Supplemental materials and methods

Human-specific regulation of MeCP2 levels in fetal brains by microRNA miR-483-5p

Kihoon Han, Vincenzo Alessandro Gennarino, Yoontae Lee, Kaifang Pang, Kazue Hashimoto-Torii, Sanaa Choufani, Chandrasekhar S. Raju, Michael C. Oldham, Rosanna Weksberg, Pasko Rakic, Zhandong Liu & Huda Y. Zoghbi

Cell culture, transfection, lysis and western blot DAOY, SK-N-SH and Neuro2a cells were cultured in DMEM (Invitrogen) with 10% FBS and penicillin/streptomycin. Control and BWS human fibroblasts were cultured in MEM Alpha (Invitrogen) with 10% FBS and penicillin/streptomycin. Cells in 6-well plates were transfected with either 70 pmol of miRNA mimic (miRIDIAN Dharmacon) or MECP2 small- interfering RNA (Ambion), or 200 pmol of miRNA hairpin inhibitor (miRIDIAN Dharmacon) using a Dharmafect reagent (Dharmacon) according to the manufacturer’s instructions. Caenorhabditis elegans miRNA cel-miR-67 mimic or inhibitor (miRIDIAN Dharmacon) was used as a control for each experiment. After 4 to 5 days, cells were lysed with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and complete protease inhibitor cocktail (Roche)) with brief sonication and boiled with Laemmli sample buffer.

Antibodies Rabbit anti-MeCP2 (1:1000, Chemicon; 1:1000, Cell Signaling), rabbit anti-HDAC4 (1:1000, Abcam), rabbit anti-TBL1X (1:1000, Abcam), mouse anti-GAPDH (1:5000, Advanced ImmunoChemical Inc.), and HRP conjugated anti-b-actin (1:10000, Abcam) antibodies were used for western blot.

Human brain samples Human embryonic (18 to 22 weeks of gestation) cortical tissues were obtained from legal interruption of pregnancies at Albert Einstein College of Medicine and at San Francisco General Hospital. The research protocol was approved by the Human Investigation Committee at Yale University and Albert Einstein College of Medicine, and the Committee on Human Research (institutional review board) at the University of California, San Francisco, respectively. No samples were associated with medical histories showing complications during pregnancy. We evaluated sample quality by immunohistochemistry and qRT-PCR on part of each tissue sample, then we selected tissues showing normal nuclear labeling as well as low level expressions of a cellular stress inducible (HSP70) and an immediate early response gene (FOS) (data not shown) (Hashimoto-Torii et al. 2011). Human cortical tissues from postnatal stages (ranging from 5 to 28 years) were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD.

Primers for qRT-PCR To distinguish spliced cDNA from genomic DNA contamination, we designed specific exon primers across introns for qRT-PCR reactions as follows (only the primer sets targeting 3’UTR regions of MECP2 were designed within a single exon) : MECP2 for -coding exons forward 5’- GATCAATCCCCAGGGAAAAGC-3’, reverse 5’- CCTCTCCCAGTTACCGTGAAG-3’; MECP2 for short and long 3’UTR forward 5’- GGATTGCAAAGCAAACCAAC-3’, reverse 5’-AATCGGGAAGCTTTGTCAGA-3’; MECP2 for long 3’UTR forward 5’-CTGTTTTGAGCCCTGAGGAG-3’, reverse 5’- CCTTGCTGCTCTCTTGCTCT-3’; mouse MeCP2 for short and long 3’UTR forward 5’- GGATTGCAAAGCAAACCAAC-3’, reverse 5’-AATCGGGAAGCTTTGTCAGA-3’; mouse MeCP2 for long 3’UTR forward 5’-ACCTCTAACCCTGCCTGGAT-3’, reverse 5’- GGCCGTGCTAGCAAAGTAAG-3’; IGF2 forward 5’-ACACCCTCCAGTTCGTCTGT-3’, reverse 5’-CGGAAACAGCACTCCTCAAC-3’; CITED2 forward 5’- CTGCAGAAGCTCAACAACCA-3’, reverse 5’-CTGGTTTGTCCCGTTCATCT-3’; ARID5B forward 5’-AGGAAAACGCCGATAGAACG-3’, reverse 5’- CCCAGTTTTTGAGCAGCTTG-3’; CNOT6 forward 5’-ACATGTTGATGGCTGTGCAA- 3’, reverse 5’-AATTTGCCATGGCTAGCTGA-3’; MAML1 forward 5’- ATGAAGCCAAAGCCAGGAAA-3’, reverse 5’-ACTGGAAGGGTTCTGCTCCT-3’; RBM14 forward 5’-TCGAGTGTGACGTGGTGAAA-3’, reverse 5’- CCTTCACTTCTTTGCCGTTG-3’; HDAC4 forward 5’-AAGCCCACACCCTCCTGTAT-3’, reverse 5’-GCGAGCGAGCCTAGAAGTTT-3’; TBL1X forward 5’- CAAAACCTTCCAGGGACACA-3’, reverse 5’-ATGCCAGCAACATTCCAGAC-3’; MEF2C forward 5’- ACGAGGATTATGGATGAACG-3’, reverse 5’- GCCTTCTTCATCAACCCAAA-3’; GAPDH forward 5’-AGAAGGCTGGGGCTCATTTG- 3’, reverse 5’-AGGGGCCATCCACAGTCTTC-3’. qRT-PCR reactions were performed using the ABI Prism 7000 Sequence Detection System with PerfeCta SYBR Green FastMix (Quanta Biosciences). The results were analyzed as described previously (Gennarino et al. 2009).

Luciferase assay Each human MECP2 (1-7,293; 1-1,006; 3,514-4,561), CNOT6 (1,101-1,750; 3,801-4,153), MAML1 (1,251-2,343), RBM14 (101-800), HDAC4 (1,266-2,041), and TBL1X (396-821) 3’UTR region was PCR amplified and subcloned into psiCHECK-2 vector (Promega). Mutagenesis reactions were performed using the QuikChange XL Site-Directed Mutagenesis kit (Stratagene). HEK293T or DAOY cells in 24-well plates were transfected with 30 ng of psiCHECK-2 construct plus 5~15 pmol of miRNA duplex using Lipofectamine 2000 (Invitrogen). After 24 hrs, luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

RNA extraction from mouse hippocampus and RT-PCR Total RNA was extracted from the hippocampus of 3-week-old mice using Trizol (Invitrogen) and 2 mg of total RNA was used for cDNA synthesis by Superscript II and Oligo-dT primers (Invitrogen). The primers for RT-PCR reactions were as follows: human MECP2 3’ UTR forward 5’-CTCAGCTCGCTGGATGGCGGAAG-3’, reverse 5’- CTCTAGAAACAGCTGACTCCCTG-3’; human and mouse MECP2 CDS forward 5’- GACTTCACGGTAACTGGGAGAGG-3’, reverse 5’-CCTTGACCTCGATGCTGACCGTC- 3’; mouse GAPDH forward 5’-TGTTGCCATCAACGACCCCTTC-3’, reverse 5’- GATGGCATGGACTGTGGTCATGAG-3’.

Hippocampal neuron culture, transfection and immunostaining Hippocampal neurons were prepared from postnatal day 1 mice and plated on poly-D- lysine/mouse laminin coated coverslips (BD biosciences) in Neurobasal medium supplemented with GlutaMAX-I (Invitrogen), B-27 and 0.5 % FBS. At DIV 5, neurons on each coverslip were transfected with 200 ng of pEGFP-C1 (Clontech), 20 pmol of miRNA duplex and 500 ng of pcDNA3.1 (as filler, Invitrogen) using Lipofectamine 2000. At DIV 10 (for MeCP2 level) or DIV 12 (for mature spine density), transfected neurons were fixed with 4 % formaldehyde/4 % sucrose in PBS, and permeabilized with 0.2 % Tx-100 in PBS. PBS with 0.1 % BSA and 3 % horse serum was used for blocking and antibody incubation. Mouse anti-GFP (1:500, Chemicon), rabbit anti-GFP (1:500, GeneTex) and rabbit anti-MeCP2 (1:500, Chemicon) antibodies were used for immunostaining. For the luciferase assay, neurons were transfected with 200 ng of psiCHECK-2 construct, 20 pmol of miRNA duplex and 500 ng of pcDNA3.1, and luciferase activities were measured as HEK293T cells.

Image acquisition and statistical analysis For western blot experiments, all images were acquired by a LAS 4000 (GE Healthcare), and quantified by an ImageJ software package. For immunostaining of cultured neurons, all z- stack images were acquired by a LSM710 (Zeiss) confocal microscope under the same parameter settings, and an average intensity of MeCP2 puncta colocalized with DAPI signal (n = 15), and mature (mushroom-shaped) spine density (n = 20 to 28) were measured by ImageJ. For measuring MeCP2 puncta, background signals were removed by threshold setting. For mature spine density, only those on the dendrites of secondary and tertiary branches were quantified. The mushroom-shaped spine was defined as a spine with head width greater than length and width of neck. All data were presented as mean ± SEM. Statistical significance was determined by Student’s t test or one-way ANOVA with post hoc Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, *** p < 0.001.

Bioinformatics analysis was performed using the DAVID web tool and default parameters (http://david.abcc.ncifcrf.gov) as described previously (Gennarino et al. 2011). After performing the analysis, only Biological Process categories with a FDR correct ≤ 5 were retained (Huang da et al. 2009). Interaction networks of MeCP2, HDAC4 and TBL1X were identified by IPA (Ingenuity Pathway Analysis, http://www.ingenuity.com). To investigate the conservation of miR-483-5p binding sites, we initially obtained 340 potential target of miR-483-5p using CoMeTa (Gennarino et al. 2012). To be more confident, we limited the binding site types to canonical, marginal and atypical categories described in (Bartel 2009), and got 169 target genes with high confident binding sites. Next, we extracted the orthologous genomic coordinates of the binding site sequences in human, chimpanzee, gorilla, orangutan, rhesus, mouse and rat using liftover and DAS from the UCSC genome browser database (Kent et al. 2002). To access the conservation of the binding sites between different species, we aligned the binding site sequences and required a conserved site to respect the parameters described in (Bartel 2009).

References Bartel DP. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136: 215-233. Gennarino VA, D'Angelo G, Dharmalingam G, Fernandez S, Russolillo G, Sanges R, Mutarelli M, Belcastro V, Ballabio A, Verde P et al. 2012. Identification of microRNA-regulated gene networks by expression analysis of target genes. Genome Res 22: 1163-1172. Gennarino VA, Sardiello M, Avellino R, Meola N, Maselli V, Anand S, Cutillo L, Ballabio A, Banfi S. 2009. MicroRNA target prediction by expression analysis of host genes. Genome Res 19: 481-490. Gennarino VA, Sardiello M, Mutarelli M, Dharmalingam G, Maselli V, Lago G, Banfi S. 2011. HOCTAR database: a unique resource for microRNA target prediction. Gene 480: 51-58. Hashimoto-Torii K, Kawasawa YI, Kuhn A, Rakic P. 2011. Combined transcriptome analysis of fetal human and mouse cerebral cortex exposed to alcohol. Proc Natl Acad Sci U S A 108: 4212-4217. Huang da W, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44-57. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The browser at UCSC. Genome Res 12: 996-1006.

Supplemental Figure 1. miR-483-3p expression is increased postnatally in human brains and miR-483-3p does not affect the luciferase activity of a construct with MECP2 long 3’UTR. (A) Stem-loop of the precursor-miRNA containing miR-483-5p and miR-483-3p. (B) Expression levels of miR-483-3p are significantly higher during postnatal stages compared to fetal stages in human brains. (C) Luciferase assays with a construct containing the long 3’UTR of MECP2 show that miR-483-5p, but not miR-483-3p, decreased the luciferase activity.

Supplemental Figure 2. miR-483-5p regulates human MeCP2 levels in cultured hippocampal neurons. (A) Luciferase assay in mouse cultured hippocampal neurons with the constructs described in (Fig. 2B) (3,514-4,561). (B) mRNAs were extracted from the hippocampus of wildtype (WT) and MECP2 Tg1 mice, and RT-PCR reactions were performed to amplify indicated products. The human MECP2 3’UTR was detected only in Tg1 samples. Note that RT-PCR products for human and mouse MECP2 coding region (CDS) were increased in Tg1 samples compared to WT. (C) Sequencing of the human MECP2 transcript from Tg1 hippocampus confirmed that the miR-483-5p binding site was intact. (D) One transfected (white box 2) and two nearby untransfected (white box 1 and 3) Tg1 neurons show selective decrease of MeCP2 protein levels (yellow arrowheads) by miR-483-5p expression. The white arrowheads indicate DAPI signals from co-cultured glial cells where MeCP2 signals were not detected under current staining conditions and parameter settings. Scale bar, 50 mm.

Supplemental Figure 3. miR-483-5p binding sites in the 3’UTRs of human CNOT6, MAML1, RBM14, HDAC4 and TBL1X. (A) (top) The colored numbers indicate position of miR-483-5p binding sequence in the 3’UTR. Base pairs between miR-483-5p and CNOT6 3’UTR are indicated by vertical lines. The red colored ‘GUC’ was changed to ‘CAG’ in mutant luciferase constructs. (bottom) Alignment of CNOT6 3’UTRs from indicated species for the miR-483-5p binding site. (B-E) The same information for MAML1 (B), RBM14 (C), HDAC4 (D) and TBL1X (E) is shown. (F) Schematic diagrams show the luciferase constructs of each gene containing putative miR-483-5p binding sites with or without mutation.

Supplemental Figure 4. Analysis of the conservation of miR-483-5p binding sites in the putative target genes predicted by CoMeTa. (A) The conservation of each binding site (total 223 binding sites in 169 genes) is shown (blue, conserved; pink, not conserved; gray, not annotated). Each row represents single miR-483-5p binding site. (B) The percentages of miR- 483-5p binding sites for each category (conserved, not conserved or not annotated) in indicated species are shown. The same color coding of (A) is used.

Supplemental Figure 5. Gene Ontology analysis for the 9 putative target genes (NRXN3, ELK1, HOXC11, SAMD4A, PRPF4B, FHL1, TYRO3, CFLAR and KIAA0913) of miR-483-5p with conserved binding sites from human to rodents. (A,B) Fold enrichments for the biological process terms (A) and the cellular component terms (B) are indicated. Supplemental Table 1. CoMeTa COOL and Gene Ontology analysis of miR-483-5p putative targets Term P value FDR miR-483-5p putative targets organization 8.82E-06 0.01 SMARCA2, CHD4, RCOR1, TLK2, MSL-1, SEH1L, SUPT16H, SMARCC1, PTGES3, SET, CHD1, H2AFV, SATB2, JARID1A, RBM14, NIPBL, HMG20A, CBX4, ING4, DNMT1, HDAC4, HMGB1 Chromatin organization 1.13E-05 0.02 SMARCA2, CHD4, RCOR1, TLK2, MSL-1, SUPT16H, SMARCC1, SET, CHD1, H2AFV, SATB2, JARID1A, RBM14, HMG20A, ING4, CBX4, DNMT1, HDAC4, HMGB1 Chromatin modification 5.15E-05 0.09 SMARCA2, CHD4, RCOR1, TLK2, MSL-1, SMARCC1, CHD1, SATB2, JARID1A, RBM14, ING4, CBX4, HMG20A, DNMT1, HDAC4 Negative regulation of 4.40E-04 0.73 NAB2, NFX1, SMARCA2, ILF3, CITED2, SATB2, TNRC6B, TRIM33, ARID5B, MECP2, TBL1X, ING4, CBX4, DNMT1, CBFA2T2, HDAC4, EIF2C1, ZNF238, HMGB1 Negative regulation of RNA metabolic process 8.98E-04 1.48 NFX1, SMARCA2, ILF3, CITED2, SATB2, ARID5B, MECP2, TBL1X, ING4, CBX4, DNMT1, CBFA2T2, HDAC4, HMGB1, ZNF238 Transcription 2.72E-03 4.41 MAML1, MAX, NFX1, CAND1, SMARCA2, HIF1AN, TCEA1, ILF3, PPARBP, CITED2, SMARCC1, MGA, CNOT6, HDGF, CTNND1, HNRPDL, TRIM33, RBM14, ISGF3G, LITAF, TBL1X, TFDP1, ZNF532, HDAC4, FRY, EIF2C1, ZNF238, POLR2J2, RCOR3, EPAS1, NAB2, PRIM2A, CHD4, RCOR1, SUPT16H, PTOV1, MAFG, JARID1A, ARID5B, MECP2, HMG20A, CBX4, DNMT1, NOTCH2, TCEA2, FOXJ2, DRG1 The miR-483-5p putative targets in each category are shown. MECP2 is labeled with green.

Supplemental Table 2. Human miR-483-5p predicted targets conservation across different species. A binary code is used to define the conservation (1) or not (0). NA = a 3'UTR/binding site for a specific gene/species is not annotated by UCSC.

gene\species human chimp gorilla orangutan rhesus mouse rat ABHD4 1 1 1 NA 1 NA NA ADAM15 1 1 1 0 NA 0 0 ADARB1 1 1 1 1 0 NA NA ADCY6 1 0 1 1 0 NA NA ADPGK 1 1 1 1 0 0 0 ADRBK1 1 0 1 0 0 0 0 ALDH3B1 1 1 1 0 NA NA NA ANKFY1 1 1 0 1 1 0 NA ARHGEF9 1 1 1 0 1 0 NA ATF7_1 1 0 0 NA NA 0 0 ATF7_2 1 0 0 NA NA 0 0 ATG9A 1 1 NA 1 1 0 0 BAG1 1 1 1 0 0 0 0 BICD2 1 1 1 1 0 0 0 BMP1 1 1 1 0 1 NA NA BSN 1 0 1 0 0 NA NA C14orf101_1 1 0 0 0 0 NA NA C14orf101_2 1 1 1 1 0 0 0 C17orf85 1 1 1 1 0 0 0 CACNB1 1 0 NA 0 1 0 0 CCDC6 1 1 1 0 0 0 0 CD55_1 1 1 1 0 0 NA NA CD55_2 1 1 1 0 0 0 0 CELSR2 1 0 1 0 0 0 0 CFLAR_1 1 1 1 1 0 0 0 CFLAR_2 1 1 1 0 1 0 1 CFLAR_3 1 1 1 1 0 1 0 CIT 1 1 0 1 1 0 0 CLCN3 1 1 1 0 0 0 0 CNOT6_1 1 1 1 0 0 0 0 CNOT6_2 1 1 1 1 1 NA NA DBN1 1 1 1 0 0 0 0 DDX3X 1 1 1 1 0 0 0 DERL1_1 1 1 NA 1 1 NA NA DERL1_2 1 0 0 0 0 0 0 DERL1_3 1 1 1 1 0 NA 0 DERL1_4 1 1 1 1 1 NA NA DERL1_5 1 1 0 1 0 0 0 DNAJB2_1 1 1 1 0 0 0 0 DNAJB2_2 1 1 1 1 1 0 0 DNMT1 1 1 1 1 1 NA NA DPY19L4 1 1 NA 1 NA NA NA DPYSL2_1 1 1 1 0 0 0 0 DPYSL2_2 1 1 1 0 0 0 0 EDA 1 1 1 1 0 NA 0 EFNB3 1 1 1 NA NA NA 0 ELAVL1 1 1 1 1 0 0 0 ELAVL3 1 NA 1 1 NA NA NA ELK1 1 1 1 1 1 1 1 EPAS1 1 1 NA 0 0 NA NA EPB41L4B 1 1 1 1 0 0 0 EPHA4_1 1 1 1 1 0 0 0 EPHA4_2 1 1 1 1 1 0 0 FBXL7 1 1 0 0 1 0 0 FHL1_1 1 1 1 1 1 1 0 FHL1_2 1 0 1 1 1 1 0 FOXJ2 1 0 1 1 0 NA NA FOXP1 1 1 1 0 0 0 0 FTO 1 0 0 0 0 NA NA GFAP 1 0 0 0 0 0 0 GNL3L_1 1 1 1 1 0 NA NA GNL3L_2 1 1 0 1 1 NA NA GOSR1_1 1 0 NA NA NA NA NA GOSR1_2 1 0 NA NA NA NA NA GOSR1_3 1 0 NA NA NA NA NA GOSR1_4 1 0 NA NA NA NA NA GPR56 1 0 1 0 0 NA NA GSK3A 1 1 1 0 0 0 NA H2AFV 1 1 1 0 0 NA NA HDAC4_1 1 0 0 0 0 0 NA HDAC4_2 1 1 1 1 1 0 0 HOXC11 1 0 NA 0 1 1 1 HTR4 1 1 1 1 1 0 0 IDS_1 1 NA 1 0 0 NA NA IDS_2 1 1 1 0 1 NA NA IDS_3 1 1 0 0 1 NA NA IGF2 1 1 0 1 0 0 0 IL1R1_1 1 1 1 1 0 NA NA IL1R1_2 1 1 1 1 0 NA 0 IL1R1_3 1 1 0 0 0 NA NA ILF3 1 1 1 1 1 0 0 INA 1 1 1 1 1 0 0 IVL 1 0 1 0 1 NA NA KCNN3 1 1 1 0 NA NA NA KIAA0913_ 1 1 1 0 0 0 0 1 KIAA0913_ 1 1 1 0 0 1 0 2 KIAA0913_ 1 1 0 0 0 1 0 3 KIAA1217_ 1 0 1 1 1 0 NA 1 KIAA1217_ 1 1 1 1 1 0 0 2 KIF3B 1 1 1 1 0 NA NA KIF4A 1 1 1 0 0 0 0 KLHDC3 1 1 1 1 1 NA NA LITAF 1 1 1 0 0 NA NA LRP1 1 0 1 1 1 0 0 MAML1_1 1 1 1 1 1 0 0 MAML1_2 1 1 1 1 1 NA NA MAML1_3 1 1 1 1 0 NA NA MAP2K7 1 1 1 1 0 NA NA MAP4 1 1 1 0 0 0 0 MARCKSL1 1 0 1 0 1 0 0 MARK4 1 1 1 0 1 NA NA MBTPS1 1 1 1 1 1 0 0 MECP2_1 1 1 1 1 1 0 0 MECP2_2 1 0 0 0 0 0 0 MEGF9_1 1 1 1 1 0 NA NA MEGF9_2 1 1 1 1 0 NA NA MID1IP1 1 1 1 0 1 NA NA MRPL52 1 0 0 1 NA NA NA MTCH1 1 1 1 1 0 NA 0 MUC3A 1 1 1 1 1 NA NA MYO1C_1 1 1 1 0 1 NA NA MYO1C_2 1 1 1 0 0 0 NA MYO9B 1 1 1 1 1 NA NA NAV1 1 1 1 0 NA 0 0 NAV2 1 1 1 0 0 0 0 NDEL1_1 1 0 1 0 0 0 0 NDEL1_2 1 0 1 1 1 0 0 NDEL1_3 1 0 1 1 1 0 0 NDRG2 1 0 0 0 0 0 0 NISCH_1 1 1 1 0 0 0 0 NISCH_2 1 1 1 0 0 0 0 NISCH_3 1 1 1 0 0 0 0 NKX3-1 1 1 NA 0 0 NA NA NMNAT2 1 1 1 0 0 NA 0 NPR3 1 0 0 0 0 NA NA NRXN3 1 1 1 1 1 1 1 NTRK2 1 1 1 1 1 NA NA OPHN1_1 1 1 0 1 0 NA NA OPHN1_2 1 1 1 0 0 NA NA PABPN1 1 1 1 1 1 0 NA PARVA 1 1 1 0 0 NA NA PCM1 1 1 1 NA 0 NA NA PDGFD 1 1 1 0 0 0 0 PDXK 1 1 1 NA 0 NA NA PIP5K1A 1 0 1 0 0 NA NA PJA2_1 1 0 0 0 0 0 NA PJA2_2 1 1 1 1 1 0 0 PLXND1 1 1 1 0 0 0 NA POLR2J2 1 0 0 0 0 NA NA POU4F2 1 1 1 0 0 0 0 PPFIA1 1 1 1 0 0 0 0 PPP2R1A 1 1 1 0 NA 0 0 PPP2R5C 1 1 1 1 1 0 0 PRO1768_1 1 1 1 1 0 0 0 PRO1768_2 1 1 1 0 0 NA NA PRPF4B 1 1 1 1 1 1 0 PSME3 1 0 0 0 1 0 0 PTOV1 1 1 1 1 1 NA NA QKI 1 0 1 1 1 NA 0 RAD23B 1 1 1 1 0 NA NA RBM14 1 1 1 0 0 NA NA RBM5 1 1 1 0 0 0 0 RCOR3_1 1 1 NA NA NA NA NA RCOR3_2 1 1 1 0 0 0 NA RFC3 1 1 1 0 1 NA NA RHOA 1 1 1 0 0 0 0 RIF1 1 1 1 0 0 0 0 RPS6KA3 1 1 0 0 0 NA NA RYR3 1 1 1 NA 0 0 0 SALL2 1 1 1 0 0 NA 0 SAMD4A 1 1 1 1 1 1 NA SATB2 1 1 1 1 1 NA NA SEC61A1_1 1 0 1 0 0 0 0 SEC61A1_2 1 1 1 0 0 NA NA SEMA4G_1 1 1 1 1 0 0 0 SEMA4G_2 1 NA 1 1 1 0 0 SEMA6D 1 0 1 0 0 0 0 SETD5 1 1 1 0 0 0 0 SFRS2B_1 1 1 0 1 1 NA NA SFRS2B_2 1 1 1 1 0 NA NA SFRS5_1 1 1 0 0 1 NA NA SFRS5_2 1 1 1 1 1 0 NA SH3PXD2A 1 1 1 1 1 NA 0 SHOX2 1 1 1 1 0 0 0 SIPA1L3 1 1 1 0 1 0 0 SLC12A5_1 1 1 1 0 0 NA NA SLC12A5_2 1 1 1 1 0 0 0 SLC12A5_3 1 1 1 1 0 0 0 SLC38A1 1 0 0 0 0 0 NA SLCO4A1 1 1 1 1 0 0 0 SMG6_1 1 1 1 1 1 NA NA SMG6_2 1 0 1 NA 0 0 0 SNAP25 1 1 1 1 1 0 0 SNPH_1 1 1 1 0 0 0 0 SNPH_2 1 1 0 0 0 NA NA SNX27 1 1 1 0 0 0 0 SORBS1 1 1 1 1 1 NA NA SORT1 1 1 1 NA 0 0 0 SOX11_1 1 1 1 0 1 0 0 SOX11_2 1 1 1 0 1 0 0 SRC 1 1 1 1 0 NA NA SRCAP_1 1 1 1 1 1 0 0 SRCAP_2 1 0 0 0 0 0 0 STC2 1 0 1 0 1 NA NA STXBP1 1 1 0 1 0 0 NA SUPT16H 1 1 1 1 0 NA NA TBL1X 1 1 1 NA 1 0 0 TCF7L1 1 1 0 0 0 NA NA TDRKH 1 NA 1 NA NA NA NA TGFB2 1 0 0 0 0 0 0 TIAM1_1 1 0 1 0 0 NA NA TIAM1_2 1 1 1 0 0 0 0 TLK2 1 1 0 1 1 0 0 TMCC1 1 0 0 0 0 NA NA TRIM33 1 0 1 0 0 0 0 TRIM38 1 1 1 0 1 NA NA TYRO3 1 1 1 0 1 0 1 UCK2 1 1 1 0 1 0 0 USP48 1 1 1 0 0 0 NA VAMP1 1 1 1 1 1 0 0 VAT1 1 0 0 NA 0 0 0 VPS16_1 1 1 1 0 0 0 0 VPS16_2 1 1 NA 0 1 NA NA WDR26 1 NA 1 0 0 NA NA WIPF2_1 1 NA NA NA NA NA NA WIPF2_2 1 0 0 0 0 NA NA WNT10B 1 0 1 1 1 0 0 WSB1_1 1 0 0 1 NA 0 0 WSB1_2 1 0 0 0 0 0 0 XYLT1_1 1 1 1 1 0 0 0 XYLT1_2 1 1 1 0 0 0 NA ZC3H7B 1 1 1 1 1 0 0 ZNF592 1 1 1 1 0 0 0