BACKGROUND
esrrb encodes nuclear receptor estrogen related receptor β , which belongs to the nuclear receptor family.
esrrb acts as a transcription factor by binding to a specific dna sequence estrogen related receptor response element , which is also known as steroid factor response element , or half site estrogen response element  <cit> .
esrrb, first cloned in  <dig>  was not intensively studied until recent years.
knocking out of esrrb was embryonic lethal due to placental malformation  <cit> .
though early studies showed a very limited range of tissues with positive esrrb expression, recent studies reported that short form esrrb alternative splicing isoform had a broad range of expression  <cit> .
esrrb was found to be a core-reprogramming factor to reprogram pluripotent stem cells  .
c-myc and klf <dig> of the oskm  core-reprogramming factors can be replaced by esrrb  <cit> .
esrrb was also recently reported to drive sox <dig> transcription and induce ipsc in a single cell system  <cit> .
tumorigenesis and tumor progression are related to esrrb.
esrrb was shown to be down-regulated in prostate cancer epithelium compared to normal prostate tissue .
its re-expression in du <dig> and lncap cells was shown to stimulate tumor suppressor cdkn1a  concentration.
also, esrrb can inhibit estrogen receptor transcriptional activity in uterine endometrial cancer cells and nrf2-keap signaling pathway in breast cancer cells  <cit> .
there are a handful of transcriptome-wide expression survey data from esrrb knockdown in both human ipscs and mouse embryonic stem cells .
known esrrb controlled genes include klf <dig>  c-myc, cdkn1a and cyp19a <dig>  but esrrb target genes in cancer cells are still not known.
this manuscript focuses on the discovery of esrrb ligand-independent and esrrb ligand-dependent target genes.
we performed rna-seq analysis to characterize esrrb regulated mrnas in a prostate cancer cell line and we found the treatment of dy <dig> expanded esrrb’s transcriptional regulation activity to many more genes.
RESULTS
establishment of the esrrb stably transfected du <dig> cells
esrrb expression vector or control pcdna <dig>   vector were transfected into du <dig> cells.
after 3 weeks of zeocine selection, we characterized the esrrb status by reverse transcriptase -pcr, qpcr and western blot analysis .
our results showed that esrrb was successfully expressed in du145-esrrb cells.
although rna-seq showed that du145-pc <dig>  cells had a very small amount of esrrb expressed , the esrrb concentration is below the detection limit of rt-pcr and western blot.
compared to hek <dig> cells, which expressed endogenous esrrb, overexpression of esrrb in du <dig> cells raised the esrrb protein concentration to a comparable physiological concentration .
in addition, our rt-pcr results and rna-seq results confirmed the estrogen related receptor gamma  was not expressed in du <dig> cells.
the absence of esrrg eliminated any possible functional contamination by esrrg in our esrrb studies .fig.
<dig> characterization of esrrb-expressing cancer cell line.
esrrb status of two independent replicates of stable transfected control du145-pc <dig>  and du145-esrrb cells are tested by a quantitative pcr b western blot and c reverse transcriptase pcr.
a relative mrna concentrations of esrrb were measured by qpcr, esrrb transcripts concentration were determined by standard curve method and esrrb concentration were first normalized to the concentration of house keeping gene gapdh, then normalized to esrrb/gapdh ratio of du145-pc <dig>  cells.
b total protein was extracted form hek <dig>  du145-esrrb and control du145-pc <dig>  cells.
protein concentration of esrrb was determined by western blot using gapdh as internal control.
c rt-pcr was performed on total rna extracted from hek <dig>  du145-esrrb and control du145-pc <dig>  cells.
esrrb was expressed in du145-esrrb cells, while esrrg is not expressed in either du145-pc <dig>  and du145-esrrb cells
esrrb expression alters mrna profile
to distinguish genes regulated by esrrb, we performed rna-seq analysis on cdna libraries constructed from two biological replicates of both du145-pc <dig>  and du145-esrrb cells.
spearman ranking correlation analysis showed that the expression of esrrb in du <dig> created a distinct transcriptome compared to control du145-pc <dig>  cells .
we found  <dig> genes  altered due to esrrb expression .
seven genes that are among the most changed genes  were confirmed by qpcr .
gene ontology  analysis shows that the products of esrrb driven differentially expressed genes fell into functional categories of regulation of cell development as well as immune responses .fig.
<dig> transcriptome correlation and esrrb altered mrnas.
a transcriptome correlation analysis was performed using spearman ranking correlation.
color represents the correlation coefficient.
dy <dig> treatment to du145-esrrb cells results in the lowest correlation coefficient with du145-pc <dig>  cells.
b
dot plot of esrrb-induced gene expression alteration.
genes expressed at adequate level are tested for differential gene expression test.
the plot was made by plotting the log2fc  against the log2 cpm  difference.
red color marks the genes that are significant differentially expressed , and the blue lines marked the log2fc cutoff value .
<dig> genes passed both thresholds
table  <dig> esrrb altered mrnas
fig.
<dig> esrrb-regulated mrna validation.
left panel qpcr validation of esrrb-regulated mrnas.
expression values were firstly normalized to gapdh and normalized ratios are further normalized to that of du145-pc <dig> .
error bars represent standard deviation.
student t test was performed for statistical analysis .
seven genes were differentially expressed in both rna-seq and qpcr,  <dig> gene, tgfbeta, is not differentially expressed in either assay and serves as a negative control.
right panel rna-seq analysis result, fold change  indicates the ratio of normalized read counts in du145-esrrb to that of du145-pc <dig>
table  <dig> gene ontology analysis of esrrb altered mrnas
dy <dig> requires esrrb to affect gene expression
to get a more comprehensive understanding of esrrb-regulated genes and characterize esrrb’s potential ligand dependent activity, control du145-pc <dig>  and du145-esrrb cells were treated with esrrb/esrrg synthetic ligand dy <dig>  since both qpcr and rna-seq show esrrb transcript concentration is extremely low in du <dig> cells and esrrg is absent, and esrrb protein concentration is also below the detection limit of western-blot analysis, it was not surprising to observe dy <dig> treatment without esrrb expressed did not result in any gene differentially expressed .
after we applied dy <dig> to du145-esrrb cells, we found dy <dig> treatment most significantly modified the transcriptome .
further comparison of du145-esrrb cells alone to dy131-treated du145-esrrb cells detected  <dig> altered mrnas .
<dig> of them overlapped with esrrb-induced mrna alterations .
we defined an esrrb agonist as a ligand that moves the mrna concentration in the direction as esrrb does; and an antagonist moves the concentration in the opposite direction as esrrb does.
by comparing the trend of the altered genes induced by esrrb expression and dy <dig> treatment, dy <dig> acts as an agonist for  <dig> of the  <dig> genes, and an antagonist for  <dig> of the  <dig> genes .
there are another  <dig> mrnas changed with both esrrb and dy <dig> treatment compared to esrrb alone, indicating their responses is ligand-dependent .fig.
<dig> mrna alteration by dy <dig> requires esrrb expression.
a dy <dig> treatment alone did not alter the expression of any gene.
in contrast, when esrrb was expressed, b dy <dig> altered  <dig> mrnas.
c venn diagram of pairwise comparisons of altered mrnas showed  <dig>  esrrb altered mrnas can be further regulated by dy <dig> treatment .
d heat map of mrna concentration of the  <dig> genes that response to both esrrb expression as well as dy <dig> treatment.
log2-transformed normalized read counts of these  <dig> genes were color coded.
dy <dig> is an agonist for  <dig> mrnas that are responsive to esrrb, while it is an antagonist of esrrb in regulating the other  <dig> mrnas
table  <dig> dy <dig> altered mrnas when esrrb is expressed
go analysis showed esrrb-dependent dy <dig> up-regulated genes were important for regulation of transcription, regulation of apoptosis and proliferation, and a majority of down-regulated genes are related to oxidation and reduction, metabolism and translation elongation .table  <dig> gene ontology analysis of esrrb-dependent dy131-altered genes
discussion
esrrb has gained lots of attention in recent years because of its biological function in stem cells and its ability to reprogram somatic cells to ipsc with oct <dig> and sox <dig> .
several other functions of esrrb have also been discovered including alteration of energy balance, estrogen receptor and glucocorticoid receptor transcription function modulation, keap1-nrf <dig> signaling inhibition, and tumorigenesis in prostate cancer and endometrial adenocarcinoma .
but transcriptome-wide esrrb function and esrrb-regulated genes in cancer cells are not well studied.
esrrb was reported by chan et al.
as a tumor suppressor in du <dig> and lncap prostate cancer cells using both in vitro and in vivo models  <cit> .
expression of esrrb induced p21/cdkn1a by directly binding to an erre in p21/cdkn1a’s promoter, arrested cell cycle at s-phase, and significantly inhibited cell growth  <cit> .
interestingly, we did not find p21/cdkn1a up-regulation after esrrb expression alone, but after we treated du <dig> cells with 3 μm dy <dig>  we observed a significant increase of p21/cdkn1a mrna .
scrutinizing the data revealed that chan’s lab cultured their cells with full serum, while we used charcoal-stripped serum for cell culture and dy <dig> treatment  <cit> .
this implies that there is a compound or factor that can be removed by charcoal treatment modulated esrrb’s activity  <cit> .
from the esrrb-regulated gene list, we found a few target genes that are related to the known function of esrrb.
kiaa <dig> encoded gene product has been shown to associate with cellular mortality.
a kiaa <dig> mutation was reported to relate to nonsyndromic hearing loss.
considering the significant effect of esrrb mutations on human hearing loss, kiaa <dig> could be a mediator of esrrb mutant related hearing loss  .
another interesting esrrb responsive gene is tagln .
it was inhibited by esrrb while dy <dig> treatment relieved the inhibition.
tagln was reported to promote du <dig> cell migration and invasion, indicating esrrb can also affect du <dig> cell behavior by affecting tagln  <cit> .
judging by the numbers of altered genes induced by esrrb with or without dy <dig>  and the result that dy <dig> did not alter any mrna in the absence of esrrb, we conclude that dy <dig> activity is esrrb-dependent.
CONCLUSIONS
in conclusion, we characterized the transcriptome alteration induced by esrrb expression as well as esrrb with its ligand dy <dig> in prostate cancer cells.
we conclude esrrb-target synthetic ligand requires esrrb to generate its gene expression modulation effect.
finally, analysis of esrrb target genes indicates esrrb may be an important factor in regulating cell proliferation.

 esrrb encodes nuclear receptor estrogen related receptor β, which belongs to the nuclear receptor family. esrrb acts as a transcription factor by binding to a specific dna sequence estrogen related receptor response element, which is also known as steroid factor response element, or half site estrogen response element. esrrb, first cloned in was not intensively studied until recent years. knocking out of esrrb was embryonic lethal due to placental malformation. though early studies showed a very limited range of tissues with positive esrrb expression, recent studies reported that short form esrrb alternative splicing isoform had a broad range of expression. esrrb was found to be a core-reprogramming factor to reprogram pluripotent stem cells. c-myc and klf of the oskm core-reprogramming factors can be replaced by esrrb. esrrb was also recently reported to drive sox transcription and induce ipsc in a single cell system. tumorigenesis and tumor progression are related to esrrb. esrrb was shown to be down-regulated in prostate cancer epithelium compared to normal prostate tissue. its re-expression in du and lncap cells was shown to stimulate tumor suppressor cdkn1a concentration. also, esrrb can inhibit estrogen receptor transcriptional activity in uterine endometrial cancer cells and nrf2-keap signaling pathway in breast cancer cells. there are a handful of transcriptome-wide expression survey data from esrrb knockdown in both human ipscs and mouse embryonic stem cells. known esrrb controlled genes include klf c-myc, cdkn1a and cyp19a but esrrb target genes in cancer cells are still not known. this manuscript focuses on the discovery of esrrb ligand-independent and esrrb ligand-dependent target genes. we performed rna-seq analysis to characterize esrrb regulated mrnas in a prostate cancer cell line and we found the treatment of dy expanded esrrb’s transcriptional regulation activity to many more genes.  establishment of the esrrb stably transfected du cells esrrb expression vector or control pcdna vector were transfected into du cells. after 3 weeks of zeocine selection, we characterized the esrrb status by reverse transcriptase -pcr, qpcr and western blot analysis. our results showed that esrrb was successfully expressed in du145-esrrb cells. although rna-seq showed that du145-pc cells had a very small amount of esrrb expressed, the esrrb concentration is below the detection limit of rt-pcr and western blot. compared to hek cells, which expressed endogenous esrrb, overexpression of esrrb in du cells raised the esrrb protein concentration to a comparable physiological concentration. in addition, our rt-pcr results and rna-seq results confirmed the estrogen related receptor gamma was not expressed in du cells. the absence of esrrg eliminated any possible functional contamination by esrrg in our esrrb studies.fig.  characterization of esrrb-expressing cancer cell line. esrrb status of two independent replicates of stable transfected control du145-pc and du145-esrrb cells are tested by a quantitative pcr b western blot and c reverse transcriptase pcr. a relative mrna concentrations of esrrb were measured by qpcr, esrrb transcripts concentration were determined by standard curve method and esrrb concentration were first normalized to the concentration of house keeping gene gapdh, then normalized to esrrb/gapdh ratio of du145-pc cells. b total protein was extracted form hek du145-esrrb and control du145-pc cells. protein concentration of esrrb was determined by western blot using gapdh as internal control. c rt-pcr was performed on total rna extracted from hek du145-esrrb and control du145-pc cells. esrrb was expressed in du145-esrrb cells, while esrrg is not expressed in either du145-pc and du145-esrrb cells esrrb expression alters mrna profile to distinguish genes regulated by esrrb, we performed rna-seq analysis on cdna libraries constructed from two biological replicates of both du145-pc and du145-esrrb cells. spearman ranking correlation analysis showed that the expression of esrrb in du created a distinct transcriptome compared to control du145-pc cells. we found genes altered due to esrrb expression. seven genes that are among the most changed genes were confirmed by qpcr. gene ontology analysis shows that the products of esrrb driven differentially expressed genes fell into functional categories of regulation of cell development as well as immune responses.fig.  transcriptome correlation and esrrb altered mrnas. a transcriptome correlation analysis was performed using spearman ranking correlation. color represents the correlation coefficient. dy treatment to du145-esrrb cells results in the lowest correlation coefficient with du145-pc cells. b dot plot of esrrb-induced gene expression alteration. genes expressed at adequate level are tested for differential gene expression test. the plot was made by plotting the log2fc against the log2 cpm difference. red color marks the genes that are significant differentially expressed, and the blue lines marked the log2fc cutoff value. genes passed both thresholds table  esrrb altered mrnas fig.  esrrb-regulated mrna validation. left panel qpcr validation of esrrb-regulated mrnas. expression values were firstly normalized to gapdh and normalized ratios are further normalized to that of du145-pc. error bars represent standard deviation. student t test was performed for statistical analysis. seven genes were differentially expressed in both rna-seq and qpcr, gene, tgfbeta, is not differentially expressed in either assay and serves as a negative control. right panel rna-seq analysis result, fold change indicates the ratio of normalized read counts in du145-esrrb to that of du145-pc table  gene ontology analysis of esrrb altered mrnas dy requires esrrb to affect gene expression to get a more comprehensive understanding of esrrb-regulated genes and characterize esrrb’s potential ligand dependent activity, control du145-pc and du145-esrrb cells were treated with esrrb/esrrg synthetic ligand dy since both qpcr and rna-seq show esrrb transcript concentration is extremely low in du cells and esrrg is absent, and esrrb protein concentration is also below the detection limit of western-blot analysis, it was not surprising to observe dy treatment without esrrb expressed did not result in any gene differentially expressed. after we applied dy to du145-esrrb cells, we found dy treatment most significantly modified the transcriptome. further comparison of du145-esrrb cells alone to dy131-treated du145-esrrb cells detected altered mrnas. of them overlapped with esrrb-induced mrna alterations. we defined an esrrb agonist as a ligand that moves the mrna concentration in the direction as esrrb does; and an antagonist moves the concentration in the opposite direction as esrrb does. by comparing the trend of the altered genes induced by esrrb expression and dy treatment, dy acts as an agonist for of the genes, and an antagonist for of the genes. there are another mrnas changed with both esrrb and dy treatment compared to esrrb alone, indicating their responses is ligand-dependent.fig.  mrna alteration by dy requires esrrb expression. a dy treatment alone did not alter the expression of any gene. in contrast, when esrrb was expressed, b dy altered mrnas. c venn diagram of pairwise comparisons of altered mrnas showed esrrb altered mrnas can be further regulated by dy treatment. d heat map of mrna concentration of the genes that response to both esrrb expression as well as dy treatment. log2-transformed normalized read counts of these genes were color coded. dy is an agonist for mrnas that are responsive to esrrb, while it is an antagonist of esrrb in regulating the other mrnas table  dy altered mrnas when esrrb is expressed go analysis showed esrrb-dependent dy up-regulated genes were important for regulation of transcription, regulation of apoptosis and proliferation, and a majority of down-regulated genes are related to oxidation and reduction, metabolism and translation elongation.table  gene ontology analysis of esrrb-dependent dy131-altered genes discussion esrrb has gained lots of attention in recent years because of its biological function in stem cells and its ability to reprogram somatic cells to ipsc with oct and sox. several other functions of esrrb have also been discovered including alteration of energy balance, estrogen receptor and glucocorticoid receptor transcription function modulation, keap1-nrf signaling inhibition, and tumorigenesis in prostate cancer and endometrial adenocarcinoma. but transcriptome-wide esrrb function and esrrb-regulated genes in cancer cells are not well studied. esrrb was reported by chan et al. as a tumor suppressor in du and lncap prostate cancer cells using both in vitro and in vivo models. expression of esrrb induced p21/cdkn1a by directly binding to an erre in p21/cdkn1a’s promoter, arrested cell cycle at s-phase, and significantly inhibited cell growth. interestingly, we did not find p21/cdkn1a up-regulation after esrrb expression alone, but after we treated du cells with 3 μm dy we observed a significant increase of p21/cdkn1a mrna. scrutinizing the data revealed that chan’s lab cultured their cells with full serum, while we used charcoal-stripped serum for cell culture and dy treatment. this implies that there is a compound or factor that can be removed by charcoal treatment modulated esrrb’s activity. from the esrrb-regulated gene list, we found a few target genes that are related to the known function of esrrb. kiaa encoded gene product has been shown to associate with cellular mortality. a kiaa mutation was reported to relate to nonsyndromic hearing loss. considering the significant effect of esrrb mutations on human hearing loss, kiaa could be a mediator of esrrb mutant related hearing loss. another interesting esrrb responsive gene is tagln. it was inhibited by esrrb while dy treatment relieved the inhibition. tagln was reported to promote du cell migration and invasion, indicating esrrb can also affect du cell behavior by affecting tagln. judging by the numbers of altered genes induced by esrrb with or without dy and the result that dy did not alter any mrna in the absence of esrrb, we conclude that dy activity is esrrb-dependent.  in conclusion, we characterized the transcriptome alteration induced by esrrb expression as well as esrrb with its ligand dy in prostate cancer cells. we conclude esrrb-target synthetic ligand requires esrrb to generate its gene expression modulation effect. finally, analysis of esrrb target genes indicates esrrb may be an important factor in regulating cell proliferation. 
orphan nuclear receptor estrogen related receptor β is well known in stem cells and early embryonic development. gene ontology analyses showed altered genes are related to transcription and translation regulation, cell proliferation and apoptosis regulation, and cellular metabolism. our results characterized mrna profiles in du prostate cancer cells driven by esrrb expression and esrrb ligand dy and provided multiple markers to characterize esrrb’s function in esrrb research. we investigated the mrna profile alterations induced by esrrb expression and its synthetic ligand dy in human prostate cancer du cells via rna-seq analysis.
 orphan nuclear receptor estrogen related receptor β is well known in stem cells and early embryonic development. however, little is known about its function in cancer. method we investigated the mrna profile alterations induced by esrrb expression and its synthetic ligand dy in human prostate cancer du cells via rna-seq analysis.  we distinguished mrnas differentially expressed by esrrb alone. although dy alone did not change any gene, treatment of dy in the presence of esrrb altered mrnas. these observations indicated esrrb had both ligand-independent and ligand-dependent activity. when esrrb was expressed, dy treatment further regulated esrrb-altered mrnas. dy acted as an antagonist for of mrnas and as an agonist for of the mrnas. gene ontology analyses showed altered genes are related to transcription and translation regulation, cell proliferation and apoptosis regulation, and cellular metabolism.  our results characterized mrna profiles in du prostate cancer cells driven by esrrb expression and esrrb ligand dy and provided multiple markers to characterize esrrb’s function in esrrb research. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords estrogen related receptorrna-seqprostate cancergene expression profiledy131transcription regulationnuclear receptorissue-copyright-statement© the author 2015 