Sex Hormone-Mediated Regulation of RORA, a Potential Contributor to Sex Bias in ASD

Background:  Expression of retinoic acid-related orphan receptor alpha (RORA) is reduced in both brain and lymphoblastoid cells from multiple cohorts of individuals with ASD.  Moreover, RORA regulates the gene for aromatase, which converts male to female hormones that, in turn, regulate RORA in opposite directions, with androgen suppressing RORA.  The regulatory feedback interactions between RORA, aromatase and sex hormones suggest that sexually dimorphic regulation of RORA may contribute to the male bias of ASD.  However, the molecular mechanisms through which androgen and estrogen differentially regulate RORA are still unknown, as are the mechanisms through which RORA deficiency may contribute to the neuropathology of ASD.

Objectives:  1) To investigate the mechanisms involved in the opposite regulation of RORA by male and female hormones, including the involvement of hormone receptors, coregulators, and epigenetic changes; 2) To identify genome-wide transcriptional targets of RORA in order to determine relevance to ASD; 3) To investigate sexually dimorphic expression of RORA and validated targets in the human brain.

Methods:  Functional knockdown of hormone receptors and coregulators with small interfering RNA (siRNA) was used to investigate their involvement in sex hormone regulation of RORA in human neuronal cells.  DNA methylation analysis is being used to analyze hormone-induced epigenetic changes at the RORA promoter.  ChIP-on-chip analysis was used for genome-wide identification of putative transcriptional targets of RORA, followed by validation of selected targets by RORA knockdown and ChIP-qPCR analyses.  Expression of validated targets was also examined in RORA-deficient brain tissues from individuals with ASD.  Gene expression data from the BrainSpan human brain atlas is being examined to determine the relative expression of RORA and validated target genes in brain tissues from normal males and females.

Results: AR and its corepressor SUMO1 are involved in the suppression RORA expression by androgen, while ERα and coactivator NCOA5 collaborate in the up-regulation of RORA by estrogen.  Over 2500 genes were identified as putative targets of RORA, of which more than 400 are listed in autism gene databases.  Gene ontology analysis of this dataset of genes reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion, synaptogenesis, synaptic transmission/plasticity, and axonogenesis, as well as higher level functions such as cognition, memory, and spatial learning. Six validated targets of RORA were also found to be reduced in RORA-deficient postmortem cortical tissues from individuals with ASD.  Epigenetic analyses and the meta-analysis of gene expression in brain are still in progress.

Conclusions:  Deficiency of RORA causes dysregulation of multiple neurologically-relevant genes, thus compromising their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD. Differential recruitment of corepressor and coactivator proteins to the RORA promoter by the androgen and estrogen receptors, respectively, not only explains the opposite regulation by male and female hormones, but also reveals another layer of complexity in gene regulation in ASD. Moreover, the specific coregulators recruited by the hormone receptors are expected to lead to epigenetic changes that suppress or activate RORA expression in response to androgen and estrogen.