Fish husbandry and transgene and morpholino injections
Zebrafish were bred and maintained as previously described [44, 45]. Microinjections were carried out as previously described . Briefly, eGFP expression vectors were injected into 1-2 cell stage embryos (n≥300). Reporter expression was assayed between 24hpf-5dpf and embryos with consistent GFP expression were selected and raised to adulthood and founders were identified. For morhpholino experiments, previously published sox10 morpholino was ordered from Gene Tools (Philomath, OR) . 6.6 ng of the morpholino was injected into each embryo by microinjection. Embryos were analyzed and imaged using a Carl Zeiss Lumar V12 Stereo microscope with AxioVision version 4.8 software. Transgenic lines for ERBB3_MCS1, ERBB3_MCS4 and ERBB3_MCS3 are listed at the Zebrafish International Resource Center (ZIRC) (http://zebrafish.org) under allele designations JH112, JH113 and JH114 respectively.
Identification of conserved non-coding sequences and transcription factor binding sites
Conserved sequences were identified at the human ERBB3 locus and upstream and downstream intergenic regions (chr12:54,724,274-54,784,370) using the PhastCons custom track on the UCSC Genome Browser (http://genome.ucsc.edu) on genome build hg18. We used the 17-way MutiZ alignment to identify the most conserved sequences within the introns of the gene and within intergenic regions surrounding the gene. The coordinates of the MCSs and the primers used to amplify them are shown in Additional file 7, Table S1.
Fasta format DNA sequence of ERBB3_MCS6 from the genome browser was used to query MatInspector , MATCH 1.0 and Jaspar databases for identifiable TFBS using default settings.
Vector construction and mutagenesis
Expression vectors were constructed using Gateway Technology (Invitrogen, Carlsbad, CA). The desired genomic regions were amplified by with attB-flanked primers and recombined into the pDONR221 vector. Successful recombination was confirmed by sequencing. Next, entry clones were recombined into the destination vectors pLGF-E1b for luciferase assays and pT2cfosGW for zebrafish injections [25, 47].
ERBB3_MCS6 was mutated using the QuikChange II XL Site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutagenesis primers were designed to change the potential transcription factor binding sites (TFBS) to Hpa1 restriction sites using the QuikChange Primer Design tool. Primers are included in Additional file 8, Table S2.
Sox10-pcDNA3.1 and Sox10-ΔSTP-pcDNA3.1 were cloned using Sox10-pCMV and Sox10-ΔSTP-pCMV as templates. Coding sequence for human SOX10 and AP2 for transactivation experiments was amplified by PCR from I.M.A.G.E clone MGC-3510 and MGC-22117 respectively and cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) using In-Fusion PCR Advantage Cloning Kit (Clontech, Mountain View, CA). The PCR primers used were designed using In-Fusion Primer Design tool and are shown in Additional file 9, Table S3. Successful cloning was verified by sequencing.
The E189X Sox10 cDNA cloned into a pCMV promoter was a kind gift from Ken Inoue, Jim Lupski and Michael Wegner [23, 38].
Cell culture and transfection
Immortalized melanocytes (melan-a) and immortalized Schwann cells were maintained as described [48, 49]. NIH 3T3 cells were grown in 10% FCS in DMEM under standard conditions. Neuro2A cells were grown in 10%FCS in MEM under standard conditions.
melan-a cells were plated in 24-well plates 24 hours prior to transfection at a density of 4 × 10^4 cells/well. 400 ng of the luciferase reporter plasmids were cotransfected with 8 ng of CMV-RL renilla expression vector (Promega, Madison, WI). 48 hours after transfection, cell lysate was collected and assayed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). For siRNA knockdown, 200 ng of luciferase reporter vectors were cotransfected with 5 pmol of each siRNA pool to a total of 10 pmol/well of a 24-well plate. Where appropriate, scrambled siRNA was added to maintain a final concentration of 10 pmol/well of siRNA.
Neuro2A cells were plated in 24-well plates 24 hours prior to transfection at a density of 5 × 10^4 cells/well. 400 ng of luciferase reporter plasmids were cotransfected with 200 ng of Sox10-pcDNA3.1 and ΔSTP-Sox10-pcDNA3.1. Where appropriate, empty pcDNA3.1 vector was added to maintain a final concentration of 800 ng/well of DNA. 8 ng of CMV-RL were added to each well. Cell lysate was collected 24 hours after transfection and assayed as mentioned above. Luciferase assays were carried out using a Tecan GENiosPro machine. All assays were performed in triplicate and repeated in at least two independent experiments.
For luciferase assays in S16 cells, 1 × 10^4 cells were plated in 96-well plates 24 hours prior to transfection with luciferase vectors. Each transfection reaction included 200 ng of experimental and control luciferase expression constructs and 2 ng of a renilla expression construct to control for transfection efficiency and cell viability. Cells lysates were collected 48 hours after transfection and luciferase assays were carried out with the Dual-Luciferase Assay System (Promega, Madison, WI) and analyzed on a Glomax Multi-Detection System (Promega, Madison, WI).
ON-TARGETplus SMARTpool siRNA was ordered against mouse Sox10 and Tfap2a from Dharmacon (Lafayette, CO). Knockdown was achieved by using 5 pmol of each siRNA in a 24-well transfection format.
Cells were trypsinized and washed twice in 1XPBS and resuspended in 2X Incomplete Lamelli buffer and passed through a QiaShredder (Invitrogen, Carlsbad, CA) to obtain whole cell lysate. Protein was quantified and run on a 10% Mini Protean TGX gel (Biorad, Hercules, CA), transferred onto a nitrocellulose membrane and blocked overnight in 5% non-fat dry milk block. Sox10 antibody was used at a dilution of 1.5 ng/ul (MAB2864, R&D Biosystems, Minneapolis, MN), AP2 antibody was used at 1:500 (ab52222, Abcam, Cambrigde, MA), ErbB3 antibody was used at 1:500 (sc285, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-tubulin at 1:3000 (CP06, Calbiochem, San Diego, CA). HRP-conjugated secondary was used and antibody binding was visualized using SuperSignal West Dura Extended Duration Substrate (34076, Thermo Fischer, Rockford, IL). Membranes were stripped using Restore PLUS Western Blot Stripping Buffer (46430, Thermo Fischer, Rockford, IL
RNA extraction, cDNA synthesis and Real-time PCR
Neuro2A cells were plated in 6-well dish at a density of 2.5 × 10^5 cells/well using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). A total of 2 ng of Sox10-pcDNA3.1 and ΔSTP-Sox10-pcDNA3.1 were transfected into the cells and RNA was collected 24 hours later using the RNeasy Mini Kit (Qiagen, Valencia, CA) using manufacturers instructions. Subsequently, cDNA was synthesized using the SuperScriptIII First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). Real-time PCR was then performed in triplicate using the Universal Gene Expression Master Mix (ABI, Carlsbad, CA) and the PrimeTime qPCR Assay designed against mouse Sox10 and ErbB3. Real-time PCR and analysis were performed on the Opticon2 (Biorad, Hercules, CA).
Electrophoretic mobility shift assay
Probes spanning the SOXE-2 binding site were designed (Additional file 10, Table S4) and labeled with the Biotin 3' End DNA Labeling Kit (Pierce, Rockford, IL) according to manufacturers instructions. Nuclear extract was made from melan-a cells using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL). EMSAs were performed using the LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL). Briefly, 12.5 fmol of labeled probe was incubated with nuclear extract in the presence of binding buffer and 1 ug of poly (dI.dC) in a 20 ul reaction for 10 min at room temperature. For competition assays, 500 and 1000 molar fold excess of unlabeled probe was added. Products were run on precast polyacrylamide gels (4-20% or 7.5%) (Biorad, Hercules, CA) and signal was developed using the LightShift Chemiluminescent kit.
Chromatin Immunoprecipitation (ChIP) and Real-time PCR
ChIP was performed in melan-a cells as previously described  with some changes. Each ChIP experiment was performed with ~1 × 108 cells. Alternative lysis buffers to those in the referenced protocol were used, as follows: Lysis buffer 1 (5 mM PIPES, 85 mM KCl, 0.5% NP-40, and 1 × Roche Complete, EDTA-free protease inhibitor), lysis buffer 2 (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, and 1 × Roche Complete, EDTA-free protease inhibitor), and lysis buffer 3 (16.7 mM Tris-HCl, 1.2 mM EDTA, 167 mM NaCl, 0.01% SDS, 1.1% Triton X-100, and 1 × Roche Complete, EDTA-free protease inhibitor). Sonication was performed with a Bioruptor (Diagenode, Denville, NJ) with the following settings: high output; 30 second disruption; 30 second cooling; total sonication time of 35 min with addition of fresh ice and cold water to water bath every 10 minutes. 2 ug of antibody specific to H3K4me1 (ab8895, Abcam Cambridge, MA) and non-specific IgG (ab46540, Abcam Cambridge, MA) was used for immunoprecipitation. IP wash conditions were also adjusted from the above referenced protocol, as follows: Each IP was washed twice with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), twice with cold LiCl wash buffer (0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris-HCl), and rinsed once with PBS, pH 7.4. Immunoprecipitated DNA was analyzed by real-time PCR using SYBR Green (ABI, Carlsbad, CA) and primers shown in Additional file 11, Table S4 and performed and analyzed on an Opticon2 (Biorad, Hercules, CA) using the % Input method. Primer sequences for PCR are given in supplementary table 5.
ChIP in S16 cells was performed as previously described . The antibodies used were anti-Sox10 (sc-17342X, Santa Cruz Biotechnology, Santa Cruz, CA) and control anti-goat IgG (sc-2808, Santa Cruz Biotechnology, Santa Cruz, CA). ChIP was analyzed in duplicate by quantitative PCR and analyzed by the % Input method. Primers used for PCR are shown in Additional file 11, Table S4.