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

Abstract

BackgroundOur 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.MethodsHere 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.ResultsOur 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.ConclusionsAR 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.

Investigation of sex differences in the expression of RORA and its transcriptional targets in the brain as a potential contributor to the sex bias in autism

“…Regardless of sex differences, the underlying mechanisms driving regional and developmental differences in RORA expression, such as those seen in the OFC, are unclear. Recently, we described differential recruitment of co-regulator proteins that are involved in the sex hormone-dependent regulation of RORA in the SH-SY5Y neuronal cell model [ 41 ]. We demonstrated that estrogen-mediated upregulation of RORA expression required both the estrogen receptor (ER-alpha) and the coactivator NCOA5, while androgen (DHT)-mediated downregulation of RORA required the androgen receptor (AR) and the corepressor function of SUMO1.…”

Understanding autism in the light of sex/gender

“…[ 29 ] look into specific sex-implicated aetiological mechanisms. Their previous studies show that autism may be associated with deficiency in the expression of the retinoic acid-related orphan receptor alpha (RORA) gene, and therefore, dysregulated feedback loops with androgen and oestrogen, as well as the many autism-associated genes regulated by RORA [ 62 , 23 , 22 ]. In their new article, Hu et al .…”

Sex differences in brain plasticity: a new hypothesis for sex ratio bias in autism

“…For instance, androgens and estrogens differentially regulate the RORA gene, a candidate susceptibility gene for autism, the expression of which is low in the frontal cortex of autistic individuals [ 171 ]. The product of this gene, which promotes the conversion of T into estrogen, also acts through co-activators, demonstrating the complexity of the gene/hormone interactions [ 172 ].…”