Hormonal Regulation and Transcriptional Targets of RORA, a Novel Candidate Gene for Autism Spectrum Disorder
Open AccessAutism spectrum disorder (ASD or autism) is a pervasive neurodevelopmental disorder characterized by deficits in social understanding and interactions, aberrant language development and/or usage, and stereotyped, repetitive behaviors. Autism is inexplicably biased towards males by a ratio of at least 4:1, with no clear understanding of whether or how sex hormones may play a role in autism susceptibility. Here, we show that dihydrotestosterone (DHT) and 17beta-estradiol oppositely regulate expression of a novel autism candidate gene, the retinoic acid-related orphan receptor alpha (RORA), in the human neuronal cell line SH-SY5Y. DHT acts through androgen receptor (AR) which may interact with the corepressor protein SUMO-1 to suppress RORA expression, whereas estradiol enhances RORA expression through estrogen receptor alpha (ERalpha) which interacts with the coactivator protein NCOA5. Moreover, we extensively identify transcriptional targets of RORA in human neuronal cells using whole-genome promoter array analysis. Several potential transcriptional targets of RORA are then selected for experimental validation by independent chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) expression analyses and shRNA-mediated knockdown analyses. We demonstrate that RORA indeed regulates expression of several genes whose functions have been associated with autism. These validated RORA targets are A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2. Quantitative reverse transcriptase-PCR analysis of brain tissues from individuals with autism, as well as sex- and age-matched unaffected individuals, reveals that expression of these targets is aberrant in the brain of individuals with autism who exhibit reduced RORA expression. Interestingly, the transcript levels of selected targets, especially CYP19A1, correlate positively with RORA transcript levels in the human brain tissues. Co-immunoprecipitation and sequential chromatin immunoprecipitation analyses performed in neuronal cells show that RORA interacts with NCOA1 and NCOA5 in the human neuronal cell line, and that RORA can form a complex with NCOA5 to modulate CYP19A1 expression. Using confocal immunofluorescence microscopic analysis of tissue arrays containing postmortem specimens from the frontal cortex of individuals with autism and age-matched controls, we further show that the protein levels of RORA and aromatase, a protein encoded by the gene CYP19A1 which is the key enzyme converting androgens to estrogens, are significantly reduced in the brain of individuals with autism relative to that of sex- and age-matched controls. Aromatase protein levels also correlate positively with RORA protein levels in the brain tissues, suggesting that aromatase expression may be regulated by RORA in the human brain. Findings from this dissertation indicate that RORA has the potential to be under feedback regulation by both androgen and estrogen through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism.
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Sarachana_gwu_0075A_11578.pdf | 2018-01-16 | Open Access |
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