct regulation of the promoter by GR-DEX. The respective sequence includes the response

element for Hepatocyte Nuclear Factor 1(HNF1), but HNF1 levels were not increased after DEX

and GR-DEX was not shown to bind to the HNF1 gene promoter. The mechanism of this

induction therefore remains unclear (37).

Our data support the presence of an additional mechanism of direct GR-DEX-induced UGT1A1

transcription mediated via the proximal promoter. Although our findings are based on rat

UGT1A1, a certain homology between rat and human can be expected. Our in silico analysis

revealed several potential GRE half sites present in the proximal region of human UGT1A1

promoter, indicating that this direct GR regulation on the UGT1A1 promoter might be

conserved. Therefore, our data point to direct regulation of UGT1A1 transcription through GR

binding to the proximal UGT1A1 promoter.

PRG is a known inducer of UGT1A1 transcription in HepG2 cells (38). In addition, UGT1A1

activity is increased during pregnancy, which has been linked to increased blood PRG levels

20

(38). Here we show that the rUGT1A1 promoter is highly activated by PRG treatment in the

presence of PR via direct PR-promoter interaction (Fig. 9). However, PR is only expressed at

minimal levels in liver (39), and the UGT1A1-inducing effect of PRG has been contributed to

PXR, a less specific PRG target (38,40). We could not reproduce the PRG-induced rUGT1A1

transcription after PXR co-transfection (Fig. 8). This might be caused by the discrepancy

between rat and human UGT1A1 regulation or the PXR-mediated PRG effect might be present at

the distal promoter region, as it is in PBREM in human.

Interestingly, our results indicate that, in addition to PR and PXR, PRG induces UGT1A1

transcription via GR activation (Fig. 7)), which has not been shown previously. PRG stimulation

of our GR co-transfected rUGT1A1 promoter, induced UGT1A1 transcription in liver cells,

whereas PRG alone did not induce UGT1A1. PR and GR are known to be homologous NRs, and

are known to respond to the same ligands. Previously, PRG has been reported to mediate its

effects via GR (41). Both the GR-DEX and PR-PRG effects disappeared after mutation of the

putative GRE, indicating that these two NRs bind to the same response element, and may thereby

activate similar pathways to induce UGT1A1 transcription. PR and GR have been reported to

bind to the same response element in other promoters (41).

We showed that GR-DEX and PR-PRG have a strong rUGT1A1-inducing effect. However, we

also showed that DEX slightly induces rUGT1A1 independent of GR-overexpression (Fig. 10).

Since this induction disappeared after GRE mutation, these results suggest the presence of

endogenous GR, that responds to ligand treatment and thereby induces a modest up-regulation.

Limitations Our experiments were performed in CV1 and HepG2 cells. However, although the latter were

originally derived from hepatocytes, they have lost many of the hepatocyte characteristics,

possibly also UGT1A1 regulation. Westerink et al. showed that UGT1A1 and UGT1A6

transcripts were between 10- and more than 1000-fold higher in primary hepatocytes, when

compared to HepG2 cells (42). Therefore, we aim to reproduce our results in primary rat

hepatocytes in future experiments.

Future perspectives

GR, PR and AR

DEX is known to induce UGT1A1 activity in vitro and in vivo (31-33). PRG has been shown to

induce UGT1A1 expression in HepG2 cells (38), but has not been tested in primary rat

hepatocytes or in vivo. In order to establish the relevance of the PRG-induced UGT1A1 up-

regulation, we would like to confirm this induction in primary hepatocytes and in rat liver. If

present, we would like to assess to what extend the PRG-effect is mediated by GR, by adding a

GR antagonist.

Additional future experiments will predominantly aim to determine the mechanism of GR- and

PR-mediated UGT1A1 transcription in vitro. We showed that GR and PR mediate their effects

on rUGT1A1 via the same GRE. To further confirm direct GR- and PR-binding to the identified

GRE in the rUGT1A1 promoter, we would like to perform electrophoretic mobility shift assay

and CHIP-sequencing.

21

GR and PR are homologues and both belong to the steroid receptor subfamily of NRs, together

with MR and AR (43). Future experiments need to confirm the AR-mediated rUGT1A1

induction, as observed in the NR-promoter screen (Figure 3B). AR or its ligand testosterone, are

not known to induce hUGT1A1 expression, but increased renal UGT1A1 expression has been

reported in rats after testosterone treatment (44). However, the mechanism of this induction has

not been determined. If AR appears to induce UGT1A1 in vitro and in vivo, additional

experiments need to show whether the AR-mediated induction is mediated via the identified

GRE. In addition, we need to assess whether androgens up-regulate UGT1A1 in combination

with GR-overexpression, as we showed for PRG. If both PRG and androgens affect UGT1A1

transcription via the identified GRE, we might have identified a new hormone responsive

fragment in the UGT1A1 promoter that could be responsible for the hormone effects on

UGT1A1 activity.

Estrogen Related Receptor 3 (ERR3)

In addition to the 3-ketosteroid receptors, ERR3 co-transfection caused a clear induction of

rUGT1A1. ERR3 belongs to the estrogen related receptors group, which is part of the same

subfamily as the steroid receptors; the Estrogen Receptor-like receptors (43). Other than GR and

PR, ERR3 has no known physiologic ligands or pharmaceutical agonists. Two inverse agonists

have been identified; 4-hydroxytamoxifen and diethylstilbestrol (DES)(45,46), but both are

reported with carcinogenic effects (47,48). Since no agonist has been identified, currently we are

not able to stimulate ERR3 activity to manipulate its effect on UGT1A1 activation for in vivo

experiments.

Steriodogenic Factor-1 (SF-1) and Liver Receptor Homologue-1 (LRH-1)

The NR-promoter screen showed a clear rUGT1A1induction after co-transfection with SF-1.

However, SF-1 in humans is not present in UGT1A1 expressing tissues and is therefore unlikely

to regulate UGT1A1 in vivo. However, liver receptor homologue-1 (LRH-1), which is a close

homologue of SF-1, is overexpressed in the liver, where it regulates bile acid synthesis and

mediates the transcription of another nuclear receptor, small heterodimer partner (SHP) (49). In

figure 3B, a slight induction of rUGT1A1 can be observed after LRH-1 co-transfection.

However, we performed the screening in CV-1 cells, using rat promoter. Possibly, LRH-1 will

show a greater induction in hepatocytes, using hUGT1A1 promoter. Although LRH-1 is still

classified as an orphan NR, phosphatidylinositols are suggested as its physiologic ligands

(50)and several small molecule synthetic agonists have been identified, that are capable of

inducing LRH-1 activity (51). The identification of ligands, makes LRH-1 a potential drug

target. Therefore, we would like to determine the interaction of LRH-1 with hUGT1A1 in future

experiments.

In addition to the 2.3 kb rUGT1A1 promoter, regulatory elements might be located further

distanced from the UGT1A1 gene. Although PBREM has been thoroughly investigated, less

distal promoter regions up to -3194 kb could still contain unknown regulatory elements. In future

experiments, we aim to screen the entire UGT1A1 promoter up to PBREM.

Our experiments were performed using rat UGT1A1 promoter, which will enable us to

extrapolate our findings in vivo in rat models. Once the identified NR-promoter interactions are

confirmed in a rats, our findings will need to be reproduced in human UGT1A1 promoter.

22

Conclusion

We have shown that GR mediates rUGT1A1 transcription after stimulation with various ligands,

including dexamethasone, cortisol and progesterone. PR mediates rUGT1A1 transcription after

stimulation with progesterone. Both GR and PR bind to a newly identified response element in

the proximal rUGT1A1 promoter, which is located at a distance of -77/-95 bp of the rUGT1A1

gene. Mutation of this response element disrupts the transcriptional regulation of rUGT1A1 by

both GR-DEX and PR-PRG. Our data thus indicate that GR/PR-ligand complexes can induce

rUGT1A1 transcription by direct binding to the proximal UGT1A1 promoter.

Implications Ultimately, understanding the transcriptional regulation of UGT1A1 will contribute to clinical

care in three ways:

1) NRs that mediate UGT1A1 transcription can be potential targets for UGT1A- inducing drugs

that can treat or prevent unconjugated hyperbilirubinemia. Although DEX and PRG have already

been identified previously as UGT1A1 inducers, at this point both ligands are not potent and

suitable to treat unconjugated hyperbilirubinemia and clinical studies report controversial results

(52). We now reveal part of the mechanism of DEX and PRG-mediated UGT1A1 induction,

which can contribute to the development of potent, targeted drugs.

2) Understanding transcriptional regulation can help to explain and predict drug interactions that

affect UGT1A1 activity. Thereby, we can avoid drugs that interfere with bilirubin breakdown

and prevent drug induced hyperbilirubinemia. If physiological NR ligands are identified,

measuring blood levels of these ligands might enable estimating bilirubin breakdown capacity,

and subsequent intervention if necessary.

3) Differences in transcriptional UGT1A1 regulation between individual infants, may cause

differences in the capacity to breakdown bilirubin or other toxic compounds. Identification of

UGT1A1-regulating NRs and the respective binding sites might contribute to the identification

of mutations or polymorphims in the UGT1A1 promoter, that might explain for differences in

severity of unconjugated hyperbilirubinemia between individuals. Identification of these

polymorphisms might enable prenatal or early antenatal screening with subsequent early

intervention if necessary.

23

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28

APPENDICES

Functional analysis of transcription

by the Nuclear Hormone Receptor family

Protocol: High Throughput Screening (48-well format)

DAY 1 Plate preparation

Cells: CV-1, 30.000 cells per well (in 200 ul medium per well)

Medium: DMEM, phenol-red free, 10 % FBS, penicillin/streptomycin

Replicates: triplicates, 3 wells per reaction

The entire screen consists of 5 plates, prepare 6 plates for additional reactions or controls

DAY 2 Transfection

Per well, a total of 160 ng DNA is used consisting of 50 ng NR dimer, 100 ng promoter and 10 ng control

(Renilla) as a transfection control (optional). For this, 0,48 of Fugene HD is required for transfection

(160ng x 3 = 0.48 µl) per well.

Depending on whether homodimeric NRs or NR/RXR heterodimers are used, use the following

combination of DNA:

Transfection using Fugene HD (Roche), ratio 3:1 (µl Fugene HD: µg DNA)

-The entire screen consists of 5x16 = 80 reactions (35 NR/RXR heterodimer and 45 NR homodimer

reactions)

-Each reaction will be prepared in a separate Eppendorf tube, each containing different NR DNA and a

certain amount of mastermix containing Promoter, Renilla, Optimem, Fugene and RXRa in case of

NR/RXR heterodimer reactions.

For reaction scheme, see attachment 1

For plate lay-out, see attachment 2

NR/RXR heterodimer Per well (48-well format)

NR 25 ng

RXRa 25 ng

Promoter 100 ng

Renilla 10 ng

Total 160 ng

NR homodimer Per well (48-well format)

NR 50 ng

Promoter 100 ng

Renilla 10 ng

Total 160 ng

29

-NR/RXRa heterodimers: Add 1,8 ul NR DNA per Eppendorf

-NR homodimers: Add 3,6 ul NR DNA per Eppendorf

-After Fugene addition, mix shortly and distribute 72 ul Mastermix 1 in the appropriate Eppendorf tubes

-Centrifuge at 2,0 rpm for 10 seconds

-Incubate for 10 minutes

-Add 20 ul from each Eppendorf in 3 wells in a droplet like fashion

Mastermix 2 (45

interactions)

Well content

(ul)

Eppi content

(ul)

Mastermix content (ul) for 45

reactions

Promoter 2 7,2 388,8

Renilla 0,5 1,8 97,2

Optimem 16,5 59,4 3207,6

Fugene 0,48 1,728 93,312

Total 19,48 70,128 3786,912

-After Fugene addition, mix shortly and distribute 70 ul of Mastermix 3 in the appropriate Eppendorf

tubes

-Centrifuge at 2,0 rpm for 10 seconds


-Add 20 ul from each Eppendorf in 3 wells in a droplet like fashion

DAY 3 Ligand treatment

24 NRs are known to need ligand for activation

For all ligand activated NRs 2 reactions are needed: + and – ligand

Ligands are dissolved in ethanol or dmso, so negative controls are ethanol or dmso

For each reaction, prepare 1 ml DMEM with stripped serum with 1 ul ligand

Ligand

Reactions Prepare

Plate I

Calculations are based on the following concentrations

Promoter 50 ng/ul

NR 50 ng/ul

Renilla 20 ng/ul

Mastermix 1 with RXRa (35

interactions)

Well content

(ul)

Eppi content

(ul)

Mastermix content (ul) for 35

reactions

Promoter 2 7,2 302,4

RXRa 0,5 1,8 75,6

Renilla 0,5 1,8 75,6

Optimem 16,5 59,4 2494,8

Fugene 0,48 1,728 72,576

Total 19,98 71,928 3020,976

For calculations, see attachment 3

30

T3 2,4,6,8 4 ul T3

4 ml medium

TTNBP 10,12,14 3 ul TTNBP

3 ml medium

WY14643 16 1 ul WY14643

1 ml medium

Plate II

BRL49653 2 1 ul BRL49653

1 ml medium

GW501516 4 1 ul GW501516

1 ml medium

LXR ligand 6,8 2 ul LXR ligand

2 ml medium

FXR ligand 10,12 2 ul FXR ligand

2 ml medium

Vitamin D 14 1 ul Vitamin D

1 ml medium

PCN 16 1 ul PCN

1 ml medium

Plate III

TCOPOBOP 2 1 ul TCOPOBOP

1 ml medium

9-cis RA 5,7,9 3 ul 9-cis RA

3 ml medium

Plate IV

b-estradiol 6,8 2 ul b-estradiol

2 ml medium

Plate IV

Dexamethasone 2 1 ul dexamethasone

1 ml medium

Cortisol 4 1 ul cortisol

1 ml medium

Progesterine 6 1 ul progesterone

1 ml medium

Androstane 8 1 ul androstane

1 ml medium

DMSO controls Plate I: 9,11,13,15 17 ul DMSO

17 ml medium Plate II: 1,3,5,7,9,11,13,15

Plate III: 1,3,4,6,8

Ethanol controls Plate I: 1,3,5,7 10 ul Ethanol

10 ml medium Plate IV: 5,7

Plate V:1,3,5,7

Medium Plate III: 10-16 27 ml medium

Plate IV: 1-4 + 9-16

Plate V: 9-16

For ligand concentrations, see attachment 4

31

-Remove medium from plate

-Wash cells with PBS

-Add 300 ul medium with ligand per well

DAY 4 Dual luciferase assay

-Prepare ±15 ml LAR II buffer and 15 ml Stop & Glow Buffer

-Trash medium and empty wells completely

-Lysis with 50 ul Passive Lysis Buffer (5X diluted) per well


-Transfer 10 ul lysate to reading plate

Use luminometer protocol: ‘Dual luciferase 50 ul Weilin’

32

List of promoters relevant to the Circadian Circuitry

Name

ACCESSIO

N Description

Bmal1 NM_001178 Transcription Factor, heterodimerizes with Clock, core clock TF

Clock NM_004898 Transcription Factor, heterodimerizes with Bmal1, core clock TF

NPAS2 NM_002518 Transcription Factor, heterodimerizes with Bmal1

Per1 NM_002616 Period 1, heterodimerizes with Cry1 and Cry2



Cry1 NM_004075 Cryptochrome 1, heterodimerizes with Per1,2, and 3

Cry2 NM_021117 Cryptochrome 2, heterodimerizes with Per1,2, and 3

Rev-erb

alpha NM_021724 Nuclear Hormone Receptor, repressor (represses Bmal1)

Rev-erb beta NM_005126 Nuclear Hormone Receptor, repressor

Rora NM_134261 Nuclear Hormone Receptor, activator (activates Bmal1)

Rorb NM_006914 Nuclear Hormone Receptor, activator

Rorc NM_005060 Nuclear Hormone Receptor, activator (activates Bmal1)

Dec1 NM_003670

Transcription Factor (bHLH family), negative regulator of molecular

clock

Dec2 NM_030762

Transcription Factor (bHLH family), negative regulator of molecular

clock

Dbp NM_001352 Transcription Factor (PAR bZIP family), circadian expression in SCN

Tef NM_003216 Transcription Factor (PAR bZIP family), circadian expression in SCN

Hlf NM_002126 Transcription Factor (PAR bZIP family), circadian expression in SCN

E4bp4 NM_005384

Transcription Factor (PAR bZIP family), negative regulator of mol.

clock

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=71852578



















33

Nuclear Receptor cDNA’s:

Full length, sequence verified, cDNA’s of all murine Nuclear Hormone Receptors (pcDNA3.1-V5H6

backbone)

Class Official Full name Short Alternative names

1A NR1A1 Thyroid receptor a TRa c-erbA-1, THRA

NR1A2 Thyroid receptor b TRb c-erbA-2, THRB

1B NR1B1 Retinoic acid receptor RARa

NR1B2 Retinoic acid receptor RARb HAP

NR1B3 Retinoic acid receptor RARg RARD

1C NR1C1

Peroxisome proliferator activated

receptor PPARa

NR1C2


receptor PPARd PPARb, NUC1, FAAR

NR1C3


receptor PPARg

1D NR1D1 Rev-Erb REVERBa EAR1, EAR1A

NR1D2 Rev-Erb REVERBb EAR1b, BD73, RVR, HZF2

1F NR1F1 RAR-related Orphan Receptor RORa RZRa

NR1F2 RAR-related Orphan Receptor RORb RZRb

NR1F3 RAR-related Orphan Receptor RORg TOR

1H NR1H2 Liver X Receptor LXRb UR, OR-1, NER1, RIP15

NR1H3 Liver X Receptor LXRa RLD1, LXR

NR1H4 Farnesoid X Receptor FXR FXR, RIP14, HRR1

NR1H5 mouse FXRb (in human pseudogene?) FXRb

1I NR1I1 Vitamin D Receptor VDR

NR1I2 Pregnane X Receptor PXR ONR1, SXR, BXR

NR1I3 Constitutive Androstane Receptor CAR MB67, CAR1, CARa

2A NR2A1 Hepatocyte Nuclear Factor HNF4a HNF4

NR2A2 Hepatocyte Nuclear Factor HNF4g HNF4G

2B NR2B1 Retinoic X Receptor RXRa

NR2B2 Retinoic X Receptor RXRb H-2RIIBP, RCoR-1

NR2B3 Retinoic X Receptor RXRg

2C NR2C1 Testicular Orphan Nuclear Receptor 2 TR2 TR2, TR2-11

NR2C2 Testicular Orphan Nuclear Receptor 4 TR4 TR4, TAK1

2E NR2E1 Tailless (homolog of drosophila) TLX TLL, XTLL

NR2E3 Photoreceptor-specific Nuclear Receptor PNR

2F NR2F1 COUP-TF1 CTF1 COUPTFA, EAR3, SVP44

NR2F2 COUP-TF2 CTF2 COUPTFB, ARP1, SVP40

NR2F6 COUP-TF3 CTF3 EAR2

3A NR3A1 Estrogen Receptor Era ERa

NR3A2 Estrogen Receptor ERb ERb

3B NR3B1 Estrogen Related Receptor ERRa ERR1

NR3B2 Estrogen Related Receptor ERRb ERR2

NR3B3 Estrogen Related Receptor ERRg ERR3

3C NR3C1 Glucocorticoid Receptor GR

NR3C2 Mineralocorticoid Receptor MR

34

NR3C3 Progesterone Receptor PR

NR3C4 Androsterone Receptor AR

4A NR4A1 NR4a1 NR4a1 NGFIB, TR3, N10, NUR77, NAK1

NR4A2 NR4a2 NR4a2 NURR1, NOT, RNR1, HZF-3, TINOR

NR4A3 NR4a3 NR4a3 NOR1, MINOR

5A NR5A1 Steroidogenic Factor 1 SF1 ELP, FTZ-F1, AD4BP

NR5A2 Liver Receptor Homolog LRH1 xFF1rA, xFF1rB, FFLR, PHR, FTF

6A NR6A1 Germ Cell Nuclear Factor GCNF1 RTR

0B NR0B1 DAX1 DAX1 AHCH

NR0B2 Small Heterodimer Partner SHP

35

Attachment 1

Plate #1

promoter:

50ng/ul NR: 50ng/ul

Co-receptor

50: ng/ul

Internal

control

20ng/ul

Sample # 100 ng 25 ng 25 ng Renilla (ng) ligand

1 MRP2 TRa1 RXRa 10 dmso

2 MRP2 TRa1 RXRa 10 T3

3 MRP2 TRa2 RXRa 10 dmso

4 MRP2 TRa2 RXRa 10 T3

5 MRP2 TRb1 RXRa 10 dmso

6 MRP2 TRb1 RXRa 10 T3

7 MRP2 TRb2 RXRa 10 dmso

8 MRP2 TRb2 RXRa 10 T3

9 MRP2 RARa RXRa 10 dmso

10 MRP2 RARa RXRa 10 TTNBP

11 MRP2 RARb RXRa 10 dmso

12 MRP2 RARb RXRa 10 TTNBP

13 MRP2 RARg RXRa 10 dmso

14 MRP2 RARg RXRa 10 TTNBP

15 MRP2 PPARa RXRa 10 dmso

16 MRP2 PPARa RXRa 10 WY14643

2ul 1.5ul 1.5ul 0.5 ul

Plate #2

promoter:

50ng/ul NR: 50ng/ul

Co-receptor

50: ng/ul

Internal control

20ng/ul

Sample # 100 ng 25 ng 25 ng Renilla (ng) ligand

1 MRP2 PPARg RXRa 10 dmso

2 MRP2 PPARg RXRa 10 BRL49653

3 MRP2 PPARd RXRa 10 dmso

4 MRP2 PPARd RXRa 10 GW501516

5 MRP2 LXRa RXRa 10 dmso

6 MRP2 LXRa RXRa 10 LXR ligand

7 MRP2 LXRb RXRa 10 dmso

8 MRP2 LXRb RXRa 10 LXR ligand

9 MRP2 FXR RXRa 10 dmso

10 MRP2 FXR RXRa 10 FXR ligand

11 MRP2 FXRb RXRa 10 dmso

12 MRP2 FXRb RXRa 10 FXR ligand

13 MRP2 VDR RXRa 10 dmso

36

14 MRP2 VDR RXRa 10 Vitamin D3

15 MRP2 PXR RXRa 10 dmso

16 MRP2 PXR RXRa 10 PCN

2ul 1.5ul 1.5ul 0.5 ul

Plate #3

promoter:

50ng/ul NR: 50ng/ul

Co-receptor

50: ng/ul

Internal control

20ng/ul

Sample # 100 25 25 Renilla (ng) ligand

1 MRP2 CAR RXRa 10 dmso

2 MRP2 CAR RXRa 10 TCOPOBOP

3 MRP2 pcDNA3.1 RXRa 10 dmso

4 MRP2 RXRa 10 dmso

5 MRP2 RXRa 10 9-cis RA

6 MRP2 RXRb 10 dmso

7 MRP2 RXRb 10 9-cis RA

8 MRP2 RXRg 10 dmso

9 MRP2 RXRg 10 9-cis RA

10 MRP2 RVRa 10 stripped serum medium

11 MRP2 RVRb 10 stripped serum medium

12 MRP2 RORa 10 stripped serum medium

13 MRP2 RORb 10 stripped serum medium

14 MRP2 RORg 10 stripped serum medium

15 MRP2 HNF4a 10 stripped serum medium

16 MRP2 HNF4g 10 stripped serum medium

2ul 3ul 1.5ul 0.5 ul

Plate #4

promoter:

50ng/ul NR: 50ng/ul

Co-receptor

50: ng/ul

Internal control

20ng/ul


1 MRP2 TR2 10 stripped serum medium

2 MRP2 TR4 10 stripped serum medium

3 MRP2 TLX 10 stripped serum medium

4 MRP2 PNR 10 stripped serum medium

5 MRP2 ERa 10 ethanol

6 MRP2 ERa 10 b-estradiol

7 MRP2 ERb 10 ethanol

8 MRP2 ERb 10 b-estradiol

37

9 MRP2 ERR1 10 stripped serum medium



12 MRP2 CTF-1 10 stripped serum medium



15 MRP2 SF-1 10 stripped serum medium

16 MRP2 pcDNA3.1 10 stripped serum medium


Plate #5

promoter:

50ng/ul NR: 50ng/ul

Co-receptor

50: ng/ul

Internal control

50ng/ul


1 MRP2 GR 10 ethanol

2 MRP2 GR 10 dexamethasone

3 MRP2 MR 10 ethanol

4 MRP2 MR 10 cortisol

5 MRP2 PR 10 ethanol

6 MRP2 PR 10 progesterine

7 MRP2 AR 10 ethanol

8 MRP2 AR 10 androstane

9 MRP2 NR4A1 10 stripped serum medium



12 MRP2 LRH-1 10 stripped serum medium

13 MRP2 GCNF-1 10 stripped serum medium

14 MRP2 DAX-1 10 stripped serum medium

15 MRP2 SHP 10 stripped serum medium

16 MRP2 pcDNA3.1 10 stripped serum medium


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