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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

Schematic diagram showing the upstream region of the RORA gene (edited from the UCSC Genome Browser). Potential AR and ER binding sites are labeled (ARbs, = AR potential binding site; ERbs, = ER potential binding site). AR, androgen receptor; ER, estrogen receptor.
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Figure 2: Schematic diagram showing the upstream region of the RORA gene (edited from the UCSC Genome Browser). Potential AR and ER binding sites are labeled (ARbs, = AR potential binding site; ERbs, = ER potential binding site). AR, androgen receptor; ER, estrogen receptor.

Mentions: AR and ERα are able to regulate transcription by binding directly to specific DNA elements in the promoter region of their target genes as well as by non-genomic mechanisms. The RORA promoter region contains multiple binding sites for AR and ER spanning a region as far as approximately 10 kb upstream of the transcription start site (TSS). In our previous study, we selected four ER binding sites (ERbs-I, -II, -III, and IV) and three AR binding sites (ARbs-I, -II, and -III) located within 10 kb upstream of the TSS of RORA for chromatin immunoprecipitation analysis of hormone receptor binding (Figure 2). We found that AR and ERα are recruited to some of these binding elements in the presence of DHT and E2, respectively [16]. However, it is still unclear which binding sites are involved in the DHT-mediated downregulation and E2-mediated upregulation of RORA. We therefore constructed several firefly luciferase vectors containing different AR/ERα binding sites in the region upstream of the TSS and then conducted dual luciferase reporter assays of promoter activity in SH-SY5Y cells treated with DHT, E2, or ethanol, using the co-transfected Renilla luciferase vector as a negative control. The firefly luminescence signal in each reaction was normalized with the signal from Renilla luciferase in the same reaction to account for the variability between transfection experiments.


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)

Schematic diagram showing the upstream region of the RORA gene (edited from the UCSC Genome Browser). Potential AR and ER binding sites are labeled (ARbs, = AR potential binding site; ERbs, = ER potential binding site). AR, androgen receptor; ER, estrogen receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Schematic diagram showing the upstream region of the RORA gene (edited from the UCSC Genome Browser). Potential AR and ER binding sites are labeled (ARbs, = AR potential binding site; ERbs, = ER potential binding site). AR, androgen receptor; ER, estrogen receptor.
Mentions: AR and ERα are able to regulate transcription by binding directly to specific DNA elements in the promoter region of their target genes as well as by non-genomic mechanisms. The RORA promoter region contains multiple binding sites for AR and ER spanning a region as far as approximately 10 kb upstream of the transcription start site (TSS). In our previous study, we selected four ER binding sites (ERbs-I, -II, -III, and IV) and three AR binding sites (ARbs-I, -II, and -III) located within 10 kb upstream of the TSS of RORA for chromatin immunoprecipitation analysis of hormone receptor binding (Figure 2). We found that AR and ERα are recruited to some of these binding elements in the presence of DHT and E2, respectively [16]. However, it is still unclear which binding sites are involved in the DHT-mediated downregulation and E2-mediated upregulation of RORA. We therefore constructed several firefly luciferase vectors containing different AR/ERα binding sites in the region upstream of the TSS and then conducted dual luciferase reporter assays of promoter activity in SH-SY5Y cells treated with DHT, E2, or ethanol, using the co-transfected Renilla luciferase vector as a negative control. The firefly luminescence signal in each reaction was normalized with the signal from Renilla luciferase in the same reaction to account for the variability between transfection experiments.

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