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Published in final edited form as: Mol Cell Endocrinol. 2013 May 22; 371(1-2): 140-147. doi:10.1016/j.mce.2012.12.029.
POD-1 binding to the E-box sequence inhibits SF-1 and StAR expression in human adrenocortical tumor cells
Monica Malheiros Françaa, Bruno Ferraz-de-Souzab, Mariza Gerdulo Santosc, Antonio Marcondes Lerarioc, Maria Candida Barisson Villares Fragosoc, Ana Claudia Latronico”, Rork D. Kuickd, Gary D. Hammere, and Claudimara F.P. Lotfia,*
aDepartment of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-900, SP, Brazil
bLaboratory of Carbohydrates and Radioimmunoassays (LIM-18), School of Medicine, University of São Paulo, São Paulo 01246-903, SP, Brazil
cLaboratory of Hormones and Molecular Genetics (LIM-42), Division of Endocrinology, School of Medicine, University of São Paulo, São Paulo 01246-903, SP, Brazil
dBiostatistics Core of the Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-2200, USA
eDepartment of Internal Medicine, Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48109-2200, USA
Abstract
Pod-1/Tcf21 is expressed at epithelial-mesenchymal interaction sites during development of many organs. Different approaches have demonstrated that Pod-1 transcriptionally inhibits Sf-1/NR5A1 during gonadal development. Disruption of Sf-1 can lead to disorders of adrenal development, while increased dosage of SF-1 has been related to increased adrenal cell proliferation and tumorigenesis. In this study, we analyzed whether POD-1 overexpression inhibits the endogenous Sf-1 expression in human and mouse adrenocortical tumor cells. Cells were transiently transfected with luciferase reporter gene under the control of Sf-1 promoter and with an expression vector encoding Pod-1. Pod-1 construct inhibited the transcription of the Sf1/Luc reporter gene in a dose- dependent manner in mouse Y-1 adrenocortical carcinoma (ACC) cells, and inhibited endogenous SF-1 expression in the human H295R and ACC-T36 adrenocortical carcinoma cells. These results were validated by chromatin immunoprecipitation assay with POD-1-transfected H295R cells using primers specific to E-box sequence in SF-1 promoter region, indicating that POD-1 binds to the SF-1 E-box promoter. Moreover, POD-1 over-expression resulted in a decrease in expression of the SF-1 target gene, StAR (Steroidogenic Acute Regulatory Protein). Lastly, while the induced expression of POD-1 did not affect the cell viability of H295R/POD-1 or ACC-T36/POD-1 cells, the most significantly enriched KEGG pathways for genes negatively correlated to POD-1/TCF21 in 33 human ACCs were those associated with cell cycle genes.
Keywords
POD-1; TCF21; NR5A1; SF-1; E-box; Human adrenocortical tumor cells
1. Introduction
Pod-1 (capsulin, epicardin, Tef21) is a basic helix-loop-helix (bHLH) transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal interactions in the developing urogenital, cardiovascular, respiratory, and gastrointestinal systems (Quaggin et al., 1998; Quaggin, 2002; Robb et al., 1998; Lu et al., 1998). Loss of Pod-1 leads to lung, kidney, and spleen abnormalities and neonatal lethality (Quaggin et al., 1999 and Lu et al., 2000).
Pod-1 is expressed in the urogenital ridge, which gives rise to gonadal and adrenal tissues later in embryonic development (Tamura et al., 2001). In adrenal glands, Pod-1 is expressed exclusively in the capsule as shown in mice with a lacZ reporter under the control of Pod-1 regulatory region (Kim et al., 2009). Different approaches have demonstrated that Pod-1 transcriptionally inhibits steroidogenic factor 1 (Sf-1/Ad4Bp/Nr5a1), an orphan nuclear receptor required for gonadal development (Tamura et al., 2001; Cui et al., 2004). During human and mouse development, SF-1 expression drives the differentiation of the steroidogenic lineages (Luo et al., 1994). Increased SF-1 dosage can augment human adrenal cell proliferation, playing a critical role in adrenocortical tumorigenesis (Doghman et al., 2007). Indeed, SF-1 expression level in ACC is a prognostic factor for this type of cancer (Pianovski et al., 2006; Almeida et al., 2010; Sbiera et al., 2010). On the other hand, POD-1 is downregulated in lung cancer and melanoma (Smith et al., 2006; Arab et al., 2011).
In this study, we examined the correlation of POD-1 expression to gene expression profiles in sporadic ACC and observed a significant decrease in expression in ACC versus benign adrenocortical adenomas (ACAs) and normal adrenocortical tissue. To determine whether a down-regulation of POD-1 expression might contribute to the elevated expression of SF-1 in a subset of ACC samples, we over-expressed POD-1 in two different human adrenocortical carcinoma cell lines, NCI-H295R and ACC-T36. POD-1 overexpression inhibited endogenous SF-1 expression through binding to the E-box sequence of SF-1 promoter in adrenocortical tumor cells. Moreover, POD-1 levels in human ACC samples inversely correlate with a number of genes associated with the cell cycle, including SF-1, supporting a regulatory role for of POD-1 in gene expression in ACC.
2. Materials and methods
2.1. Cell cultures
Y1 mouse adrenocortical tumor cell line (Yasumura et al., 1966) and TM3 mouse Leydig cell line (Mather, 1980) were grown, respectively, in DMEM supplemented with 12.5% horse serum and 2.5% fetal bovine serum, and 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 5% horse serum and 2.5% bovine serum. The H295R human adrenocortical tumor cell line (Gazdar et al., 1990) was cultured in RPMI (Gibco, USA)
supplemented with 2% fetal bovine serum (Gibco, USA) and 1% Insulin-Transferrin- Selenium (Gibco, USA). ACC-T36 cells were generated by serial passage (ten passages) of a primary culture of an adrenocortical carcinoma presenting in a 38 year-old woman. Adrenal tumor fragments were obtained in the Clinical Medicine Department of Medicine School, Hospital das Clínicas from University of São Paulo. The tumor fragments were transported in sterile ice-cold DMEM (Dulbeco’s Modified Eagle’s Medium - Gibco) with antibiotics (25 mg/l of ampicillin and 100 mg/l of streptomycin - Sigma) washed several times in Phosphate Buffer Saline (PBS) at 37 ℃ and dissected from fat and necrotic tissues in a sterile environment. After digestion by sequential 4 mg/ml collagenase plus 1 µg/ml DNasel (Gibco) at 37 ℃ in a humidified atmosphere of 95% air/5% CO2, cells were mechanical disaggregated with gentile movements in a volumetric pipette. The digested material was then filtered in a 100 um nylon filter, to retain non-digested material and pelleted at 700 rpm for 10 min. Dispersed cells were suspended in DMEM containing 10% FBS (fetal bovine serum), 25 mg/l of ampicillin and 100 mg/l of streptomycin, and then plated in 12.5 cm2 Falcon flasks (Becton Dickinson, Franklin Lakes, NJ, USA). Medium renewal or subculturing was carried every 2 or 3-days. The protocol was approved by the Ethics Committee for Research Projects Analysis (CAPPesq) from Medicine School and by Ethics Committee for Research in Humans of Institute of Biomedical Sciences of University of São Paulo with protocol number 072/06. All cultures were maintained at 37 ℃ in a humidified atmosphere containing 95% air and 5% CO2.
2.2. Transfection assay
Y1 and TM3 cells were plated and transfected using 2 µg pcDNA3/Pod-1 or pcDNA3 in combination with Fulgene HD (Roche, Germany). NCI-H295R cell line and ACC cells were transiently transfected with pCMVMyc-Pod1, kindly provided by Dr. Masataka Nakamura (Tokyo Medical University, Japan) as described earlier (Funato et al., 2003) or with pcDNA3Pod1. 1.5 × 105 cells were plated and transfected with 2 µg of plasmid DNA in combination with 2 ul X-tremeGENE HP-DNA transfection reagent® (Roche, Germany). After 24 or 48 h of transfection, total RNA was extracted using Trizol® reagent (Invitrogen, USA) and treated with TURBO DNA-free™ (Ambion) before obtaining cDNA.
2.3. Luciferase reporter gene assays
Y1 and TM3 cells were plated in triplicate and 24 h after plating were transfected with 0.5 µg or lug of pDNA3/Pod-1 and 1 µg pDNA3Sf-1/Luc in combination with Fulgene 6 (Roche, Germany). Cells were lysed and assayed for both Firefly and Renilla Luciferase using the Dual-Luciferase Reporter Assay System (Promega).
2.4. RT-qPCR
After RNA extraction with Trizol® reagent (Invitrogen, USA), cDNA was generated from 1 µg of total RNA using Oligo dT, RNase-OUT, and M-MLV Reverse Transcriptase (Invitrogen, USA). RT-qPCR was performed on a RotorGene6000 Corbett (Quiagen, USA) sequence detector using Platinum SYBR qPCR SuperMix-UDG (Invitrogen, USA). A cycle threshold (Ct) value in log range of amplification was selected for each sample in triplicate and normalized to B-actin expression levels. Data were analyzed using the 2-44Ct method (Livak and Schmittgen, 2001).
Mol Cell Endocrinol. Author manuscript; available in PMC 2018 January 02.
2.5. Chromatin immunoprecipitation (ChIP)
H295R cells transfected with pCMVMyc-Pod1 were fixed with 1% formaldehyde for 10 min. ChIP assays were performed using ChIP-IT Express kit (Active Motif, Rixensart, Belgium) following the manufacturer’s instructions. Chromatin was fragmented by sonication with six 10 s pulses at 25 um amplitude in a VCX130 PB ultrasonic processor (Sonics & Materials, CT, USA). Most resulting chromatin fragments ranged from 200 bp to 600 bp. Sheared chromatin was incubated with 3 µg anti-MYC (Clontech) or with 1 µg IgG as negative control (ChIP-IT control Kit-Human, Active Motif).
2.6. ChIP-PCR
The 5.0-kb upstream sequences of human steroidogenic factor-1 (NR5A1, transcript ENST00000373588, Ensembl release 64, GRCh37) and androgen receptor (AR, transcript ENST00000374690) were analyzed for putative E-box binding sites using MatInspector (Cartharius et al., 2005). Based on this analysis and on the location of predicted E-box sites in mice (Funato et al., 2003; Hong et al., 2005), the following primers were designed using Primer3 (Untergasser et al., 2007): AR E-box forward 5’-CTCTGA TTCTTGGGGCTGAG-3’, and reverse 5’CATGACCAAGCCAGCAGATA 3’ (113 bp amplicon); SF-1 E-box-552, forward 5’CCCACAGCAGATG AGGACAT 3’, and reverse 5’TGCTGCTTCCACCTCTTTTT 3’ (132 bp amplicon); SF-1 E-box-55, forward 5’- ACCAACAAAGAAGGCGAGAG-3’, and reverse 5’-CTGTCCGTCCGTCCTCCT-3’ (109 bp amplicon). As a negative control for POD-1 binding, primers amplifying sequences in intron 1-2 of AR (forward 5’-TTGTCAAAGTCTTTTCCAGTTAATTT-3’ and reverse 5’- TTAACCCTACCAAGTAAATTTGTTC-3’, 114 bp amplicon) and 50-kb downstream of NR5A1 (forward 5’TGGAAGGGGAAGAATGAATG-3’and reverse 5’- CTGACGTCACACA TCCCATC-3’, 135 bp amplicon) were used.
Anti-MYC IP, IgG-IP, and 0.1% input DNA samples were used as templates for PCR amplification. PCR reactions were performed using 5 U/ul Platinum Taq DNA Polymerase (Invitrogen) and 5 uM primers for 35 cycles of amplification with an annealing temperature of 60 °C.
2.7. MTS assay
104 Cells/well were plated into 96-well plates and after 24 h were transfected with pCMVMycPod1 or pcDNA3Pod1. After 48 h, 20 ul of tetrazolium compound (MTS) was added for 4 h at 37 ℃. After transfection, cells were analyzed daily during 5 days. The optical density was read at 490 nm using an ELISA plate reader (Bio-Tek Instruments, Winooski, VT, USA).
2.8. Functional enrichment analysis
We collapsed probe-sets to distinct genes using Entrez gene identifiers, and calling a gene negatively correlated to TCF21 if any probe-set for the gene was negatively correlated to TCF21 with p< 0.01. We tested for enrichment of Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) using one-sided Fisher’s exact tests. We estimated false discovery rates by permuting the gene labels 100 times.
2.9. Statistic analysis
Data were presented as the mean ± standard deviation (SD). Statistical significance was determined using paired T-tests on log-transformed data except where otherwise noted. Results were considered statistically significant when p< 0.05.
3. Results
3.1. Pod-1 construct inhibited a luciferase reporter gene under the control of Sf-1 promoter, in a dose-dependent manner
Previous study indicated that Pod-1 repressed the transcription from the Sf-1 reporter gene in I-10 cell line, which was derived from the Leydig cells (Tamura et al., 2001). We examined this possibility in Y1 mouse adrenocortical tumor cell line and TM3 mouse Leydig cell line transiently transfected with luciferase reporter gene under the control of Sf-1 promoter (pDNA3/Sf1Luc) and with an expression vector encoding Pod-1 (pPod1). As shown in Fig. 1, transfected Pod-1 decreased expression of the Sf-1 reporter construct in Y1 cells in a dose-dependent manner [0.5 mcg pDNA3/Pod-1: 0.77 ± 0.02 or 22% repression (p = 0.003); 1.0 mcg pDNA3/Pod-1: 0.55 ± 0.04 or 45% repression (p=0.005)]. Similar results were obtaining in the TM3 mouse Leydig cell line [0.5 mcg pDNA3/Pod-1: 0.73 ± 0.19 or 27% repression (p=0.16); 1.0 mcg pDNA3/Pod-1: 0.44 ± 0.08 or 56% repression (p= 0.015). Thus, Pod-1 could specifically repress expression of the reporter gene, which carried the regulatory region of the Sf-1 gene.
3.2. POD-1 is markedly downregulated in adrenocortical carcinoma and negatively correlated to SF-1
We used our published array data from Gene Expression Omnibus (GEO) series GSE10927, consisting of mRNA abundance assays of 33 ACCs, 22 ACAs, and 10 normal adrenal cortex samples, that used Affymetrix HG-U133_Plus_2 arrays, which hold 54675 probe-sets representing approximately 20,000 genes (Giordano et al., 2009; Heaton et al., 2012). We used the log-transformed abundance estimates as given. The data set held two probe-sets measuring TCF21/POD-1 that were highly correlated (r=0.83), which we averaged, and used to compute the correlation of POD-1 to every other probe-set, using just the 33 ACCs. The analysis of POD-1 in the cohort of adrenocortical tissues showed that this gene is expressed about 2-fold less in ACA vs NLs, and another 2-fold in ACCs (Fig. 2). We also found that the probe-set for SF-1/NR5A1 was negatively correlated to POD-1 in the 33 ACCs (Fig. 3). These results are consistent with POD-1 serving a repressive role in SF-1- mediated transcription and as a potential negative regulator of adrenocortical tumorigenesis.
3.3. pcDNA3Pod1 and pCMVMycPod1 transfection resulted in increased POD-1 expression in adrenocortical tumor cells
The transient transfection of pcDNA3Pod1 or pCMVMycPod1 in H295R and ACC-T36 cells resulted in an expected increased POD-1 expression in both cell types. H295R and ACC-T36 cells transfected with POD-1 containing pcDNA3 vectors showed a 4.2-fold (p= 0.04) and 1.5-fold (p=0.004), increase in POD-1 expression, respectively (Fig. 4A). Similarly, transfection with POD-1-containing pCMVMyc promoted a 24.6 x 103-fold (p=
5 × 10-6) and 6.4 × 103-fold (p=0.0006) increase in POD-1 expression in H295R and ACC- T36 cells, respectively (Fig. 4B).
3.4. Forced expression of POD-1 reduced the endogenous SF-1 expression in adrenal tumor cells
To investigate whether induced expression of POD-1 affects the endogenous expression of SF-1, we performed a RT-qPCR for the analysis of SF-1 expression in cells transiently transfected with expression vectors pcDNA3Pod1 or pCMVMycPod1. pcDNA3Pod1 transfection in H295R and ACC-T36 cells showed SF-1 reduction of 0.45 (p= 0.042) and 0.49 (p=0.025) of control levels (Fig. 4A), whereas pCMVMycPod1 transfection reduced SF-1 expression to 0.34 (p=0.009) and 0.34 (p=0.017) of controls, respectively (Fig. 4B). While the level of transfected POD-1 varied in each cell lines and did not correlate with degree of inhibition across the two cell lines, the data indicate that POD-1 inhibited the expression of SF-1 in adrenal tumor cells.
3.5. Validation of chromatin enrichment and characterization of POD-1 binding to SF-1 E- box
In order to investigate whether the repressive effect of POD-1 overexpression on SF-1 expression was mediated through POD-1 binding to E-box elements in the SF-1 promoter region, chromatin immunoprecipitation (ChIP) assays were performed in H295R cells transfected with pCMVMycPod1 using an anti-Myc antibody (Fig. 5). To validate this approach, ChIP-PCR assays were performed using primers amplifying an E-box sequence in the promoter region of AR; it has been previously shown in mice that Pod-1 binds to an E- box element in the promoter of Ar to repress transcription (Hong et al., 2005). As shown in Fig. 5, immunoprecipitated chromatin was enriched for this region, whereas intron 1-2 of AR, used as a local negative control and not expected to bear E-box elements, failed to amplify. The 5.0-kb upstream sequence of human SF-1 was analyzed for putative E-box elements using MatInspector (Cartharius et al., 2005) and two such elements were identified, located 552 and 55 base pairs upstream of the transcription start site, respectively. ChIP-PCR assays using specifically designed primers showed that both “E-box -552” and “E-box -55” sequences were amplified from Myc-immunoprecipitated DNA from NCI-H295R- pCMVMycPod1 cells, confirming chromatin enrichment by ChIP, while a negative control sequence, located 50 kb downstream of SF-1, failed to amplify (Fig. 5). Altogether, these results confirm POD-1-binding to E-box elements in the human SF-1 promoter.
3.6. Decreased SF-1 expression induced by POD-1 overexpression affect the StAR expression of adrenal tumor cells
To investigate the effects of POD-1-mediated SF1 repression on cell viability and steroidogenic function of adrenal tumor cells, colorimetric MTS assay and RT-qPCR for analysis of StAR expression was performed with NCI-H295R and ACC-T36 cells transiently transfected with pcCMVMycPod1. The viability of cells transfected with POD-1 was similar to that of cells transfected with empty vectors (Fig. 6). However, in both tumor cell types, StAR expression (Fig. 7) was downregulated following the transfection of POD-1, consistent with a decrease in SF-1-mediated StAR transcription.
3.7. Negatively correlated genes to POD-1 are enriched for cell cycle
We selected probe-sets on the arrays measuring human mRNA levels in ACCs that were correlated to POD-1 expression with p-values smaller than 0.01, which gave 732 positively and 527 negatively correlated probe-sets. To perform enrichment testing we collapsed the 54,675 probe-sets to 19,230 distinct genes, which now had 492 positively and 380 negatively correlated genes to POD-1. We tested 232 pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) obtained in May 2011, using one-sided Fisher Exact tests to ask if the gene intersections were significantly larger than expected by chance. The top three pathways for the negatively correlated genes are shown in Table 1, the most significant of which was for “Cell cycle”, suggesting that tumors with higher POD-1 expression have lower proliferation rates. Negatively correlated cell cycle genes selected include among others CDK1 (Cyclin-dependent kinase) and BUB1B (budding uninhibited by benzimidazoles 1 homolog beta). CDK1 is regulated by CKIs (cyclin-dependent kinase inhibitors) such as p57Kip2, which is often downregulated coordinately with H19 in ACC (Gicquel et al., 1997; Heaton et al., 2012). BUB1B is overexpressed in ACC and the difference in the expression of BUB1B and PINK1 (PTEN-induced putative kinase 1) was strong predictor of overall survival in a group of adult ACCs (de Reynies et al., 2009; Fragoso et al., 2012).
4. Discussion
In this study, we found that POD-1/TCF21 acts as a transcriptional suppressor of SF-1 gene expression in adrenocortical carcinoma cells. A possible mechanism involved in the repression of SF-1 expression is through POD-1 binding to the E-box sequence in the SF-1 promoter region.
In Pod-1 KO embryos, Sf1 expression was increased at the boundary between the gonad and mesonephros, regions where Pod-1 is normally expressed (Quaggin et al., 1999). Also, Sf1 was co-expressed with the ß-galactosidase reporter that replaced the first exon of the Podl gene, showing ectopic expression of Sf1 in Pod1-expressing cells. These results support the model that Pod-1 normally represses SF1 expression in these development sites (Cui et al., 2004). Consistent with this model, our results show that induced expression of Pod-1 inhibited, in a dose-dependent manner, the Sf-1 promoter activity in mouse Y1 and TM3 sterodoigenic cell lines. Indeed, induced expression of Pod-1 has been shown to inhibit the expression of Sf-1 in the Leydig cell-derived I-10 cells (Tamura et al., 2001). Also, Pod-1 suppressed Sf-1 expression by inhibiting the binding of Usf1, an activator of Sf-1 expression (Daggett et al., 2000), to the Sf-1 E-box during gonadogenesis. Moreover, Pod-1 knockout mice showed enhanced expression of Sf-1 in Leydig cells (Cui et al., 2004).
SF-1 is a key regulator of adrenal tumorigenesis and has been shown to be over-expressed in adrenocortical tumors (Figueiredo et al., 2005; Pianovski et al., 2006; Almeida et al., 2010). On the other hand POD-1 is markedly downregulated in adrenocortical carcinoma (ACC) in comparison to adrenocortical adenoma (ACA) and normal adrenal tissue. Abnormal expression of POD-1 has been demonstrated in other types of tumors. POD-1 is aberrantly methylated and downregulated in head and neck carcinomas and in non-small-cell lung cancer (Smith et al., 2006). In melanoma, POD-1 has been suggested to be functionally
involved in tumor progression (Arab et al., 2011). In this study, we show that POD-1 is able to decrease endogenous SF-1 expression in adrenocortical tumor cells, providing insight into the role of POD-1 in adrenal tumors. Furthermore, our results suggest that POD-1 binds directly to the SF-1 promoter, inhibiting its activity. While such interaction did not affect the proliferation of adrenocortical tumor cells in culture, POD-1 levels inversely correlate with gene involved in cell cycle progression. Moreover, POD-1 over-expression decreases StAR expression, consistent with a down-regulation of SF-1 mediated steroidogenesis.
Acknowledgments
5. Funding
MMF is the recipient of a scholarship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, the State of São Paulo Research Foundation); CFPL received funding from FAPESP, from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development), and from Pró-Reitoria de Pesquisa da Universidade de São Paulo (Dean’s Office of the University of São Paulo for Research Projects). GDH is the recipient of NIH grant R01-CA-134606 which supported the genomic studies in this work.
We are grateful to Joelcimar Martins da Silva (Laboratory of Neuroanatomy, Institute of Biomedical Science, University of São Paulo), Dr. Miriam Nishi, Andrea G. Fernandes (Laboratory of Hormones and Molecular Genetics (LIM42), Division of Endocrinology, School of Medicine, University of São Paulo) for technical assistance, and Rosana D. Prisco for statistical analysis.
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Relative Luciferase Activity
Y1
Relative Luciferase Activity
TM3
1.2
1.2
1.0
1.0
p =. 003
p =. 16
0.8
4
0.8
0.6
p =. 005
0.6
T
p =. 015
0.4
0.4
T
0.2
0.2
SF-1 Luciferase reporter
SF-1 Luciferase reporter
empty vector 0.5
POD-1 0.5
POD-1
1.0
empty
vector 1.0
empty vector 0.5
POD-1
POD-1
0.5
1.0
empty
vector 1.0
pcDNA3 vector
pcDNA3 vector
Transfected constructs
Transfected constructs
10
POD-1 (Tcf21) base-2 log expression
9
000
☐
☒
8
☒
☐
0 ☐
☒
☒
7
☒
☒
*
*
6
NL
ACA
ACC
n=10
n=22
n=33
11
SF-1 (NR5A1) base-2 log expression
r =- 0.54; p=0.0012
10
9
8
7
6
6
7
8
9
POD-1 (Tcf21) base-2 log expression
A
1.2
1.0
SF-1 mRNA
relative expression
0.8
p =. 042
p =. 025
0.6
0.4
0.2
empty POD-1
empty POD-1
pcDNA3 vector
5.0
H295R ☒
p =. 04
POD-1 mRNA
relative expression
4.0
ACC-T36
☐
3.0
2.0
p =. 004
1.0
empty POD-1
empty POD-1
pcDNA3 vector
B
1.2
SF-1 mRNA relative expression
1.0
0.8
0.6
p =. 009
p =. 017
0.4
T
T
0.2
empty POD-1
empty POD-1
pCMVMyc vector
H295R ☒
27×103
p =. 0006
24×103
ACC-T36 ☐
21×103
18×103
15×103
12×103
9×103
p =. 000005
3
empty POD-1
empty POD-1
pCMVMyc vector
-1135bp
Input
IP
lgG
H20
AR Ebox -1135
Ebox
+1600bp
AR negative control
-552bp
SF-1 Ebox -552
Ebox
-55bp ☐
SF-1 Ebox -55
Ebox
SF-1 negative control
+50kb
A 1,6
B
1,6
NCIpcDNA3
1,4
NCIpCMVMyc
1,4
1,2
NCIpPod1
1,2
NCIpCMVMycPod1
OD (490nm)
1
OD (490nm)
1
0,8
0,8
0,6
0,6
0,4
0,4
0,2
0,2
0
0
48h
72h
96h
120h
144h
168h
24h
48h
72h
96h
120h
144h
C
2
D
2
1,6
1,6
OD (490nm)
OD (490nm)
1,2
1,2
0,8
ACCpcDNA3
0,8
ACCpCMVMyc
0,4
ACCpcDNA3Pod1
0,4
ACCpCMVMycPod1
0
48h
72h
96h
120h
0
48h
72h
96h
120h
H295R ☒
ACC-T36 ☐
1.2
1.0
p =. 026
StAR mRNA
relative expression
0.8
p =. 045
0.6
0.4
0.2
empty POD-1
empty POD-1
pCMVMyc vector
| KEGG pathway | Number of genes in pathway | Number negatively correlated to TCF21 (of 380) | P-value, one-sided Fisher's exact test | Estimated false discovery rate (Q- value) | Genes in intersection |
|---|---|---|---|---|---|
| Cell cycle | 123 | 9 | 0.0008 | 0.050 | YWHAQ, CHEK2, E2F2, ORC2, SKP2, BUB1B, CCNA2, CCNB2, CDK1 |
| mRNA surveillance pathway | 80 | 7 | 0.0010 | 0.045 | HBS1L, PAPOLA, CSTF1, CPSF2, PPP2R5C, RNGTT, RBM8A |
| Glycosaminoglycan biosynthesis-heparan sulfate | 26 | 4 | 0.0016 | 0.050 | B3GALT6, EXTL1, EXTL2, HS2ST1 |