Background
Methods
Cell culture
Frozen human brain tissues
Chromatin immunoprecipitation (ChIP)-on-chip analysis
Analysis of RORA binding sites on Affymetrix promoter tiling arrays
Rationale for selection of putative target genes for further confirmation
Prediction of RORA binding elements
ChIP-quantitative PCR analysis
Short hairpin RNA (shRNA) transfection
RNA isolation and quantitative RT-PCR analysis
Statistical analysis
Pathway and gene ontology analyses
Results
Identification of RORA transcriptional targets using whole-genome promoter array analysis and gene ontology analyses
Annotation cluster 1: enrichment score 5.107 | ||||
---|---|---|---|---|
GO term | Count | P Value | Benjamini* | Genes |
GO:0030182-neuron differentiation | 35 | 1.52E-08 | 3.56E-05 | RAB3A, CDK5R1, ADORA2A, PAX3, RORA, RTN1, EPHB2, ARX, ATP2B2, BDNF, SLC1A3, LAMB2, CD44, CXCR4, ANK3, DMD, ROBO3, LHX8, LMX1B, MDGA2, PTPRR, NTNG1, RPGRIP1, NUMBL, SOD2, PTPN11, CTNNA2, SLITRK1, NTRK1, NTRK2, FOXG1, CNTN4, CACNA1F, CUX1, NTM |
GO:0048666-neuron development | 28 | 2.96E-07 | 3.48E-04 | RAB3A, CDK5R1, ADORA2A, EPHB2, ARX, ATP2B2, BDNF, SLC1A3, LAMB2, CD44, ANK3, CXCR4, DMD, ROBO3, LHX8, LMX1B, NTNG1, RPGRIP1, CTNNA2, PTPN11, SOD2, NUMBL, SLITRK1, FOXG1, NTRK2, CNTN4, CACNA1F, NTM |
GO:0000904-cell morphogenesis involved in differentiation | 22 | 1.86E-06 | 1.45E-03 | RAB3A, CDK5R1, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, ATP2B2, DAB2, BDNF, SLC1A3, LAMB2, CXCR4, ANK3, LAMA5, FOXG1, CNTN4, ROBO3, CACNA1F, FN1 |
GO:0000902-cell morphogenesis | 27 | 2.53E-06 | 1.48E-03 | RAB3A, CDK5R1, ADORA2A, EPHB2, ARX, ATP2B2, BDNF, DAB2, SLC1A3, LAMB2, ANK3, CXCR4, DMD, MKKS, ROBO3, FN1, NTNG1, MARK2, CTNNA2, PTPN11, NUMBL, SLITRK1, LAMA5, FOXG1, CNTN4, CACNA1F, CDC42BPB |
GO:0032989-cellular component morphogenesis | 28 | 6.15E-06 | 2.88E-03 | RAB3A, CDK5R1, ADORA2A, EPHB2, ARX, ATP2B2, DAB2, BDNF, SLC1A3, LAMB2, ANK3, CXCR4, DMD, OBSL1, MKKS, ROBO3, FN1, NTNG1, MARK2, CTNNA2, PTPN11, NUMBL, SLITRK1, LAMA5, FOXG1, CNTN4, CACNA1F, CDC42BPB |
GO:0048858-cell projection morphogenesis | 21 | 7.30E-06 | 2.14E-03 | RAB3A, CDK5R1, ADORA2A, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, BDNF, LAMB2, CXCR4, ANK3, LAMA5, DMD, FOXG1, MKKS, CNTN4, ROBO3, CACNA1F |
GO:0048667-cell morphogenesis involved in neuron differentiation | 19 | 1.01E-05 | 2.63E-03 | RAB3A, CDK5R1, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, ATP2B2, BDNF, SLC1A3, LAMB2, CXCR4, ANK3, FOXG1, CNTN4, ROBO3, CACNA1F |
GO:0048812-neuron projection morphogenesis | 19 | 1.31E-05 | 3.07E-03 | RAB3A, CDK5R1, ADORA2A, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, BDNF, LAMB2, CXCR4, ANK3, DMD, FOXG1, CNTN4, ROBO3, CACNA1F |
GO:0030030-cell projection organization | 26 | 1.39E-05 | 2.96E-03 | MTSS1, RAB3A, CDK5R1, DNAH9, ADORA2A, EPHB2, ARX, ATP2B2, BDNF, LAMB2, CD44, ANK3, CXCR4, DMD, MKKS, ROBO3, FGD3, NTNG1, CTNNA2, PTPN11, NUMBL, SLITRK1, LAMA5, FOXG1, CNTN4, CACNA1F |
GO:0032990-cell part morphogenesis | 21 | 1.40E-05 | 2.73E-03 | RAB3A, CDK5R1, ADORA2A, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, BDNF, LAMB2, CXCR4, ANK3, LAMA5, DMD, FOXG1, MKKS, CNTN4, ROBO3, CACNA1F |
GO:0031175-neuron projection development | 20 | 4.65E-05 | 6.80E-03 | RAB3A, CDK5R1, ADORA2A, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, BDNF, LAMB2, CD44, CXCR4, ANK3, DMD, FOXG1, CNTN4, ROBO3, CACNA1F |
GO:0007409-axonogenesis | 17 | 4.98E-05 | 6.86E-03 | RAB3A, CDK5R1, NTNG1, PTPN11, CTNNA2, EPHB2, NUMBL, ARX, SLITRK1, BDNF, LAMB2, CXCR4, ANK3, FOXG1, CNTN4, ROBO3, CACNA1F |
GO:0007411-axon guidance | 9 | 7.12E-03 | 2.67E-01 | ARX, CDK5R1, BDNF, CXCR4, ANK3, FOXG1, CNTN4, ROBO3, EPHB2 |
Biological functions, disorders, and pathways | P value | Number of molecules |
---|---|---|
Physiological system development and function | ||
Organismal survival | 3.56E-08 to 6.82E-04 | 305 |
Nervous system development and function | 5.83E-08 to 4.28E-03 | 344 |
Tissue morphology | 5.83E-08 to 4.72E-03 | 419 |
Cardiovascular system development and function | 8.89E-08 to 4.72E-03 | 268 |
Embryonic development | 9.01E-08 to 4.72E-03 | 370 |
Diseases and disorders
| ||
Hereditary disorder | 8.09E-06 to 1.56E-03 | 204 |
Neurological disease | 8.09E-06 to 4.72E-03 | 292 |
Psychological disorders | 8.09E-06 to 8.09E-06 | 107 |
Cancer | 1.65E-05 to 4.77E-03 | 731 |
Hematological disease | 1.65E-05 to 4.67E-03 | 166 |
Top canonical pathways
|
Ratio
| |
Superpathway of inositol phosphate compounds | 4.97E-04 | 37/227 |
Production of nitric oxide and reactive oxygen species in macrophages | 1.29E-03 | 35/210 |
Axonal guidance signaling | 2.47E-03 | 69/468 |
3-phosphoinositide biosynthesis | 2.52E-03 | 29/175 |
Small cell lung cancer signaling | 2.60E-03 | 17/89 |
Function annotation (number of molecules) | P value |
---|---|
Nervous system development and function | |
Quantity of neurons (67) | 5.83E-08 |
Development of brain (102) | 9.01E-08 |
Morphology of nervous system (161) | 3.96E-07 |
Morphology of nervous tissue (118) | 5.44E-07 |
Development of central nervous system (122) | 1.50E-06 |
Axonogenesis (43) | 1.68E-05 |
Morphology of nerves (30) | 7.32E-05 |
Cell-cell adhesion of neurons (6) | 1.48E-04 |
Morphology of central nervous system (98) | 1.90E-04 |
Morphology of rhombencephalon (35) | 2.41E-04 |
Quantity of sensory neurons (19) | 2.81E-04 |
Development of nerves (19) | 2.81E-04 |
Abnormal morphology of neurons (51) | 2.85E-04 |
Morphology of neurons (55) | 3.37E-04 |
Antinociception (13) | 3.84E-04 |
Development of neurons (37) | 3.98E-04 |
Morphology of brain (88) | 4.40E-04 |
Development of interneurons (7) | 4.56E-04 |
Migration of neurons (46) | 4.79E-04 |
Abnormal morphology of cerebellum (21) | 5.58E-04 |
Development of metencephalon (24) | 5.88E-04 |
Loss of neurons (29) | 8.01E-04 |
Long-term potentiation of granule cells (4) | 8.18E-04 |
Abnormal morphology of cranial nerve (19) | 8.60E-04 |
Neuritogenesis (83) | 1.11E-03 |
Development of cerebellum (23) | 1.22E-03 |
Development of forebrain (45) | 1.26E-03 |
Fasciculation of nervous tissue (9) | 1.34E-03 |
Abnormal morphology of granule cells (12) | 1.42E-03 |
Development of globus pallidus (3) | 1.56E-03 |
Activation of hippocampus (3) | 1.56E-03 |
Abnormal morphology of hair cells (9) | 1.84E-03 |
Morphogenesis of neurites (58) | 1.85E-03 |
Adhesion of neuronal cells (14) | 1.88E-03 |
Abnormal morphology of outer hair cells (6) | 2.01E-03 |
Sensory system development (23) | 2.09E-03 |
Development of rhombencephalon (28) | 2.15E-03 |
Chemotaxis of granule cells (4) | 2.23E-03 |
Guidance of axons (31) | 2.24E-03 |
Morphology of mechanosensory neurons (10) | 2.49E-03 |
Cell viability of neurons (39) | 2.51E-03 |
Analgesia (18) | 2.62E-03 |
Synaptic transmission of cerebral cortex cells (11) | 3.03E-03 |
Memory (39) | 3.52E-03 |
Neurotransmission (71) | 3.75E-03 |
Development of cranial nerve (11) | 3.79E-03 |
Cell viability of granule cells (8) | 4.28E-03 |
Neurological disease
| |
Schizophrenia (107) | 8.09E-06 |
Chorea (117) | 3.23E-04 |
Huntington's Disease (116) | 4.09E-04 |
Movement Disorders (183) | 9.11E-04 |
Dyskinesia (118) | 1.27E-03 |
Neurological signs (120) | 1.45E-03 |
Familial transthyretin amyloidosis (3) | 1.56E-03 |
Seizure disorder (60) | 3.92E-03 |
Disorder of basal ganglia (135) | 4.31E-03 |
Polymicrogyria (4) | 4.72E-03 |
Behavior
| |
Behavior (180) | 2.74E-07 |
Cognition (79) | 1.70E-05 |
Learning (74) | 3.41E-05 |
Circling behavior (12) | 1.11E-03 |
Emotional behavior (42) | 3.04E-03 |
Memory (39) | 3.52E-03 |
Spatial learning (33) | 4.23E-03 |
Autism gene database | Total number of genes in database | Total promoter regions on array | Total IP RORA-binding promoter regions | RORA-binding autism genes (overlap) | P value |
---|---|---|---|---|---|
AutDB (SFARI gene) | 328 | 25,500 | 2,544 | 49 | 0.0028 |
AutismKB (syndromic + non-syndromic) | 3,050 | 25,500 | 2,544 | 426 | <0.001 |
AutDB + AutismKB | 3,158 | 25,500 | 2,544 | 438 | <0.001 |
Network prediction of selected transcriptional targets to assess relevance to autism spectrum disorder (ASD)
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 |
Confirmation of RORA binding to selected transcriptional targets in human neuronal cells
RORA suppression reduces expression of selected transcriptional targets in human neuronal cells
Expression of transcriptional targets of RORA is relatively reduced in the frontal cortex of individuals with autism in comparison with age-matched controls
Sample comparison (A vs. C) | Age (A, C) | RORA | CYP19A1 | ITPR1 | A2BP1 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Control | Autistic | A/C | Control | Autistic | A/C | Control | Autistic | A/C | Control | Autistic | A/C | ||
5144A vs. 5251C | 20, 19 | 6.4 | 2.0 | 0.316 | 1.7E-04 | 5.2E-05 | 0.307 | 127.7 | 70.8 | 0.554 | 553.1 | 105.3 | 0.190 |
5173A vs. 5873C | 30, 28 | 38.4 | 25.4 | 0.661 | 1.2E-02 | 5.9E-03 | 0.487 | 246.4 | 27.3 | 0.111 | 830.1 | 74.2 | 0.089 |
6337A vs. 5718C | 22, 22 | 7.9 | 0.4 | 0.046 | 7.7E-06 | 1.0E-07 | 0.013 | 217.4 | 176.3 | 0.811 | 246.0 | 64.6 | 0.263 |
Average ratio (A/C) | 17.6 | 9.3 |
0.527
| 4.1E-03 | 2.0E-03 |
0.484
| 197.2 | 91.5 |
0.464
| 543.1 | 81.4 |
0.150
| |
P value (unpaired t test) | 0.562 | 0.660 | 0.137 | 0.052 | |||||||||
HSD17B10
|
NLGN1
|
NTRK2
| |||||||||||
Control
|
Autistic
|
A/C
|
Control
|
Autistic
|
A/C
|
Control
|
Autistic
|
A/C
| |||||
5144A vs. 5251C | 20, 19 | 128.7 | 15.7 | 0.122 | 178.3 | 23.8 | 0.133 | 3218.9 | 544.6 | 0.169 | |||
5173A vs. 5873C | 30, 28 | 126.3 | 41.1 | 0.325 | 255.3 | 44.9 | 0.176 | 2600.1 | 1077.2 | 0.414 | |||
6337A vs. 5718C | 22, 22 | 47.6 | 39.2 | 0.824 | 84.8 | 61.5 | 0.725 | 929.8 | 1265.1 | 1.361 | |||
Average ratio (A/C) | 100.9 | 32.0 |
0.317
| 172.8 | 43.4 |
0.251
| 2249.6 | 962.3 |
0.428
| ||||
P value (unpaired t test) | 0.069 | 0.062 | 0.147 |