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

Cell culture

The human neuroblastoma cells SH-SY5Y (ATCC, Manassas,VA, USA) were cultured in 1:1 Modified Eagle’s Medium (MEM) and Ham’s F12 media (MediaTech, Manassas, VA, USA) supplemented with 15% (v/v) fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin/streptomycin (MediaTech). Cells were maintained at 37°C with 5% CO₂, and split 1:2 every 3 or 4 days when the cells reached ~80% confluency. For harvesting, the cells were treated with trypsin-ethylenediamine tetraacetic acid (MediaTech) for 2 to 3 minutes to release them from the surface of the culture flask. Complete growth medium was then added to the flask containing suspension cells to inactivate trypsin. Cells were transferred to a sterile centrifuge tube and pelleted by spinning at 800 rpm for 5 minutes at 4°C and gently washed twice with ice-cold PBS.

Frozen human brain tissues

Frozen postmortem prefrontal cortex (BA9/10) tissues from male individuals with autism (n = 3) and from age-matched typically developing males (n = 3) were obtained through the Autism Tissue Program (San Diego, CA, USA). All frozen brain tissues were preserved by the Harvard Brain Tissue Resource Center, Harvard Medical School. The list of human brain tissues used in this study is shown in Additional file1, along with information on the age of the donor, the postmortem interval (PMI) between death and tissue collection, and the cause of death when known.

Chromatin immunoprecipitation (ChIP)-on-chip analysis

Chromatin immunoprecipitation (ChIP) for promoter array analysis was performed using the Millipore EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Briefly, the human neuronal cell line SH-SY5Y was cultured in complete growth medium in a T-175 flask until ~80% confluency (approximately 1.5 × 10⁷ cells). The medium was then carefully removed without disturbing the cells. The cells were fixed with 37% formaldehyde for exactly 10 minutes to crosslink chromatin. The crosslinking reaction was terminated by addition of 10% glycine, after which ice-cold PBS supplemented with protease inhibitor cocktail was added to the culture flask to wash and chill the cells. The crosslinked cells were dislodged from the flask by scraping gently with a cell scraper and then transferred to a pre-chilled centrifuge tube. Crosslinked cells were pelleted and nuclear extraction was performed using the Active Motif Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocol. Nuclear pellets were resuspended in SDS Lysis Buffer and sonicated on wet ice to shear crosslinked chromatin to 200–1,000 bp using a Heat Systems-Ultrasonics W-380 sonicator (Heat Systems-Ultrasonics/Misonix, Farmingdale, NY, USA) set to 30% of maximum output power for 20 × 10 seconds with 1-minute intervals. Sonicated chromatin was divided into several aliquots for immunoprecipitation reactions and stored at −80°C until use. Each immunoprecipitation reaction was conducted using 1 μg of goat anti-RORA1 or normal goat IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoprecipitated chromatin was then reverse-crosslinked by adding 5 M NaCl to the final concentration of 0.2 M and incubated at 65°C overnight. DNA from the immunoprecipitated chromatin was isolated and purified using Millipore DNA purification columns (Millipore) and then submitted to the Genomics Core Facility at The George Washington University for analysis on Affymetrix human promoter tiling arrays (Affymetrix, Santa Clara, CA, USA).

Analysis of RORA binding sites on Affymetrix promoter tiling arrays

Purified DNA from the chromatin immunoprecipitated with anti-RORA1 or control IgG antibodies was amplified using the Whole Genome Amplification 2 Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. The amplification product of each immunoprecipitation reaction was purified and hybridized on Affymetrix GeneChip Human Promoter 1.0R microarrays. Probes significantly enriched in RORA1-immunoprecipitated DNA relative to IgG-immunoprecipitated DNA were identified using the Partek Genomics Suite software (Partek, St. Louis, MO, USA), as described in detail below.

The Affymetrix Human Promoter 1.0R array contains over 4.6 × 10⁶ probes tiled over 25,500 promoter regions of annotated genes. Each gene promoter is thus interrogated on average by over 150 probes or “tiles”. For this study, DNA immunoprecipitated from SH-SY5Y cells with either a RORA1-specific antibody or a non-specific IgG as a control was hybridized on separate chips in triplicate, and the amount of probe hybridized to each element on the chips was determined. Partek Genomics Suite Software was used to analyze the intensities of the probe elements across the promoter array using the workflow recommended by Affymetrix for tiling arrays. In brief, the data normalization procedures include adjusting for probe sequence, RMA background correction, quantile normalization, and log (base 2) transformation of intensity data. Two-way analysis of variance was then used to determine differences between the RORA1-co-immunoprecipitated DNA and IgG-co-immunoprecipitated control DNA in hybridizing to specific probes. The MAT (model-based analysis of tiling arrays) peak-seeking algorithm[32] was subsequently used to detect enriched regions in the RORA1-co-immunoprecipated DNA versus IgG control samples. Nominal P value cutoff ≤0.05 (corresponding to false discovery rate (FDR) <7%), MAT score >0, minimum number of probes for each region >10, and average length for the ChIP fragment >600 bp were used as the peak detection parameters in this discovery phase of high-throughput screening for potential RORA targets. The microarray data from this study have been deposited into the Gene Expression Omnibus repository [GEO: GSE45756]. Positive MAT scores for a specific region indicate that RORA binding is enriched in that region relative to IgG controls. The genes mapping to the RORA-enriched promoter regions were identified using the gene annotation database (hg18) provided by Affymetrix. To further aid in the selection of gene promoters for confirmation analyses, an additional level of analysis was performed to determine the average intensity of probes across the regions hybridizing to the RORA-immunoprecipitated DNA relative to that hybridizing to the control DNA. These intensities were used to calculate the fold-change or enrichment across each gene promoter region for binding of the RORA-immunoprecipitated DNA over that of the IgG-precipitated DNA. Among the RORA-enriched promoter sites, the site corresponding to ITPR1 was used as a positive control since Itpr1 had been identified as a transcriptional target of Rora in mice[29]. Because the ITPR1 promoter region exhibited a fold-change of 1.29, we used this value as a minimum average fold-change in selecting target genes for further confirmation.

Rationale for selection of putative target genes for further confirmation

Of the 2,544 genes identified as putative targets of RORA by the ChIP-on-chip analysis (see Results), we selected ITPR1, CYP19A1, HSD17B10, A2BP1 (RBFOX1), NLGN1, and NTRK2 for further confirmation by ChIP-quanititative PCR analyses and by functional knockdown of RORA. Among these putative target genes, A2BP1, ITPR1, NLGN1, and NTRK2 have been previously identified as candidate genes for ASD[33–35]. The protein product of CYP19A1, also known as aromatase, is involved in the enzymatic conversion of androgen to estrogen via testosterone intermediates. Although CYP19A1 had previously been reported to be a target of RORA in human breast cancer cell lines[36], we recently demonstrated that its promoter region was a site of RORA binding in a human neuronal cell line[12]. Here, we wished to further confirm CYP19A1 as a transcriptional target of RORA by functional knockdown studies. Like CYP19A1, HSD17B10 also codes for an enzyme involved in the conversion of androgen to estrogen, but via 19-hydroxyandrostenedione, 19-oxyandrostenedione and estrone intermediates. Because of our earlier study demonstrating significant and correlated reductions in RORA and CYP19A1 protein levels in the autistic brain[12], we were interested in determining whether a deficiency in RORA could also impact this alternate biochemical pathway through down-regulation of HSD17B10, thus potentially reinforcing the buildup of androgen and reduction of estrogen in neuronal cells and tissues. The relevance of these genes to ASD is further elaborated in the Discussion.

Prediction of RORA binding elements

Putative binding elements of RORA in the promoter regions of A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2 were predicted using PROMO 3.0[37, 38], JASPAR[39], and the EpiTect ChIP Search Portal of SABiosciences (Valencia, CA, USA). For each putative target gene, a total of three or four predicted transcription factor binding sites within 10 kb upstream of the transcription start site were selected for ChIP-quantitative PCR analyses.

ChIP-quantitative PCR analysis

Chromatin immunoprecipitation for ChIP-quantitative PCR analysis of RORA binding elements in the promoter region of putative RORA targets was conducted using the ChIP-IT Express Enzymatic Kit (Active Motif) following the manufacturer’s instructions. Briefly, confluent SH-SY5Y cells (approximately 1.5 × 10⁷ cells in a T-175 flask) were fixed with 10% formaldehyde for exactly 10 minutes and the fixation reaction was stopped by adding 10% glycine to the flask. The cells were washed with 10 ml ice-cold PBS for 5 seconds, then 6 ml ice-cold PBS supplemented with 0.5 mM (final concentration) phenylmethylsulfonylfluoride supplied in the kit was added to the culture flask to wash and chill the cells. The crosslinked cells were transferred from the flask to a pre-chilled 15 ml centrifuge tube by scraping gently with a cell scraper. Crosslinked cells were pelleted by centrifugation for 10 minutes at 2,500 rpm (720 relative centrifugal force) at 4°C, and then resuspended in 1 ml ice-cold Active Motif Lysis Buffer (Active Motif). The crosslinked cells were transferred to an ice-cold dounce homogenizer and the nuclei were released from the cells by douncing with a tight pestle. Optimal cell lysis was assessed under a phase contrast microscope using a hemacytometer. The nuclei were then transferred to an ice-cold 1.7 ml microcentrifuge tube and pelleted by centrifugation for 10 minutes at 5,000 rpm (2,400 relative centrifugal force) at 4°C. Chromatin was then isolated from the nuclear pellets and sheared into 150 to 1,000 bp fragments by incubating with 10 U/ml (final concentration) Enzymatic Shearing Cocktail (Active Motif) at 37°C for exactly 10 minutes. The enzymatic shearing reaction was stopped by adding ethylenediamine tetraacetic acid to a final concentration of 10 mM and chilling the reaction tube on ice for 10 minutes. Optimal shearing was assessed by agarose gel electrophoresis. For each ChIP reaction, enzymatically-sheared chromatin containing ~7 to 25 μg chromatin DNA was immunoprecipitated using 1 μg antibody and 25 μl Protein G Magnetic Beads (Active Motif). The list of antibodies for ChIP analyses is shown in Additional file2. Immunoprecipitated chromatin was reverse-crosslinked according to the ChIP-IT Express Enzymatic Kit protocol and DNA was isolated and purified from the chromatin using the ChIP DNA Purification Kit (Active Motif).

Real-time quantitative PCR analysis was conducted using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) to determine the enrichment of each RORA binding element in immunoprecipitated DNA. Primers for quantitative PCR analysis were designed using Primer3 software[40] and were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Input DNA was diluted into five 10-fold serial dilutions and included in quantitative PCR analyses. Relative enrichment values of RORA binding elements in each immunoprecipitated chromatin were calculated using standard curves obtained from the enrichment of RORA binding elements in the 10-fold serial dilutions of respective input DNA. The list of primers is shown in Additional file3.

Short hairpin RNA (shRNA) transfection

SH-SY5Y cells were cultured in a six-well culture plate containing complete growth medium without antibiotics (approximately 2.5 × 10⁵ cells per well) until cells were ~70% confluent. For each well, the cells were transfected with 2.50 μg RORA1 shRNA (Santa Cruz Biotechnology) or 2.50 μg negative control shRNA (Santa Cruz Biotechnology) using Lipofectamine LTX and PLUS reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Briefly, shRNA (2.50 μg) was diluted in 500 μl Opti-MEM I Reduced Serum Medium (Invitrogen) and 1.25 μl PLUS reagent was added to the diluted shRNA solution. Lipofectamine LTX (25 μl) was then added to the shRNA-PLUS solution, incubated for 25 minutes at room temperature to form shRNA-Lipofectamine-LTX-PLUS complexes, and added to the cells. The cells were then incubated with the shRNA-Lipofectamine-LTX-PLUS complexes at 37°C and 5% CO₂ for 24 hours before harvesting. The list of shRNAs is shown in Additional file2.

RNA isolation and quantitative RT-PCR analysis

Quantitative RT-PCR analyses were performed as previously described[41]. Total RNA from the shRNA-transfected cells was isolated using TRIzol (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA) following the manufacturers' instructions. Human brain tissues were homogenized in the Bullet Blender Homogenizer (Next Advance, Averill Park, NY, USA) using nuclease-free glass beads, after which total RNA from homogenized brain tissues was isolated using the RNeasy Mini Kit (Qiagen). A total of 1 μg purified total RNA was used for cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA) according to the manufacturer’s protocols. The reaction (20 μl) was incubated at 25°C for 5 minutes, followed by 42°C for 30 minutes, and ending with 85°C for 5 minutes. After reverse transcription, the cDNA reaction mixture was diluted to a volume of 50 μl with nuclease-free water and used as a template for quantitative PCR analyses. Real-time PCR analyses were conducted using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems). Primers for quantitative RT-PCR analyses designed by Primer3 software are listed in Additional file3. The relative quantity of transcripts in each sample was calculated using standard curves based on the relative quantity of 18S RNA transcript in 10-fold serial dilutions of the respective sample.

Statistical analysis

A paired, two-sided, Student’s t test was performed to determine significance of the differences in numerical data obtained by quantitative PCR analyses. P <0.05 was considered statistically significant.

Hypergeometric distribution analyses[42] were used to determine the statistical significance of enrichment in autism candidate genes among the transcriptional targets identified by ChIP-on-chip analysis, relative to the genes present in AutDB[34], AutismKB[35], or a combination of the two databases of autism-associated genes.

Pathway and gene ontology analyses

Network prediction, functional, and gene ontology analyses were accomplished using licensed Ingenuity Pathway Analysis (IPA) and Pathway Studio7.0 software, as well as the open-access DAVID Bioinformatics Resources 6.7[43, 44]. For functional and network analyses, Fisher exact P values were calculated using the entire set of genes in the Ingenuity Knowledge Base as the reference set.

Identification of RORA transcriptional targets using whole-genome promoter array analysis and gene ontology analyses

Network prediction of selected transcriptional targets to assess relevance to autism spectrum disorder (ASD)

We selected six potential gene targets, ITPR1, CYP19A1, A2BP1, HSD17B10, NLGN1, and NTRK2, for confirmation by independent ChIP-quantitative PCR and functional analyses based on the reasons given in Methods. The probe enrichment data and genomic locations for these genes are shown in Table 5. Functional analysis of these six genes using the Pathway Studio 7.0 network prediction program revealed an association of these genes with neurological disorders, including autism and ataxia, as well as autism-related neurological functions, including synaptogenesis, synaptic transmission, long-term potentiation, learning, and memory (Figure 1). Interestingly, other autism candidate genes, including NLGN3, NRXN1, RELN, and GABAA, were also included in the predicted gene network, thus supporting the investigation of our selected genes as ASD-relevant transcriptional targets of RORA.

Nearest gene	Entrez gene name	MAT score on T (R vs. G)	P value (region)	FDR (%)	Average intensity fold-change (R vs. G)	Chromosome (strand)	Region start	Length (bps)	Number of probes in region
ITPR1	Inositol 1,4,5-triphosphate receptor, type 1	17.19	0.005	<5	1.29	chr3 (+)	4,724,792	2,804	71
HSD17B10	Hydroxysteroid (17-β) dehydrogenase 10	14.11	0.007	<5	11.86	chrX (−)	53,483,92	1,723	42
A2BP1 (RBFOX1)	RNA binding protein, fox-1 homolog	12.86	0.009	<5	1.29	chr16 (+)	7,315,418	2,636	68
NLGN1	Neuroligin 1	7.30	0.036	<5	3.27	chr3 (+)	174,777,672	639	18
CYP19A1 (aromatase)	Cytochrome P450, family 19, subfamily A, polypeptide 1	7.02	0.040	<5	1.61	chr15 (−)	49,415,636	2,111	54
NTRK2	Neurotrophic tyrosine kinase, receptor, type 2	6.72	0.044	<7	2.17	chr9 (+)	86,412,498	966	21

FDR false discovery rate, G intensity of probes hybridized with DNA fragments immunoprecipitated with nonspecific IgG, MAT model-based analysis of tiling arrays, R intensity of probes hybridized with DNA fragments immunoprecipitated with anti-RORA antibody, T t-statistic.

Confirmation of RORA binding to selected transcriptional targets in human neuronal cells

To confirm that RORA protein binds to the promoter region of these six potential transcriptional targets, we conducted ChIP using anti-RORA1 or nonspecific IgG antibody, followed by quantitative PCR analysis to determine the enrichment of RORA binding sites in the respective promoter regions of the selected genes. Figure 2 shows that RORA binding sites in the promoter region(s) of each of these potential targets were significantly enriched in chromatin samples immunoprecipitated by anti-RORA1 antibody relative to IgG-immunoprecipitated chromatin, indicating that RORA protein indeed binds to promoter regions of A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2.

RORA suppression reduces expression of selected transcriptional targets in human neuronal cells

To further validate that RORA regulates expression of A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and to examine whether reduction of RORA expression leads to reduced expression of these selected RORA transcriptional targets, shRNA-mediated knockdown of RORA was performed in SH-SY5Y cells using RORA1 shRNA, and quantitative RT-PCR analyses were conducted to measure expression of RORA1 and the potential targets. Expression of RORA1 in SH-SY5Y cells was significantly down-regulated by approximately 50% in comparison with cells transfected with negative control shRNA, indicating that shRNA-mediated knockdown of RORA1 was successful. Notably, expression of all selected RORA transcriptional targets was significantly reduced (Figure 3), indicating that expression of these genes is regulated by RORA and that these genes indeed are RORA transcriptional targets in human neuronal cells. As a negative control, the expression level of GAPDH, which is not a transcriptional target of RORA, is unaffected by transfection of the SH-SY5Y cells with RORA1 shRNA.

Expression of transcriptional targets of RORA is relatively reduced in the frontal cortex of individuals with autism in comparison with age-matched controls

Relevance of A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2 to the pathobiology of autism

A2BP1 (ataxin 2-binding protein 1), also known as RBFOX1, is an RNA-binding protein that regulates neuron-specific alternative splicing[62]. Several studies, including genetic and gene expression analyses, have established the link between this gene and autism. Using fluorescent in situ hybridization and quantitative PCR analyses, Martin and colleagues found a cryptic deletion of the A2BP1 gene in a female with autism, epilepsy, and global development delay[33]. Reduction of the A2BP1 transcript level was also observed in the lymphocytes of this individual, suggesting that the deletion causes aberrant expression of this gene in this autism case. By genotyping 27 SNPs across this gene in 206 parent–child trios, they identified two regions exhibiting a nominal association with autism. Moreover, a recent noise-reduction genome-wide association study (GWAS) of two autism GWAS datasets (with 597 and 696 families) from the Autism Genetic Resource Exchange revealed 1,535 significant linkage disequilibrium blocks overlapping 431 genes[63]. Interestingly, regions in the A2BP1 gene were identified among the most significant linkage disequilibrium blocks (P = 3.6×10^-5). Furthermore, Voineagu and colleagues also conducted transcriptomic analysis of postmortem frontal and temporal cortex tissues from 19 individuals with autism and 17 controls using microarrays[64]. They found that A2BP1 expression was significantly reduced in both frontal and temporal cortex tissues from individuals with autism relative to controls. Using high-throughput RNA sequencing and semi-quantitative RT-PCR analyses, they also demonstrated that splicing of A2BP1-dependent alternative exons in the brain of individuals with autism was dysregulated[64], suggesting that aberrant expression of A2BP1 results in dysregulation of alternative splicing in autism. Findings from several copy number variation (CNV) studies[65–70] and linkage studies[71–75] have also reported that A2BP1 is associated with autism. Aside from its association with ASD, recent studies on the physiological function of A2BP1/RBFOX1 demonstrate its involvement in synaptic transmission and neuronal excitation[76] as well as its role in the regulation of transcriptional networks involved in neuronal development[77].

CYP19A1 encodes aromatase, a key enzyme that converts androstenedione to estrone and testosterone to estradiol. Although its neuronal function is most often associated with the regulation of reproductive behaviors through the sex hormones, more recent studies have revealed unexpected functions of aromatase in the brain, including neurogenesis, neuronal differentiation, synaptic activity and plasticity, and protection against oxidative stress[50, 78–82]. Another interesting observation is the colocalization of aromatase with oxytocin in several regions of the rat brain, including the periventricular nucleus of the hypothalamus and the zona incerta, which suggests a role for this gene in oxytocinergic neurons in the limbic system[83], which may have implications for social cognition. CYP19A1 has also been associated with autism by genetic studies. Allen-Brady and colleagues conducted genome-wide screening of 70 families (192 individuals with autism and 461 unaffected relatives) and identified three regions shown to be highly significant in the linkage analysis[84]. Interestingly, one of the three regions is 15q21.1-15q22.2 (heterogeneity logarithm (base 10) of the odds = 5.31) where the CYP19A1 gene is located. Moreover, Chakrabarti and colleagues studied SNPs in 68 candidate genes for autism to identify common genetic variations associated with autistic traits and Asperger syndrome, a high-functioning subgroup of autism. A case–control association analysis of individuals with Asperger syndrome (n = 174) revealed regions in 14 genes, including CYP19A1, that showed a nominally significant association with Asperger syndrome[85].

HSD17B10 encodes 3-hydroxyacyl-CoA dehydrogenase type-2, a mitochondrial enzyme involved in mitochondrial integrity and oxidation of fatty acids and steroids. As mentioned earlier, like CYP19A1, HSD17B10 is involved in the conversion of androgens to estradiol via androstenedione and estrone intermediates. A reduction in HSD17B10 would thus be expected to lead to increased androgen and reduced estrogen, either of which may have serious consequences for brain development[86]. Indeed, reduced expression of HSD17B10 causes X-linked mental retardation, choreoathetosis, language impairment, and abnormal behavior[87–89]. These associated disorders are particularly interesting since we have identified RORA deficiency (specifically, reduced expression) in the phenotypic subtype of ASD associated with severe language impairment[13]. Using chromosome microarray analysis, Edens and colleagues reported duplications involving Xp11.22-p11.23, a region where HSD17B10 gene is located, in two females with autism and epilepsy[90].

ITPR1 encodes inositol 1,4,5-triphosphate receptor type 1, a ligand-gated ion channel that is activated by cytosolic calcium and inositol triphosphate, and that is involved in synaptogenesis and formation of dendritic contacts[91]. Computational modeling also suggests that the biochemical and biophysical properties of ITPR1 coupled with its high density and lower sensitivity to IP₃ in cerebellar Purkinje cells are critical to its postulated role in long-term depression[92]. With respect to ASD, transcriptomic analysis of frontal and temporal cortex tissues from 16 individuals with autism and 16 controls revealed down-regulation of ITPR1[64]. Bremer and colleagues used high-resolution whole genome array-based comparative genomic hybridization to screen 223 individuals with autism to identify gene dose alterations associated with autism susceptibility. They found regions in chromosome 3p25.3, where ITPR1 is located, significantly enriched in individuals with autism[93]. Another CNV study of 1,124 families, each of which included a single proband, his/her unaffected parents and, in most cases, an unaffected sibling, revealed as many as 234 CNV regions in several genes, including ITPR1, to be associated with autism[69]. Moreover, Levy and colleagues studied genomic copy-number variation in a large cohort of families with a single affected child and at least one unaffected sibling. Among significant regions identified to be de novo CNVs were regions containing the ITPR1 gene[94].

NLGN1 encodes neuroligin-1, a neuronal cell surface adhesion molecule that interacts with neurexin molecules. NLGN1 is involved in formation and remodeling of synapses in the central nervous system[95]. In addition to synaptogenesis, a recent study has also demonstrated a role for NLGN1, acting together with NRXN1β, in neuritogenesis and the formation of neuronal circuits upon activation of the fibroblast growth factor receptor-1[96]. In the context of ASD, Glessner and colleagues conducted a whole-genome CNV analysis of 859 individuals with autism and 1,409 typically developing children of European ancestry who were genotyped with ~550,000 SNP markers to comprehensively identify CNVs conferring susceptibility to autism[97]. Positive findings were evaluated in an independent cohort of 1,336 individuals with autism and 1,110 controls of European ancestry. Interestingly, a CNV region in the NLGN1 gene was significantly enriched in individuals with autism. Moreover, a family-based association analysis of 100 families with autism found a modest significant association at a region in NLGN1 gene[98]. A high-density SNP, genome-wide linkage study also showed that the chromosomal region 3q26.31-q27.3 was significantly associated with autism[99]. Deletion of this gene was also reported to cause impaired spatial memory and increased repetitive behavior[100].

NTRK2 encodes neurotrophic tyrosine kinase receptor type 2 (also known as TRKB) that is a catalytic receptor for several neurotrophins, including BDNF, NT4, and NT3. NTRK2 is involved in axon guidance signaling as well as synapse formation and plasticity[101–104]. Its role in ASD is implicated by genetic association analysis of 146 children with autism and 50 typically developing controls which identified six SNPs and multiple haplotypes in the NTRK2 gene[105]. This study also showed that BDNF levels were significantly increased in children with autism relative to that in controls, supporting a role of NTRK2/BDNF signaling as a susceptibility factor for the disorder.

Study limitations and future directions

Because little is known with regard to transcriptional targets of RORA, particularly in human neuronal cells, this ChIP-on-chip analysis was undertaken as a discovery tool to screen for potential transcriptional targets at the genome-wide level. As a result, we used a less stringent P-value of 0.05 as a filter to select probes enriched for RORA binding without applying correction for multiple testing (which is not part of the standard Partek analytical workflow for tiling arrays). However, all of the regions identified using P ≤0.05 are retained in the dataset even after imposing FDR ≤7%. Thus, although the list of 2,544 genes that are identified as putative targets of RORA should be interpreted with caution pending independent validation, we suggest that this list may be used as a basis for hypothesis generation with regard to the genes, pathways and functions that may be impacted by RORA deficiency. Another caveat is that the list of RORA targets does not replicate all of the several targets identified in the mouse cerebellum by ChIP-quantitative PCR analyses[29] other than ITPR1. We do not know the precise reason for this difference, but it is possible that the target selection may be a function of the specific tissues and cell types as well as species, since the mouse targets were identified in primary cerebellar cells, and the human targets were identified in the SH-SY5Y human neuroblastoma cell line. Indeed, co-regulator availability is also tissue-specific, and transcriptional regulation by RORA requires association with gene-specific and tissue-specific co-regulator proteins, thus adding another layer of complexity to target selection.

Another potential complication is the possibility that target selection may be directed by circadian rhythm, as RORA is a regulator of the circadian cycle. Thus, it would be interesting to conduct ChIP-on-chip analysis on cells synchronized according to stage in the circadian cycle. Finally, as noted earlier, although the average expression levels of the RORA targets were reduced in the frontal cortex of individuals with ASD relative to that of controls as expected, the differences were not statistically significant. Notably, the protein expression levels of the genes exhibiting the least significant expression differences (that is, highest P values) in this study, RORA and CYP19A1, were found to be significantly different in our previous study in which 12 autism tissue samples were compared with 22 controls[12], thus emphasizing the need for an expanded investigation of RORA targets involving additional brain tissues from individuals with ASD and controls, including samples from different brain regions.

In summary, using a high-throughput genome-wide method for identifying potential transcriptional targets of RORA followed by independent validation of six targets with functional relevance to ASD, we have demonstrated that RORA deficiency can lead to a cascade of transcriptional deregulation that impacts a number of genes known to be associated with ASD. Figure 4 summarizes the principal findings from this study.