Genome-wide identification of transcriptional targets of RORA reveals direct regulation of multiple genes associated with autism spectrum disorder

Abstract

BackgroundWe have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans.MethodsHere we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression.ResultsThe ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as development of the cortex and cerebellum, cognition, memory, and spatial learning. Independent ChIP-quantitative PCR analyses confirm binding of RORA1 to promoter regions of selected ASD-associated genes, including A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2, whose expression levels (in addition to HSD17B10) are also decreased in RORA1-repressed human neuronal cells and in prefrontal cortex tissues from individuals with ASD.ConclusionsFindings from this study indicate that RORA transcriptionally regulates A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and strongly suggest that reduction of this sex hormone-sensitive nuclear receptor in the brain causes dysregulated expression of these ASD-relevant genes as well as their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD.

RNA expression profiling in depressed patients suggests retinoid-related orphan receptor alpha as a biomarker for antidepressant response

“…24 RORa polymorphisms have further been implicated in posttraumatic stress disorder 22 and autism spectrum disorder. 45,46 We found rs809736 to be nominally associated with RORa expression in the present sample with the same direction of effect, that is, the allele associated with response in STAR*D was associated with lower RORa expression in the present study. Nevertheless, rs809736 was not replicated in the large PGC pharmacogenetic sample, hence, the nominal significant association of rs809736 with RORa gene expression in our cohort needs further replication.…”

A Regulatory Role for the Insulin- and BDNF-Linked RORA in the Hippocampus: Implications for Alzheimer's Disease

“…This makes RORA an important gene/gene product in the etiology/pathology of AD. Indeed recent ChIP-on-chip studies have indicated an over-representation of genes linked with learning, memory and cognition among probable regulatory targets of RORA [57]. Thus, on the basis of the data presented here, we here posit an important link between hippocampal RORA and AD.…”

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

“…Figure 8 presents a working model that integrates the results of these studies with those of our earlier studies that demonstrated the opposite regulation of RORA by male and female hormones and the regulation of CYP19A1 by RORA [16,38]. In this model, a reduction in RORA expression, which may be induced by increased methylation, which we have demonstrated previously in cell lines from individuals with ASD [12] is expected to lead to a decrease in CYP19A1 (aromatase), which, in turn, would result in the accumulation of its substrate testosterone.…”