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Differential recruitment of coregulators to the RORA promoter adds another layer of complexity to gene (dys) regulation by sex hormones in autism.

Sarachana T, Hu VW - Mol Autism (2013)

Bottom Line: We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ERα interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression.AR and SUMO1 are involved in the suppression RORA expression by androgen, while ERα and NCOA5 collaborate in the up-regulation of RORA by estrogen.While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Sciences, 2300 I Street NW, Washington, DC 20037, USA. valhu@gwu.edu.

ABSTRACT

Background: Our independent cohort studies have consistently shown the reduction of the nuclear receptor RORA (retinoic acid-related orphan receptor-alpha) in lymphoblasts as well as in brain tissues from individuals with autism spectrum disorder (ASD). Moreover, we have found that RORA regulates the gene for aromatase, which converts androgen to estrogen, and that male and female hormones regulate RORA in opposite directions, with androgen suppressing RORA, suggesting that the sexually dimorphic regulation of RORA may contribute to the male bias in ASD. However, the molecular mechanisms through which androgen and estrogen differentially regulate RORA are still unknown.

Methods: Here we use functional knockdown of hormone receptors and coregulators with small interfering RNA (siRNA) to investigate their involvement in sex hormone regulation of RORA in human neuronal cells. Luciferase assays using a vector containing various RORA promoter constructs were first performed to identify the promoter regions required for inverse regulation of RORA by male and female hormones. Sequential chromatin immunoprecipitation methods followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses of RORA expression in hormone-treated SH-SY5Y cells were then utilized to identify coregulators that associate with hormone receptors on the RORA promoter. siRNA-mediated knockdown of interacting coregulators was performed followed by qRT-PCR analyses to confirm the functional requirement of each coregulator in hormone-regulated RORA expression.

Results: Our studies demonstrate the direct involvement of androgen receptor (AR) and estrogen receptor (ER) in the regulation of RORA by male and female hormones, respectively, and that the promoter region between -10055 bp and -2344 bp from the transcription start site of RORA is required for the inverse hormonal regulation. We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ERα interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression.

Conclusions: AR and SUMO1 are involved in the suppression RORA expression by androgen, while ERα and NCOA5 collaborate in the up-regulation of RORA by estrogen. While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD. Inasmuch as coregulators are capable of interacting with a multitude of transcription factors, aberrant expression of coregulator proteins, as we have seen previously in lymphoblasts from individuals with ASD, may contribute to the polygenic nature of gene dysregulation in ASD.

No MeSH data available.


Related in: MedlinePlus

siRNA-mediated knockdown of SUMO1 and NCOA5 show functional involvement of these coregulators in the regulation of RORA expression by AR and ERα, respectively. (A) SH-SY5Y cells were transfected with siSUMO1 or vehicle control for 24 hours and treated with 1 nM DHT or ethanol for 2 hours. RORA expression was then measured by qRT-PCR analysis (n = 3). Relative RORA quantity in each sample was calculated using a standard curve obtained from 18S expression levels in 10-fold serial dilutions of that sample. (B) NCOA5 knockdown was performed in the same manner as for SUMO1 and cells were treated with 1 nM E2. Error bars indicate SEM. Statistical significance of the differences between groups was determined by ANOVA (P <0.001 for each knockdown experiment) followed by post hoc t tests. **P <0.01. AR, androgen receptor; DHT, 4,5α-dihydrotestosterone; E2, 17β-estradiol; ERα, estrogen receptor alpha; SEM, standard error of the mean.
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Figure 5: siRNA-mediated knockdown of SUMO1 and NCOA5 show functional involvement of these coregulators in the regulation of RORA expression by AR and ERα, respectively. (A) SH-SY5Y cells were transfected with siSUMO1 or vehicle control for 24 hours and treated with 1 nM DHT or ethanol for 2 hours. RORA expression was then measured by qRT-PCR analysis (n = 3). Relative RORA quantity in each sample was calculated using a standard curve obtained from 18S expression levels in 10-fold serial dilutions of that sample. (B) NCOA5 knockdown was performed in the same manner as for SUMO1 and cells were treated with 1 nM E2. Error bars indicate SEM. Statistical significance of the differences between groups was determined by ANOVA (P <0.001 for each knockdown experiment) followed by post hoc t tests. **P <0.01. AR, androgen receptor; DHT, 4,5α-dihydrotestosterone; E2, 17β-estradiol; ERα, estrogen receptor alpha; SEM, standard error of the mean.

Mentions: To further confirm that SUMO1 is required for DHT-mediated regulation of RORA, SUMO1 expression in SH-SY5Y cells was suppressed using siSUMO1 and the transfected cells were then treated with 1 nM DHT. Using qRT-PCR analysis to measure RORA expression in siRNA-transfected cells, we found that the suppressive effect of DHT on RORA expression was completely abolished in cells transfected with siSUMO1 (Figure 5A), indicating that SUMO1 is required for DHT-mediated suppression of RORA. Similarly, we conducted siNCOA5 transfection and E2 treatment to confirm that NCOA5 is involved in E2-mediated regulation of RORA. The enhancing effect of E2 on RORA expression was significantly reduced (but not completely) in siNCOA5-transfected cells (Figure 5B), indicating that NCOA5 is involved in E2-mediated upregulation of RORA.


Differential recruitment of coregulators to the RORA promoter adds another layer of complexity to gene (dys) regulation by sex hormones in autism.

Sarachana T, Hu VW - Mol Autism (2013)

siRNA-mediated knockdown of SUMO1 and NCOA5 show functional involvement of these coregulators in the regulation of RORA expression by AR and ERα, respectively. (A) SH-SY5Y cells were transfected with siSUMO1 or vehicle control for 24 hours and treated with 1 nM DHT or ethanol for 2 hours. RORA expression was then measured by qRT-PCR analysis (n = 3). Relative RORA quantity in each sample was calculated using a standard curve obtained from 18S expression levels in 10-fold serial dilutions of that sample. (B) NCOA5 knockdown was performed in the same manner as for SUMO1 and cells were treated with 1 nM E2. Error bars indicate SEM. Statistical significance of the differences between groups was determined by ANOVA (P <0.001 for each knockdown experiment) followed by post hoc t tests. **P <0.01. AR, androgen receptor; DHT, 4,5α-dihydrotestosterone; E2, 17β-estradiol; ERα, estrogen receptor alpha; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016566&req=5

Figure 5: siRNA-mediated knockdown of SUMO1 and NCOA5 show functional involvement of these coregulators in the regulation of RORA expression by AR and ERα, respectively. (A) SH-SY5Y cells were transfected with siSUMO1 or vehicle control for 24 hours and treated with 1 nM DHT or ethanol for 2 hours. RORA expression was then measured by qRT-PCR analysis (n = 3). Relative RORA quantity in each sample was calculated using a standard curve obtained from 18S expression levels in 10-fold serial dilutions of that sample. (B) NCOA5 knockdown was performed in the same manner as for SUMO1 and cells were treated with 1 nM E2. Error bars indicate SEM. Statistical significance of the differences between groups was determined by ANOVA (P <0.001 for each knockdown experiment) followed by post hoc t tests. **P <0.01. AR, androgen receptor; DHT, 4,5α-dihydrotestosterone; E2, 17β-estradiol; ERα, estrogen receptor alpha; SEM, standard error of the mean.
Mentions: To further confirm that SUMO1 is required for DHT-mediated regulation of RORA, SUMO1 expression in SH-SY5Y cells was suppressed using siSUMO1 and the transfected cells were then treated with 1 nM DHT. Using qRT-PCR analysis to measure RORA expression in siRNA-transfected cells, we found that the suppressive effect of DHT on RORA expression was completely abolished in cells transfected with siSUMO1 (Figure 5A), indicating that SUMO1 is required for DHT-mediated suppression of RORA. Similarly, we conducted siNCOA5 transfection and E2 treatment to confirm that NCOA5 is involved in E2-mediated regulation of RORA. The enhancing effect of E2 on RORA expression was significantly reduced (but not completely) in siNCOA5-transfected cells (Figure 5B), indicating that NCOA5 is involved in E2-mediated upregulation of RORA.

Bottom Line: We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ERα interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression.AR and SUMO1 are involved in the suppression RORA expression by androgen, while ERα and NCOA5 collaborate in the up-regulation of RORA by estrogen.While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Sciences, 2300 I Street NW, Washington, DC 20037, USA. valhu@gwu.edu.

ABSTRACT

Background: Our independent cohort studies have consistently shown the reduction of the nuclear receptor RORA (retinoic acid-related orphan receptor-alpha) in lymphoblasts as well as in brain tissues from individuals with autism spectrum disorder (ASD). Moreover, we have found that RORA regulates the gene for aromatase, which converts androgen to estrogen, and that male and female hormones regulate RORA in opposite directions, with androgen suppressing RORA, suggesting that the sexually dimorphic regulation of RORA may contribute to the male bias in ASD. However, the molecular mechanisms through which androgen and estrogen differentially regulate RORA are still unknown.

Methods: Here we use functional knockdown of hormone receptors and coregulators with small interfering RNA (siRNA) to investigate their involvement in sex hormone regulation of RORA in human neuronal cells. Luciferase assays using a vector containing various RORA promoter constructs were first performed to identify the promoter regions required for inverse regulation of RORA by male and female hormones. Sequential chromatin immunoprecipitation methods followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses of RORA expression in hormone-treated SH-SY5Y cells were then utilized to identify coregulators that associate with hormone receptors on the RORA promoter. siRNA-mediated knockdown of interacting coregulators was performed followed by qRT-PCR analyses to confirm the functional requirement of each coregulator in hormone-regulated RORA expression.

Results: Our studies demonstrate the direct involvement of androgen receptor (AR) and estrogen receptor (ER) in the regulation of RORA by male and female hormones, respectively, and that the promoter region between -10055 bp and -2344 bp from the transcription start site of RORA is required for the inverse hormonal regulation. We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ERα interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression.

Conclusions: AR and SUMO1 are involved in the suppression RORA expression by androgen, while ERα and NCOA5 collaborate in the up-regulation of RORA by estrogen. While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD. Inasmuch as coregulators are capable of interacting with a multitude of transcription factors, aberrant expression of coregulator proteins, as we have seen previously in lymphoblasts from individuals with ASD, may contribute to the polygenic nature of gene dysregulation in ASD.

No MeSH data available.


Related in: MedlinePlus