Objectives: To comprehensively identify direct transcriptional targets of RORA in human neuronal cells using high-throughput whole-genome promoter analysis. Here we focus on RORA1 which is the major isoform of RORA in the brain.
Methods: Chromatin was isolated from the human neuronal cell line SH-SY5Y and immunoprecipitated (IP) using anti-RORA1 or IgG antibody. DNA was purified from the IP chromatin and used for microarray analysis (ChIP-on-chip) using Affymetrix's Genechip Human Promoter 1.0R Arrays (n = 3 for specific and nonspecific IgG), each of which comprises over 4.6 million probes tiled through over 25,500 human promoter regions. Probes significantly enriched in RORA1-IP DNA were identified using Partek software. Selected potential targets of RORA1 were then further validated by chromatin immunoprecipitation, followed by qPCR (ChIP-qPCR) analysis. To further demonstrate that reduction of RORA1 expression could lead to aberrant transcription of novel RORA1 targets, we also conducted shRNA-mediated knockdown of RORA1 and performed RT-qPCR analysis to determine expression of selected RORA1 targets in RORA1-deficient neuronal cells. Biological networks and functions associated with RORA1 transcriptional targets were predicted using Ingenuity Pathway Analysis (IPA) and Pathway Studio 7 programs.
Results: The ChIP-on-chip analysis revealed that as many as 1,338 probes corresponding to promoter regions of 1,274 genes across the human genome were significantly enriched in RORA1-IP DNA (p-value < 0.05; log2 ratio > 0.3). Among these potential targets were genes known to have biological functions negatively impacted in ASD, including neuronal adhesion and survival, synaptogenesis, as well as development of cortex and cerebellum. ChIP-qPCR analysis confirmed binding of RORA1 to promoter regions of selected potential targets, including A2BP1, NLGN1, HSD17B10, and NTRK2. Knockdown of RORA1 in human neuronal cells resulted in reduction of expression of these genes.
Conclusions: These findings indicate that RORA1 transcriptionally regulates a number of downstream targets, including A2BP1, NLGN1, HSD17B10, and NTRK2, all of which are known to have biological functions associated with ASD.