High-Throughput Analysis of Promoter Occupancy Reveals Direct Neural Targets of FOXP2, a Gene Mutated in Speech and Language Disorders
2007; Elsevier BV; Volume: 81; Issue: 6 Linguagem: Inglês
10.1086/522238
ISSN1537-6605
AutoresSonja C. Vernes, Elizabeth Spiteri, Jérôme Nicod, Matthias Groszer, Jennifer M. Taylor, Kay E. Davies, Daniel H. Geschwind, Simon E. Fisher,
Tópico(s)Language Development and Disorders
ResumoWe previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders. We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders. Neurodevelopmental disorders that disrupt language acquisition tend to be complex at the genetic level, potentially involving a large number of different susceptibility loci, such that identification of the relevant molecular pathways remains challenging.1Fisher SE Lai CS Monaco AP Deciphering the genetic basis of speech and language disorders.Annu Rev Neurosci. 2003; 26: 57-80Crossref PubMed Scopus (116) Google Scholar, 2Wassink TH Brzustowicz LM Bartlett CW Szatmari P The search for autism disease genes.Ment Retard Dev Disabil Res Rev. 2004; 10: 272-283Crossref PubMed Scopus (79) Google Scholar In earlier studies, we discovered that heterozygous mutations of the human FOXP2 gene (MIM 605317) are responsible for a rare monogenic communication disorder, primarily characterized by difficulties in learning to make the coordinated sequences of articulatory gestures that underlie speech (developmental verbal dyspraxia [MIM 602081]).3Lai CS Fisher SE Hurst JA Vargha-Khadem F Monaco AP A forkhead-domain gene is mutated in a severe speech and language disorder.Nature. 2001; 413: 519-523Crossref PubMed Scopus (1309) Google Scholar, 4MacDermot KD Bonora E Sykes N Coupe AM Lai CS Vernes SC Vargha-Khadem F McKenzie F Smith RL Monaco AP et al.Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits.Am J Hum Genet. 2005; 76: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar The disorder also involves deficits in many aspects of linguistic processing, affecting both oral and written abilities, across expressive and receptive domains.5Watkins KE Dronkers NF Vargha-Khadem F Behavioural analysis of an inherited speech and language disorder: comparison with acquired aphasia.Brain. 2002; 125: 452-464Crossref PubMed Scopus (262) Google Scholar To date, speech and language impairments have been documented in two different multigenerational families segregating missense and nonsense point mutations of FOXP2,3Lai CS Fisher SE Hurst JA Vargha-Khadem F Monaco AP A forkhead-domain gene is mutated in a severe speech and language disorder.Nature. 2001; 413: 519-523Crossref PubMed Scopus (1309) Google Scholar, 4MacDermot KD Bonora E Sykes N Coupe AM Lai CS Vernes SC Vargha-Khadem F McKenzie F Smith RL Monaco AP et al.Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits.Am J Hum Genet. 2005; 76: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar as well as in several cases of gross chromosomal rearrangements (translocations and deletions) that disturb the integrity of the FOXP2 genomic locus in 7q31.3Lai CS Fisher SE Hurst JA Vargha-Khadem F Monaco AP A forkhead-domain gene is mutated in a severe speech and language disorder.Nature. 2001; 413: 519-523Crossref PubMed Scopus (1309) Google Scholar, 6Feuk L Kalervo A Lipsanen-Nyman M Skaug J Nakabayashi K Finucane B Hartung D Innes M Kerem B Nowaczyk MJ et al.Absence of a paternally inherited FOXP2 gene in developmental verbal dyspraxia.Am J Hum Genet. 2006; 79: 965-972Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Shriberg LD Ballard KJ Tomblin JB Duffy JR Odell KH Williams CA Speech, prosody, and voice characteristics of a mother and daughter with a 7;13 translocation affecting FOXP2.J Speech Lang Hear Res. 2006; 49: 500-525Crossref PubMed Scopus (114) Google Scholar, 8Zeesman S Nowaczyk MJ Teshima I Roberts W Cardy JO Brian J Senman L Feuk L Osborne LR Scherer SW Speech and language impairment and oromotor dyspraxia due to deletion of 7q31 that involves FOXP2.Am J Med Genet A. 2006; 140: 509-514Crossref PubMed Scopus (98) Google Scholar People who are affected with Silver-Russell syndrome (MIM 180860), associated with uniparental disomy of the maternal copy of chromosome 7, can also display verbal dyspraxia, which appears to relate to reductions in FOXP2 expression.6Feuk L Kalervo A Lipsanen-Nyman M Skaug J Nakabayashi K Finucane B Hartung D Innes M Kerem B Nowaczyk MJ et al.Absence of a paternally inherited FOXP2 gene in developmental verbal dyspraxia.Am J Hum Genet. 2006; 79: 965-972Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar FOXP2 encodes a regulatory protein belonging to the forkhead-box (FOX) group of transcription factors.3Lai CS Fisher SE Hurst JA Vargha-Khadem F Monaco AP A forkhead-domain gene is mutated in a severe speech and language disorder.Nature. 2001; 413: 519-523Crossref PubMed Scopus (1309) Google Scholar Members of this class of protein share a distinctive type of DNA-binding motif, the FOX domain, and are prominent regulators of eukaryotic gene expression, associated with a wide variety of cellular and developmental processes.9Carlsson P Mahlapuu M Forkhead transcription factors: key players in development and metabolism.Dev Biol. 2002; 250: 1-23Crossref PubMed Scopus (662) Google Scholar FOX gene dysfunction has been implicated in a range of disease states, including developmental eye disorders, ovarian failure, immune deficiency, and carcinogenesis.10Lehmann OJ Sowden JC Carlsson P Jordan T Bhattacharya SS Fox’s in development and disease.Trends Genet. 2003; 19: 339-344Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 11Katoh M Katoh M Human FOX gene family (review).Int J Oncol. 2004; 25: 1495-1500PubMed Google Scholar Several FOX transcription factors are key players in CNS development; for example, Foxg1 regulates proliferation and differentiation of progenitor cells of the telencephalon,12Hanashima C Li SC Shen L Lai E Fishell G Foxg1 suppresses early cortical cell fate.Science. 2004; 303: 56-59Crossref PubMed Scopus (280) Google Scholar whereas Foxb1 is critical for normal development of diencephalon and midbrain.13Wehr R Mansouri A de Maeyer T Gruss P Fkh5-deficient mice show dysgenesis in the caudal midbrain and hypothalamic mammillary body.Development. 1997; 124: 4447-4456Crossref PubMed Google Scholar FOXP2 itself belongs to a functionally divergent subgroup of the FOX proteins, characterized by a shorter DNA-binding domain and the presence of other defining motifs, including glutamine-rich stretches, dimerization domains, and an acidic C-terminus.14Wang B Lin D Li C Tucker P Multiple domains define the expression and regulatory properties of Foxp1 forkhead transcriptional repressors.J Biol Chem. 2003; 278: 24259-24268Crossref PubMed Scopus (168) Google Scholar Much of our current knowledge of the neural correlates of FOXP2 disruption comes from intensive phenotypic studies of a single human family (the “KE” family) in which 15 people have verbal dyspraxia due to a missense mutation in the FOX domain.3Lai CS Fisher SE Hurst JA Vargha-Khadem F Monaco AP A forkhead-domain gene is mutated in a severe speech and language disorder.Nature. 2001; 413: 519-523Crossref PubMed Scopus (1309) Google Scholar The mutation in this family is associated with bilateral abnormalities in gray-matter density, including significant decreases in the inferior frontal gyrus (including Broca’s area), caudate nucleus, and cerebellum and increases in the posterior temporal gyrus (including Wernicke’s area), angular gyrus, and putamen,15Watkins KE Vargha-Khadem F Ashburner J Passingham RE Connelly A Friston KJ Frackowiak RS Mishkin M Gadian DG MRI analysis of an inherited speech and language disorder: structural brain abnormalities.Brain. 2002; 125: 465-478Crossref PubMed Scopus (257) Google Scholar as well as altered patterns of neural activity during linguistic processing.16Liegeois F Baldeweg T Connelly A Gadian DG Mishkin M Vargha-Khadem F Language fMRI abnormalities associated with FOXP2 gene mutation.Nat Neurosci. 2003; 6: 1230-1237Crossref PubMed Scopus (250) Google Scholar Intriguingly, the neural sites of structural and/or functional abnormalities in the KE family are concordant with regions of high FOXP2 expression in the developing human brain.17Lai CS Gerrelli D Monaco AP Fisher SE Copp AJ FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder.Brain. 2003; 126: 2455-2462Crossref PubMed Scopus (266) Google Scholar We recently used human cell lines to demonstrate that the KE family's missense mutation and a nonsense mutation causing verbal dyspraxia in a second multiplex family4MacDermot KD Bonora E Sykes N Coupe AM Lai CS Vernes SC Vargha-Khadem F McKenzie F Smith RL Monaco AP et al.Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits.Am J Hum Genet. 2005; 76: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 18Vernes SC Nicod J Elahi FM Coventry JA Kenny N Coupe AM Bird LE Davies KE Fisher SE Functional genetic analysis of mutations implicated in a human speech and language disorder.Hum Mol Genet. 2006; 15: 3154-3167Crossref PubMed Scopus (123) Google Scholar dramatically interfered with transcription factor function.18Vernes SC Nicod J Elahi FM Coventry JA Kenny N Coupe AM Bird LE Davies KE Fisher SE Functional genetic analysis of mutations implicated in a human speech and language disorder.Hum Mol Genet. 2006; 15: 3154-3167Crossref PubMed Scopus (123) Google Scholar Overall, the combined findings from phenotypic evaluation, neuroimaging studies, expression analyses, and functional genetic assays suggest that a reduced dosage of functional FOXP2 has an impact on the development and function of a subset of distributed neural circuits, including those important for speech and language acquisition. Thus, the FOXP2 gene provides a unique molecular window into the neural basis of human communication.19Fisher SE Marcus GF The eloquent ape: genes, brains and the evolution of language.Nat Rev Genetics. 2006; 7: 9-20Crossref PubMed Scopus (144) Google Scholar In particular, its role as a transcription factor, modulating the expression of target genes, offers elegant functional genomic routes for dissecting the associated neurogenetic pathways. However, at present, there are no neural targets of FOXP2 reported in the literature. The aim of the present study was to discover downstream targets directly regulated by FOXP2 in neurons, by exploiting emerging strategies based on the chromatin-immunoprecipitation (ChIP) method. This is a powerful technique for studying protein-DNA interactions inside the nucleus under physiological conditions,20Kim TH Ren B Genome-wide analysis of protein-DNA interactions.Annu Rev Genomics Hum Genet. 2006; 7: 81-102Crossref PubMed Scopus (127) Google Scholar allowing characterization of genomic sites bound by a protein of interest in the native chromatin of living cells. Here, we develop FOXP2 ChIP, couple it to high-throughput screening of microarrays (ChIP-chip), and identify occupied promoters in native chromatin of human neuron-like cells. We focus on a subset of targets uncovered via this approach, demonstrating that altered FOXP2 levels yield significant changes in their expression and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Finally, we identify significant quantitative differences in target expression in the embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work, along with that of Spiteri et al.,21Spiteri E Konopka G Coppola G Bomar J Oldham M Ou J Vernes SC Fisher SE Ren B Geschwind DH Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.Am J Hum Genet. 2007; 81 (in this issue): 1144-1157Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar(in this issue) represents the first identification and validation of neural targets regulated by FOXP2 and suggests roles for this gene in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance. SH-SY5Y cells were grown in Dulbecco's modified Eagle medium (DMEM):F12 media (Sigma), and HEK293T cells in DMEM media (Sigma). Media was supplemented with 10% fetal calf serum (Sigma), 2 mM l-glutamine (Sigma), and 2 mM penicillin/streptomycin (Sigma). Cells were grown at 37°C in the presence of 5% CO2. Stable SH-SY5Y cell lines overexpressing FOXP2 or nonexpressing controls were generated by transfection with pcDNA3.1/FOXP2 (isoform I–untagged) or the empty vector, respectively, by use of Lipofectamine Plus (Invitrogen) in accordance with the manufacturer's instructions. Cells were cultured in complete medium supplemented with 500 βg/ml G418 (Calbiochem) as a selective agent. Resistant single colonies were isolated 20 d after transfection and then were cultured and expanded independently in the presence of G418 (500 βg/ml). Quantitative RT-PCR (qRT-PCR) (see “qRT-PCR” section) and western blotting (performed as described elsewhere18Vernes SC Nicod J Elahi FM Coventry JA Kenny N Coupe AM Bird LE Davies KE Fisher SE Functional genetic analysis of mutations implicated in a human speech and language disorder.Hum Mol Genet. 2006; 15: 3154-3167Crossref PubMed Scopus (123) Google Scholar) confirmed expression of recombinant FOXP2. A clone with a high and consistent level of expression was chosen for use in further experiments. Transient transfections of SH-SY5Y or HEK293T cells were performed using Transfast (Promega) or GeneJuice (Novagen) transfection reagents, respectively, in accordance with the manufacturer's instructions and were harvested 48 h after transfection. FOXP2 detection was performed using N-terminal (Santa Cruz Biotechnology) or C-terminal (Serotec) goat polyclonal antibodies.18Vernes SC Nicod J Elahi FM Coventry JA Kenny N Coupe AM Bird LE Davies KE Fisher SE Functional genetic analysis of mutations implicated in a human speech and language disorder.Hum Mol Genet. 2006; 15: 3154-3167Crossref PubMed Scopus (123) Google Scholar SH-SY5Y cells stably expressing FOXP2 isoform I were cross-linked using 1% formaldehyde in cross-linking buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, and 0.5 mM ethylene glycol tetraacetic acid [EGTA]) at room temperature. Cells were incubated for 10 min in ice-cold ChIP lysis buffer (10 mM Tris, 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, and protease inhibitors) and were centrifuged at 10,000 g at 4°C for 5 min to pellet nuclei. Nuclei from 3×107 cells were resuspended in 1 ml Sonication Buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitors) with 0.1 g of 212–300-βm glass beads (Sigma) before undergoing 10 rounds of 20-s sonication pulses at 65% power, with 2 min on ice between each round (with use of Bandelin SONOPULS HD2070 Ultrasonic Homogenisor and MS72 2-mm titanium tip with 200-βm SS amplitude). Cells were centrifuged at 10,000 g at 4°C for 5 min to remove glass beads and cell debris. Then, 1 βg of FOXP2 N-terminal antibody (Santa Cruz Biotechnology) was incubated with the sonicated supernatants in IP buffer (0.1 M Tris, 10 mM EDTA, 150 mM NaCl, 0.2 % Triton X-100, 1% phenylmethylsulfonyl fluoride, and protease inhibitors), rotating overnight at 4°C. Immune complexes were captured by addition of 5 βg sonicated salmon sperm DNA and 50 βl Protein G–sepharose beads, incubated for 3 h at 4°C. Protein was eluted from beads first by 1.5% SDS buffer (1.5% SDS, 1× TE [pH 7.5], and 30 mM NaCl) and then by 0.5% SDS buffer (0.5% SDS, 1× TE [pH 7.5], and 30 mM NaCl), with incubation of the beads with each in turn at room temperature for 15 min. Pooled supernatants were incubated at 65°C overnight to reverse cross-links. DNA was isolated via phenol-chloroform extraction followed by ethanol precipitation. Concentration and purity of the DNA was evaluated by spectrophotometry, and size was assessed via gel electrophoresis. Protein samples were extracted in parallel via precipitation with use of trichloroacetic acid (Sigma), and western blotting was used to confirm immunoprecipitation of the FOXP2 protein. In vivo Foxp2 ChIP with use of embryonic brains from wild-type or homozygous mutant mice was performed according to the protocol described by Spiteri et al.21Spiteri E Konopka G Coppola G Bomar J Oldham M Ou J Vernes SC Fisher SE Ren B Geschwind DH Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.Am J Hum Genet. 2007; 81 (in this issue): 1144-1157Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar(in this issue) In each case, whole-brain tissue at embryonic day 16 (E16) was pooled from six mice. Mutant mice carry an early nonsense mutation in Foxp2, which leads to both nonsense-mediated RNA decay and protein instability, resulting in an absence of detectable Foxp2 protein, as confirmed using both N- and C-terminal antibodies (M.G., J.N., and S.E.F., unpublished data). Wild-type and mutant mice were littermates, to maximize the homogeneity of the genomic background. Despite a lack of Foxp2 protein, homozygous mutants show no gross anomalies in anatomy or brain development during embryogenesis. Postnatally, they display developmental delays and reduced cerebellar growth, dying ∼3–4 wk after birth for as-yet unknown reasons (M.G., J.N., and S.E.F., unpublished data). All animal studies were performed conforming to the regulatory standards of the U.K. Home Office, under Project Licence 30/2016. Purified chromatin was amplified via ligation-mediated PCR in accordance with published protocols.22Oberley MJ Farnham PJ Probing chromatin immunoprecipitates with CpG-island microarrays to identify genomic sites occupied by DNA-binding proteins.Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar Size and purity of DNA was assessed via spectrophotometry and gel electrophoresis. Two hundred nanograms of amplified immunoprecipitated chromatin or total input DNA was fluorescently labeled with Cy5 or Cy3, respectively, by use of random primers provided in the BioPrime DNA labeling system (Invitrogen), in accordance with the manufacturer's instructions. The labeling reaction was allowed to proceed for 16 h at 37°C, before purification by sodium-acetate precipitation. A total of 2 βg of labeled DNA was hybridized to high-density human promoter arrays (Aviva Biosystems).23Li Z Van Calcar S Qu C Cavenee WK Zhang MQ Ren B A global transcriptional regulatory role for c-Myc in Burkitt’s lymphoma cells.Proc Natl Acad Sci USA. 2003; 100: 8164-8169Crossref PubMed Scopus (396) Google Scholar Three biological replicates were performed. Array images were scanned using the Axon GenePix 4000B. Data were retrieved, and initial quality control was performed using the GenePix Pro 6.0 software package (Molecular Devices). Microarray data analysis was performed using the Limma package for R.24Smyth GK Speed T Normalization of cDNA microarray data.Methods. 2003; 31: 265-273Crossref PubMed Scopus (1436) Google Scholar, 25Smyth GK Linear models and empirical Bayes methods for assessing differential expression in microarray experiments.Stat Appl Genet Mol Biol. 2004; 3: Article3Crossref PubMed Scopus (8463) Google Scholar Print-tip loess normalization and background correction was performed within each array. Data were normalized between arrays by use of quantile normalization, and the median value was calculated from triplicate experiments for each probe, for use in further analyses. Probes that displayed statistically significant differences of abundance (P<.05) were ranked according to both fold change and P value. The P values were adjusted for multiple testing by use of the false-discovery–rate method in the p.adjust package in R.26Benjamini Y Hochberg Y Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc Ser B. 1995; 57: 289-300Google Scholar All microarray data can be found in the tab-delimited ASCII files of, 2 , 3 , 4 , and 5 . The GOTree Machine (GOTM),27Zhang B Schmoyer D Kirov S Snoddy J GOTree Machine (GOTM): a Web-based platform for interpreting sets of interesting genes using gene ontology hierarchies.BMC Bioinformatics. 2004; 5: 16Crossref PubMed Scopus (362) Google Scholar part of WebGestalt (Web-based gene set analysis toolkit), was used to visualize gene-function relationships. This program queries the Genekey database incorporating the Locuslink, Ensembl, Swiss-Prot, HomoloGene, Unigene, Gene Ontology Consortium, and Affymetrix databases. Statistical significance of overrepresentation in the target gene list of 303 genes was calculated using the entire probe set as a reference data set, via a hypergeometric test, where significance is defined as P<.05. Functional annotation was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).28Dennis Jr, G Sherman BT Hosack DA Yang J Gao W Lane HC Lempicki RA DAVID: database for annotation, visualization, and integrated discovery.Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar Ingenuity pathway analysis software was used to identify interactions between target genes (Ingenuity Systems). All 303 enriched genes with a P value <.05 (table 1) were included in this analysis, and both direct and indirect interactions were considered. The full set of genes from the array was used as a reference data set.Table 1Genes Displaying Significant Enrichment (P<.05) in FOXP2 High-Throughput Location AnalysisGeneGenBank Accession NumberPABCG2NM_004827.027ACSL5NM_016234.0063ACSS2NM_018677.017ADAM28NM_021777.022ADMRNM_007264.036AKAP6NM_004274.031ALDOANM_000034.0015ARL1NM_001177.026ARL4ANM_005738.050ATF6NM_007348.0075ATP1A2NM_000702.040ATP1B4NM_012069.044BAIAP3NM_003933.019BUD31NM_003910.025C13orf24NM_006346.021C1orf38NM_004848.032C20orf24NM_018840.037CABP1NM_031205.037CACNG3NM_006539.0053CALCRLNM_005795.015CCKNM_000729.045CCKARNM_000730.035CCL19NM_006274.0088CCNKNM_003858.012CD164NM_006016.014CD180NM_005582.0042CD5NM_014207.015CDH5NM_001795.0025CER1NM_005454.0019CGBNM_033142.029CHST11NM_018413.035CILPNM_003613.026CLEC10ANM_182906.039CLNS1ANM_001293.035CLPXNM_006660.049CNBPNM_003418.041COL8A1NM_001850.0060COL9A1NM_001851.047COPS5NM_006837.048COX11NM_004375.000099CRISP3NM_006061.012CRYBA4NM_001886.027CXCL2NM_002089.011CYB5BNM_030579.032DCTN2NM_006400.010DGAT1NM_012079.0028DLG4NM_001365.027DLL3NM_016941.025DPAGT1NM_001382.034DUSP12NM_007240.020DYRK1BNM_004714.0062EBI2NM_004951.00062EBPNM_006579.016EIF3S10NM_003750.0019ELL2NM_012081.022ENTPD7NM_020354.042EPHX2NM_001979.013EPORNM_000121.012ERO1LNM_014584.000051F8NM_019863.046FADDNM_003824.014FBN1NM_000138.0051FLT1NM_002019.00037FMO4NM_002022.0038FOLR1NM_016725.038FRYNM_023037.015FTH1NM_002032.010FTSJ2NM_013393.0055FUT2NM_000511.023GABBR1NM_001470.044GAS7NM_005890.048GBASNM_001483.041GDF5NM_000557.036GENX-3414NM_003943.032GGHNM_003878.049GNAZNM_002073.0045GPR160NM_014373.031GPR17NM_005291.047GPR75NM_006794.019GRHPRNM_012203.038GUCA1BNM_002098.049HAPLN1NM_001884.0028HAS1NM_001523.0019HATNM_004262.014HIST1H2AGNM_021064.021HNRPKNM_002140.031HOXB6NM_156036.0023HRSP12NM_005836.0000062HSPA2NM_021979.018HSPB7NM_014424.0022HTRA2NM_012103.050HUWE1NM_031407.042HYAL2NM_003773.012IFI30NM_006332.00024IGLL1NM_020070.017IL18NM_001562.034IL1BNM_000576.042IL4RNM_000418.035ISLRNM_005545.010ITPK1NM_014216.0071KCNB1NM_004975.041KCNE1LNM_012282.0079Kifap3NM_014970.047KLK8NM_007196.035KRT17NM_000422.031LBRNM_002296.0036LDHANM_005566.013LECT1NM_007015.0054LENEPNM_018655.0085LILRA2NM_006866.043LILRB5NM_006840.043LILRP2NR_003061.017LIM2NM_030657.044LNPEPNM_005575.00012LTBNM_002341.012LTFNM_002343.020LY6G6ENM_024123.035LYPLA1NM_006330.033MAD2L2NM_006341.030MAEANM_005882.021MAPK14NM_001315.046MAPK7NM_139032.0042MAPK8IP1NM_005456.0021MAPRE3NM_012326.036MARK2NM_004954.019MDFINM_005586.023MESTNM_002402.019MFGE8NM_005928.045MORF4L2NM_012286.027MOSNM_005372.044MPONM_000250.026MPP3NM_001932.034MPV17NM_002437.043MYOTNM_006790.0057NCOR2NM_006312.0013NDUFA2NM_002488.046NDUFA8NM_014222.030NEDD8NM_006156.039NEU2NM_005383.020NEUROD2NM_006160.010NEUROG1NM_006161.018NRTNNM_004558.045NRXN3NM_004796.016NUDT1NM_002452.011NXF1NM_006362.019OPN1SWNM_001708.0046ORC6LNM_014321.025OXR1NM_018002.037P115NM_003715.043PAMNM_000919.0084PAX1NM_006192.0013PAX3NM_000438.0084PCCANM_000282.015PCSK1NM_000439.029PCSK6NM_002570.013PCYT1BNM_004845.042PDCD8NM_004208.026PDE1CNM_005020.030PDE4CNM_000923.045PDE6BNM_000283.012PEX1NM_000466.013PEX16NM_057174.034PIGCNM_153747.014PKP1NM_000299.036PLA2G4BNM_005090.046PLA2R1NM_007366.049PLAURNM_002659.012PM5NM_014287.018PMF1NM_007221.024PNKPNM_007254.030POLSNM_006999.017POU4F3NM_002700.032PPP2R3ANM_002718.0098PPP2R5DNM_006245.037PRKAG3NM_017431.046PRSCNM_006587.031PRSS12NM_003619.023PRSS22NM_022119.018PRSS8NM_002773.037PSEN2NM_000447.013PSMA3NM_002788.017PSMD1NM_002807.018PTCH2NM_003738.026PTGER1NM_000955.0026PTGER3NM_000957.021PTK9NM_198974.0091PYCR1NM_153824.012RAB18NM_021252.013RAB27ANM_004580.035RAB5CNM_004583.021RABGGTANM_004581.016RAD51AP1NM_006479.014RAI1NM_030665.015RALANM_005402.0031RALBP1NM_006788.0015RARBNM_000965.040RBM4NM_002896.039RBP2NM_004164.046RCN2NM_002902.0051RECQL5NM_004259.033RFPL3NM_006604.010RGNNM_004683.041RGS2NM_002923.047ROR2NM_004560.033RPL10NM_006013.023RPL22NM_000983.0040RPL28NM_000991.0098RQCD1NM_005444.035RRAGBNM_016656.048RRP9NM_004704.011RYR3NM_001036.021S100A3NM_002960.028SAS10NM_020368.050SCGB2A2NM_002411.030SCRG1NM_007281.028SCRN1NM_014766.033SELENBP1NM_003944.030SEMA3BNM_004636.0071SFRP4NM_003014.027SFRS11NM_004768.047SIRT3NM_012239.0053SLBPNM_006527.032SLC17A3NM_006632.0093SLC20A1NM_005415.0047SLC22A14NM_004803.0099SLC22A3NM_021977.042SLC25A3NM_002635.0041SLC2A4NM_001042.0058SLC4A4NM_003759.036SLC4A8NM_001039960.0028SLC5A1NM_000343.025SLC5A6NM_021095.026SLIT1NM_003061.025SMAD3NM_005902.020SMARCB1NM_003073.0042SMC2NM_006444.0022SMCXNM_004187.044SMSNM_004595.038SNFTNM_018664.00025SNRPGNM_003096.020SNW1NM_012245.016SOCS1NM_003745.050SOD3NM_003102.00046SORBS3NM_005775.0047SOX15NM_006942.023SOX21NM_007084.031SPEGXM_001131579.0024SPOCK1NM_004598.011SPOCK3NM_016950.018SRD5A2NM_000348.029SRPK1NM_003137.018SSX2IPNM_014021.012ST3GAL5NM_003896.012STC1NM_003155.0098STC2NM_003714.042SYKNM_003177.0022SYNJ1NM_203446.031TACSTD2NM_002353.020TAGLNNM_003186.0034TCEB1NM_005648.020TCF12NM_207037.0020TDO2NM_005651.0046TGNM_003235.0088TGM2NM_004613.014THBS1NM_003246.041THPONM_000460.028TIAF1NM_004740.033TIAM1NM_003253.0013TITF1NM_003317.024TJP2NM_004817.026TLL2NM_012465.0015TMEM4NM_014255.043TNFRSF14NM_003820.032TNFSF9NM_003811.038TNNI1NM_003281.0038TNS1NM_022648.034TOPBP1NM_00702
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