Nuclear Receptor Rev-erb Alpha (Nr1d1) Functions in Concert with Nr2e3 to Regulate Transcriptional Networks in the Retina

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.


Introduction
Nuclear receptors represent an evolutionarily conserved group of transcription factors that regulate genes involved in diverse functions such as homeostasis, reproduction, development, metabolism and immune response. Nuclear receptors bind to lipophilic-ligands such as steroid hormones, thyroid hormone, vitamin D and retinoids, which modulate transcriptional activity [1][2][3][4][5]. Nuclear receptors also function with co-activators or corepressors to activate or repress the transcription of genes involved in the development and maintenance of specific cell types. In the mouse retina, nuclear receptors regulate the development and patterning of many cell types, including photoreceptor cells consisting of rods (responsible for night vision) and cones (responsible for color vision and visual acuity). The development and patterning of cone cells in mice is regulated by the nuclear receptor thyroid hormone receptor b2 (Trb2, Nr1a2b), which controls the dorsal-to-ventral development and the terminal differentiation of medium (M)-opsin expressing cone cells [6][7]. Retinoid X receptor gamma (Rxrc) and Retinoic acid-related orphan receptor beta (Rorb) control the generation of short (S)-opsin-expressing cones (also referred to as blue cones) [8][9]. RORb also induces rod photoreceptor differentiation by activating the rod-selective transcription factor Neural retina leucine zipper (Nrl) [10]. Rod photoreceptor function and survival is also regulated by the estrogen-related receptor b (Errb) [11].
NR2E3 (mouse Nr2e3) is a nuclear receptor critical for the development and maintenance of rod and cone photoreceptor cells, and loss of NR2E3 results in disease. Mutations in human NR2E3 cause several forms of retinal degeneration including autosomal recessive enhanced S-cone syndrome [12][13], Goldmann-Favre syndrome [14][15][16][17][18], clumped pigmentary retinal degeneration [14,17], and autosomal dominant and recessive retinitis pigmentosa [19][20][21]. Loss of NR2E3 results in a decrease in rod, long-(L) cone and M-cone function, and hypersensitivity to blue light, presumably due to an over generation of S-opsin expressing cone cells [12]. Mice lacking Nr2e3 (Nr2e3 rd7/rd7 , rd7) have supernumerary blue cone cells and slow, progressive loss of rods and cones [22][23]. In addition, mice lacking a functional Nr2e3 clinically display retinal spotting observable at eye opening (postnatal day 14.5), and retinal dysplasia with whorls present at postnatal day 12.5 [24]. In recent studies, NR2E3 is expressed during development in mitotic retinal progenitor cells, and functions as a suppressor of the cone generation program [24]. NR2E3 has been shown to regulate gene networks involved in photoreceptor development and function, and loss of Nr2e3 causes mis-regulation of cone and rod genes [25][26][27][28][29].
NR2E3 functions with co-factors such as RetCoR and REV-ERB alpha (NR1D1) to regulate gene expression in the retina [25,30]. In vitro, NR2E3 forms complexes with NRL, cone rod homeobox (CRX) and NR1D1 to regulate the expression of the rod phototransduction genes Rhodopsin and Guanine nucleotide-binding protein 1 (Gnat1) [25]. Recent studies also demonstrate that Nr1d1 is up-regulated in rd7 mice during development and in the adult retina [29]. These studies also determined that Nr1d1 is a direct target of NR2E3 by chromatin immunoprecipitation, suggesting that these factors function in the same transcriptional network [29].
Nr1d1 appears to have a diverse role in regulating gene networks in many tissue types and several biological processes. Additionally, activated Nr1d1 is able to function as a monomer or a dimer, and is capable of repressing or activating gene expression, suggesting that Nr1d1 has a diverse role in gene regulation [31][32][33]. Little is known about the role of Nr1d1 in the mammalian retina, however its expression has been studied in a variety of tissues including skeletal muscle, liver, adipose, and eye, with its highest expression in the eye [3,29,[34][35][36][37][38]. Nr1d1 has been widely studied for its role in transcriptional regulation of processes such as circadian rhythm and metabolism, with heme functioning as an endogenous ligand [39][40]. Nr1d1 was first identified as being encoded on the opposite strand of the thyroid hormone receptor, and was shown to bind to thyroid hormone receptor response elements, suggesting a potential role in the Trb2-driven differentiation of M-opsin expressing cone cells (green cones) [6,[41][42]. While the role of Nr1d1 in the retina is not well understood, recent studies indicate that it may have a crucial role in retinal development, and functions in the same transcriptional network as Nr2e3 [29].
In this study, we determined the normal expression pattern of NR1D1 in the retina, identified co-targets of NR1D1 and NR2E3, and determined the effect of loss of Nr1d1 in the retina. We determined that NR1D1 and NR2E3 have a similar expression profile, and that NR1D1 is expressed in retinal progenitor cells during development, and in ganglion cells and photoreceptors of the adult retina. We further identified several genes involved in phototransduction, signaling and development that are co-targeted by NR1D1 and NR2E3, in addition to genes that were individually targeted by either nuclear receptor. Acute knockdown of Nr1d1 resulted in retinal abnormalities such as pan-retinal spotting and decreased response to light. These studies highlight an important role for Nr1d1 in the retina and in gene regulation in concert with Nr2e3.

Results
Rev-erb alpha/NR1D1 is expressed in the developing and adult retina Quantitative RT-PCR shows that Nr1d1 is first expressed at embryonic day 18.5 with increasing expression in the adult retina (Figure 1 A). The onset of Nr2e3 expression is also embryonic day 18.5, with a peak in expression at postnatal day 6.5 (Figure 1 A). Because Nr1d1 has been studied for its role in circadian rhythm, we examined the expression of Nr1d1 and Nr2e3 over a twenty-four hour period (8 am-8 am). Both Nr1d1 and Nr2e3 mRNAs showed a cycling pattern with a peak of expression at 4 pm (Figure 1 B).
To determine the localization of NR1D1 protein in the mouse retina, we first performed Western blotting to confirm antibody specificity. NR1D1 protein (67 kD) was detected by Western blot in C57BL/6 (B6) postnatal day 30.5 retinal lysate ( Loss of Rev-erb alpha/NR1D1 results in pan-retinal spotting, and a decrease in retinal response to light Loss of NR1D1 expression in the retina was evaluated using small hairpin (sh)RNA delivery by in vivo electroporation. Efficiency of gene silencing was first confirmed in vitro by quantitative RT-PCR ( Figure S1). shRNA constructs were injected subretinally into neonate B6 eyes, and in vivo electroporation was used to transfect DNA into retinal cells. Mice were aged to postnatal day 30.5 for clinical, functional and histological analysis. Clinically, Nr1d1 knockdown at postnatal day 0.5 produced panretinal spotting of the fundus (Figure 3 I A). Delivery of shRNA directed against crystallin Aa (Cryaa) resulted in lens opacity . Functionally, mice injected with shNr1d1 had 50% lower response from photoreceptor cells and second order neurons in both light-adapted (photopic-to test cone function) and dark-adapted (scotopic-to test rod function) conditions (Figure 3 II). Histological evaluation with hematoxylin and eosin staining on shNr1d1-injected mice at postnatal day 30.5 did not reveal any gross histological abnormalities (Figure 4), as are present with loss of Nr2e3. To identify abnormalities in gene expression, we examined shNr1d1-injected retinas with retinal cell type-specific markers at postnatal day 30.5 ( Figure 5). This did not reveal any marked abnormality in abundance and distribution for any particular cell type within the retina.
Transcriptional networks co-targeted by NR1D1 and NR2E3 are involved in phototransduction, signaling, and gene regulation Microarray analysis was used to identify differentially expressed genes between B6 and rd7 mice. The time points of embryonic day 18.5 and postnatal day 30.5 were selected as the period in which Nr2e3 expression begins, and as an adult time point in which Nr2e3 is expressed. Microarray results were confirmed by quantitative RT-PCR (Table S1). Recently identified target genes of NR2E3 [29], or genes identified as differentially expressed between wild type B6 and mutant rd7 retinas by microarray analysis, were evaluated by chromatin immunoprecipitation to identify genes that are co-targeted by both NR1D1 and NR2E3. Chromatin immunoprecipitation was conducted with anti-NR1D1 and anti-NR2E3 antibodies in parallel, and samples were analyzed by quantitative RT-PCR to identify potential co-targets ( Figure 6). At embryonic day 18.5, the potential regulatory regions of a total of eight out of 31 genes (,25%) were bound by NR1D1, NR2E3 or both nuclear receptors, and at postnatal day 30.5 six out of 36 genes (,18%) were positively targeted by one or both nuclear receptors. The genes targeted by NR1D1 and NR2E3 at embryonic day 18.5 are involved in: phototransduction (Interphotoreceptor retinoid-binding protein (Irbp), Retinal G Protein coupled receptor (Rgr), Recoverin (Rcvn), Rhodopsin); cell differentiation (RAR-related orphan receptor C (Rorg/c)); signaling (retinoic acid receptor responder protein 2 (Rarres2)); transcriptional regulation (nuclear receptor subfamily 2, group C, member 1 (Nr2c1)); or are novel (Bc7393). Nr2c1, Rarres2, Rcvn and Rgr were co-targeted by NR1D1 and NR2E3 at embryonic day 18.5. Bc7393 was targeted by NR2E3 alone, while Irbp, Rorg/c and Rhodopsin were targeted by NR1D1 alone. The genes targeted by NR1D1 and NR2E3 in the adult retina are involved in: cell growth (Nuclear protein one (Nupr1)), phototransduction (phosphodiesterase 8A (Pde8a)), signaling (signal transducer and activator of transcription 1 (Stat1)), transcription (myocyte enhancer factor 2C (Mef2c)), or novel retinal function (otopetrin 3 (Otop3), olfactomedin 1 (Olfm1)). Nupr1 and Pde8a were co-targeted by NR1D1 and NR2E3; Mef2c, Otop3 and Stat1 were targeted by NR1D1 alone; and Olfm1 by NR2E3 alone. To determine the transcriptional activity of NR1D1 and NR2E3 on the potential regulatory regions characterized by chromatin immunoprecipitation, we cloned these sequences, as well as proximal promoter regions of the respective genes into a luciferase reporter plasmid and tested them in HEK293T cells. We tested the individual transcriptional activity of NR1D1 and NR2E3, as well as their function in a gene complex with photoreceptor-specific transcription factor CRX, and rod-specific  transcription factor NRL which has previously been published for regulation of the Rhodopsin promoter [25]. The following genes were tested by luciferase assay: Irbp, Nupr1, Rgr, Rarres2, M-opsin, (Mw-ops), cone arrestin (Arrestin4), and, as a positive control, Rhodopsin (Figure 7).
With the exception of the M-opsin proximal promoter that was activated over three-fold by CRX alone, expression of all other promoter constructs was significantly induced by NRL alone. CRX alone repressed the Rarres2 proximal promoter, and significantly decreased NRL-driven transactivation. Additionally, concurrent expression of CRX and NRL significantly increased the activity of promoter constructs. Transcriptional activity of NR1D1 and NR2E3 depended on the promoter context. For Irbp, the activity of the putative response site (IrbpRE) was barely modulated by the expression of transcription factors, but on the proximal promoter sequence, NR2E3 and NR1D1, alone and together, activated promoter activity by respectively 2-, 2.9-and 2.6-fold. In addition, CRX/NRL-driven transactivation was significantly increased to over 3-fold in the presence of both NR2E3 and NR1D1. The proximal promoter of Rgr was activated about 4-fold by NR1D1, and this activity could not be repressed by NR2E3. In presence of NRL, no other transcription factor could significantly modulate promoter activity. The proximal Rhodopsin promoter activity was increased by about 14-fold in presence of both CRX and NRL. This CRX/NRL-driven promoter activity was further increased to more than 20-fold in the presence of NR2E3, and even 35-fold in presence of both NR2E3 and NR1D1 The 2.6-fold CRX/NRL-driven transactivation of the Nupr1 response element (NuprRE) was significantly increased by NR2E3 to 3.6-fold and NR1D1 to 4.2-fold. This increase in activity was suppressed when NR2E3 was added to the complex of CRX/NRL/NR1D1. A similar repressive role of NR2E3 was observed with M-opsin promoter activity. We also identified that NR1D1 and NR2E3 together were able to drive the expression of Arrestin4 to a far greater extent than either nuclear receptor could individually, but that NR1D1 alone was able to further increase CRX/NRL-driven transactivation from 3.5-to 4.4-fold. These results illustrate that NR2E3 is able to both repress and activate gene expression and that the presence of co-factors and additional transcription factors further modulates gene expression. Furthermore, our results also indicate that NR1D1 serves a role in the activation of many photoreceptor specific genes.

Discussion
In this study we investigated the role of NR1D1 and NR2E3 in gene regulation, and the impact of loss of Nr1d1 on the retina. The implications of these findings aid in our understanding of retinal development and function, and identify a role for Nr1d1 in the mammalian retina. The onset of Nr1d1 and Nr2e3 expression correlated with the end of cone cell generation in mice, a time period when transcriptional regulation is necessary to prevent the continued emergence of cones. While loss of Nr2e3 resulted in continued generation of cone cells, knockdown of Nr1d1 did not result in the over production of cones. This may indicate that genetic compensation by Nr2e3 was able to suppress continued cone generation. Knockdown of Nr1d1 expression did result in reduction of rod and cone function, suggesting that loss of Nr1d1 alters the development and/or function of these cell types. Clinically, the pan-retinal spotting produced by loss of Nr1d1 was highly similar to the pan-retinal spotting observed with loss of Nr2e3 in mice. While loss of Nr1d1 produced a similar clinical phenotype, histologically we did not observe gross morphological changes, as are present with loss of Nr2e3. While no retinal whorls were noted in our shNr1d1-injected mice, there may be additional ultra-structural abnormalities that were not detected in our studies, which could explain the observed retinal spotting and functional deficits. Additionally, our studies only examined injected mice at P30.5 and lack of Nr1d1 may result in slow or delayed retinal degeneration. Thus, aging the mice beyond one month may result in notable histological changes, or a more severe fundus phenotype. Loss of Nr1d1 may alter, for example, signaling pathways within photoreceptor cells, therefore limiting their capacity to fire to second order cells and decreasing retinal function. However, changes such as these may not alter the morphological integrity of the retina. Our studies identified a physiologically important role for NR1D1 in the regulation of photoreceptor gene expression. Previous in vitro luciferase assay experiments had shown that the gene complex consisting of CRX/NRL/NR2E3/NR1D1 had a strong, combinatorial effect in rod-specific gene regulation that was not apparent when evaluating any single factor [25]. In our studies, we also observed combinatorial effects of these transcription factors and determined that NR1D1 and NR2E3 have dual roles in the activation and suppression of gene expression. Consistent with the previously reported suppressor activity of NR2E3 on cone-specific genes, expression of the cone-specific Mopsin and Arrestin4 promoters was increased in presence of CRX/ NRL/NR1D1, but NR2E3 ablated or diminished this activity [26][27]. For the Irbp proximal promoter, which had been shown previously that binding of NR2E3 was not dependent on the presence of CRX [27], NR1D1 was more efficient in driving promoter activity than NR2E3. NR1D1 also acted as a transcriptional activator on the Rgr proximal promoter, suggesting a more general role for NR1D1 in transcriptional activation of photoreceptor gene expression. In the heterologous cell transactivation studies, NR1D1 and NR2E3 activated the expression of the Rhodopsin promoter and highly potentiated CRX/NRL-driven activity. However, we did not identify NR2E3 targeting to the upstream regulatory region of Rhodopsin at the onset of Nr2e3 expression in vivo. This could indicate that NR2E3 does not regulate the expression of Rhodopsin at embryonic day 18.5, but may at later time points. On the other hand, and based on previous studies [25], this also suggests that transcriptional regulation by NR2E3 is at least in part independent of binding to target promoters. Taken together, our studies demonstrate that interaction of transcription factors can have highly variable effects in the activation or suppression of gene expression depending on the regulatory region. Whereas NR1D1 preferentially activates gene expression, NR2E3 was able to both repress and activate gene expression. The presence of co-factors and additional transcription factors further modulate gene expression.
Our earlier studies with NR2E3 chromatin immunoprecipitation at postnatal day 2.5 indicated that NR2E3 targets a large subset of genes critical for photoreceptor development and function [29], suggesting gene regulation is dynamic during development. We observed that during development NR1D1 and NR2E3 co-targeted genes involved in phototransduction, signaling and transcription, indicating that both genes have the capacity to modulate genes important for retinal development. For example, during development, NR1D1 and NR2E3 co-targeted Recoverin (Rcvn), a calcium binding protein that has a critical role in the inhibition of rhodopsin kinase in rod phototransduction. The cotargeting of this gene indicates that NR1D1 and NR2E3 could suppress the expression of Rcvn in developing cone cells, and may explain why mice lacking Nr2e3 develop hybrid cones that aberrantly express rod genes [43]. In the adult retina, genes cotargeted by NR1D1 and NR2E3 were involved in phototransduction and cell growth, suggesting that improper regulation of genes involved in these pathways could cause the increase in blue cone population observed in Nr2e3 rd7/rd7 mice.
Pathways involving phototransduction, signaling, development and cell growth are major developmental networks within the retina, and mutations in nuclear receptor genes regulating these pathways can have far reaching significant consequences. Although no human mutations in NR1D1 have yet been reported in association with retinal degeneration, few studies have examined patients for mutations in this gene [44]. Examination of more patient cohorts may find mutations in NR1D1 associated with retinal disease. Our studies identified a role for Nr1d1 in gene regulation, as well as in the disease process. More studies will be needed to determine additional factors associated with the NR1D1/NR2E3 complex, as well as the dynamic gene regulation occurring in the developing and adult retina.

Ethics Statement
The mice used in this study were bred and maintained under standard conditions in the research vivarium at the University of Nebraska Medical Center under protocol number 04-086 approved by the Animal Care and Use Committee at the University of Nebraska Medical Center. Mice were housed in microisolator cages and provided with food and water ad libitum. The University of Nebraska Medical Center is in compliance with NIH policy on the use of animals in research (Animal Welfare Act P.L. 89-544, as amended by P.L. 91-679 and P.L. 94-279) and the Guide for Care and Use of Laboratory Animals, NIH Publication number 86-23. Tissues were harvested from B6.Cg-Nr2e3 rd7/rd7 (rd7) and C57BL/6J (B6) mice at embryonic day 18.5 and postnatal day 30.5. A minimum of 3 Nr2e3 rd7/rd7 and three B6 mice were analyzed for each time point. Mice were genotyped for the Nr2e3 rd7/rd7 mutation and phenotyped by indirect ophthalmoscopy for retinal spotting as previously described [13,45].

Cloning of mouse NR1D1 shRNA vectors
A distinct 19-nucleotide shRNA sequence against mouse Nr1d1 was generated, using the small interference RNA selection program of the Whitehead Institute (http://jura.wi.mit.edu/bioc/siR-NAext/home.php). LVmNr1d1Aforward and LVmNr1d1Areverse oligonucleotides for the construct were incubated at 100uC for 15 min and annealed by cooling to room temperature. Ligation was done in the presence of 100 ng of annealed oligonucleotides, and 10 ng of the pLVTHM lentiviral vector cut with MluI and ClaI (a kind gift from Dr. Didier Trono, Lausanne, Switzerland). To limit recombination of homologous plasmid sequences, bacterial transformation was done in HB101 cells. Bacterial colonies were screened by PCR using primers LVscreenfor and LVscreenrev, resulting in an amplified fragment of 271 bp in the empty pLVTHM plasmid and 325 bp in the shNr1d1 construct. Positive clones were verified by sequencing with primer LVscreenfor. An shRNA sequence directed against crystallin Aa was used as a control.

In vivo electroporation
Eyes of postnatal day 0.5 B6 mice were subretinally injected with 1 mg of shNr1d1, shCryaa, or empty vector and in vivo electroporation was performed as described previously [46][47] (n.20). Plasmid DNA diluted in sterile H 2 O was pulse electroporated into the retina at 80 volts. Mice were aged to postnatal day 30.5 and phenotyped by indirect ophthalmoscopy, electroretinogram, hematoxylin and eosin staining, and immunohistochemistry as described previously [24]. Uninjected B6 mice were used as a control in electroretinogram experiments as ERGs are unaffected by plasmid DNA and procedure [48].

Microarray analysis
RNA was isolated at E18.5 and P30.5 from B6 and rd7 mouse retinas. Briefly, eyes were enucleated and placed in PBS on ice. Retinas were dissected using a stereo-microscope (Zeiss Stemi SV 11) and RNA was isolated by TRIzolH extraction. A total of 30 retinas were collected from B6 and rd7 mice at similar time points during the day. Equimolar amounts of RNA isolated from ten retinas were pooled into three separate pools from both strains and time point. RNA was hybridized to Mouse 420A 2.0 (Affymetrix, Santa Clara, CA) chips by the UNMC Microarray Core Facility according to manufacturer specifications (Affymetrix, Santa Clara, CA). Data quality was assessed using the affyPLM package for the R programming language. Consistencies of expression levels were confirmed by validation across multiple redundant probe sets. Differential expression analysis was performed using the Linear Models for Microarray Analysis portion of Bioconductor. Genes found to be differentially expressed for each pair wise comparison using a FDR-adjusted p-value of 0.001 and at least a 2-fold change were combined and used to perform clustering analysis. A selforganizing map (SOM) clustering algorithm was applied to genes showing significant expression differences as judged by mean log2 intensity per strain. The gap statistic was used to estimate the optimal number of clusters. Additional analysis was performed using BRB Array Tools for Excel 2007, as well as the Stanford Statistical Analysis of Microarrays (SAM) plug-in for Excel 2007. Microarray results were confirmed by quantitative RT-PCR.

Chromatin Immunoprecipitation
Potential gene targets identified through subtractive hybridization [29] and microarray studies, were searched for classic nuclear receptor response element binding sites. Binding sites consisted of the homologous sequence ''AAGTCA (n1-4) AAGTCA'' with less than two base pair alterations from the consensus (or binding sites identified in our previous studies [29]) were selected at a maximum distance of 30 kB upstream of the ATG start site. Sixty seven genes were identified as containing testable response elements, and chromatin immunoprecipitation was performed on these putative gene targets, as described previously [29], using embryonic day 18.5 and postnatal day 30.5 retinas from B6 and rd7 mice. Immunoprecipitation was performed overnight with 1mg of Nr1d1 antibody (Sigma), Nr2e3 antibody (Haider lab) or preimmune Ig antibody (R&D Systems). Rd7 retinal tissue was immunoprecipitated with Nr2e3 antibody as a negative control. Quantitative RT-PCR was performed using 1ml of 1:100 dilution (Input) and 1:10 dilution (samples and Ig G control) using conditions described previously [49]. All sample data was normalized to Ig control. Primers are listed in Table S1.

Efficiency of shNr1d1
To test the efficiency of the short hairpin (sh) RNA vector in silencing mouse (m) Nr1d1 expression ( Figure S1), HEK293T cells were transiently transfected in 100-mm plates at a confluence of 30% with 2mg of pcDNA3.1-mNr1d1, a mammalian expression vector for mouse Nr1d1. Additionally, 2mg of either pLVTHM empty vector, or pLVTHM encoding a shRNA for mNr1d1 (shNr1d1) or crystallin Aa (shCryaa) were co-transfected. After 48 h of transfection, the transfection efficiency was estimated under a fluorescent microscope (Zeiss Axiovert 200) with a 20x objective, by counting the number of cells expressing the co-transfected pnlsGFP (100 ng/dish) [50]. Four regions were counted and average values used as a normalisation factor. Total RNA was then extracted with TRI ReagentH (Molecular Research Center Inc., Cincinnati, OH) according to manufacturer's instructions. Total RNA was treated with DNAse I (Applied Biosystems/Ambion, Austin, TX). Reverse transcription was performed on 500 ng of total RNA (1st strand cDNA synthesis kit for RT-PCR (AMV); Roche, Basel, Switzerland). Quantitative PCR was performed on an Mx3000p sequence analyzer (Stratagene, La Jolla, CA) using Fast Start Universal SYBRHGreen Master Mix (Rox) (Roche). To assess mouse Nr1d1 expression, primers mNr1d1for and mNr1d1rev were used. As internal standard, Gapdh expression was assessed with primers hGapdhfor and hGapdhrev (for primer sequences see Table S1). Statistical analysis was performed with Prism 4.0.2 (GraphPad Software Inc., La Jolla, CA).

Supporting Information
Table S1 (XLS) Figure S1 Efficient silencing of mouse Nr1d1 expression. In human HEK293T cells transiently expressing mouse NR1D1 (mNr1d1), a short hairpin RNA (shRNA) construct targeting mNr1d1 mRNA (shNr1d1), decreased relative mRNA levels by about 30% (p = 0.0051). A control shRNA sequence directed against crystallin Aa did not interfere with mNr1d1 mRNA expression (p = 0.0838). Results correspond to three independent transfections in duplicates. Statistical significance was tested with a two-tailed paired t-test. (DOC)