Supporting Information
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Supporting Information Wang et al. 10.1073/pnas.1619917114 SI Materials and Methods pended after centrifugation. Then, sonication and the following In Vitro RNA Probe Synthesis and RNA Pull-Down. In vitro RNA steps were carried out as previously described (18). synthesis procedures using MEGAscript T7 Transcription Kit for target probe and MEGAscript SP6 Transcription Kit for antisense Luciferase Plasmid Constructs. The promoter region of FOXE1 was control probe (Life Technologies) were performed according to the PCR-amplified from human BAC clone RP11-746L3 and cloned manufacturers’ instructions. Template DNAs were prepared using into the KpnI and SacI sites of the PGL4.10 vector (Promega). T7/SP6 primer and specific primers by PCR of cDNA clones con- Two sets of plasmids were constructed with a long fragment – + taining PTCSC2 isoform C or isoform D transcripts, respectively. ( 2,062 to 1 of the FOXE1 upstream regulatory region from + + ′ This was followed by purification with QIAquick Gel Extraction TSS and 1to 458 of 5 UTR) and a short fragment from – + Kit (QIAGEN) after the agarose gel band had been dissolved in ( 1,149 to 1oftheFOXE1 upstream regulatory region from TSS + + ′ RNase free water. Sequences were verified by Sanger sequenc- and 1to 458 of 5 UTR). Another two sets of plasmids with ing. Synthesized RNAs were analyzed qualitatively and quanti- inverted promoter regions were made from their corresponding tatively by electrophoresis and Nanodrop 2000 and then stored forward plasmids by end blunting of the insertion and religation at –80 °C. into the vector. Orientation-specific clones were screened by PCR, Primers for Target RNA probe are as follows: forward (T7), and all of the constructs were validated by Sanger sequencing. The TAATACGACTCACTATAGGGACAACGTCA-GGGCGG- expression vectors were pCMV-mCherry-MHC-IIA (Addgene GGAGGGCGC; reverse, ATGAATTAAGAGTCCTTTATTAGC. plasmid 35687, a gift from Venkaiah Betapudi, Case Western Primers for antisense control RNA probe are as follows: forward Reserve University, Cleveland) (47), pcDNA3-PTCSC2 (15), (Sp6), ATTTAGGTGACACTATAGAAATGAATTAA-GAGT- and pcDNA3 (Thermo Fisher Scientific), which was used as the CCTTTATTAGC; reverse, ACAACGTCAGGGCGGGGAGG- empty vector control. GCGC. The whole protein lysate from human nontumorous thyroid Quantitative Real-Time PCR Assay. Real-time qRT-PCR assay was sample was extracted using T-PER Tissue Protein Extraction performed in three biological replicates on ABI Prism 7900 HT Reagent (Thermo Fisher Scientific). Protein concentration was Sequence Detection System (Applied Biosystems) according to ’ determined by using Bio-Rad Protein Assay Dye Reagent Con- the manufacturer s protocol. The Taqman assays were carried centrate (Bio-Rad) kit. RNA pull-down assay was performed out using Taqman probe sets for PTCSC2 Isoform C (15), using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo FOXE1 (Life Technologies, Hs00916085_sl), and 18S (Life Fisher Scientific) according to the standard instructions. Briefly, Technologies, 4333760T) with TaqMan Fast Universal PCR the target RNA and antisense control RNA were labeled with Master Mix (Thermo Fisher Scientific). THBS1, IGFBP-3, and Biotin at the 3′ end and purified using Pierce RNA 3′ End all of the primer sets used in ChIP assay (Table S3) were de- Desthiobiotinylation Kit (Thermo Fisher Scientific). Labeled tected by Fast SYBR Green Master Mix kit (Thermo Fisher RNA probe (50 pmol) was used for the binding to Streptavidin Scientific). Magnetic Beads after incubation in 1× RNA Capture Buffer for 30 min at room temperature. In total, 200 μg protein was used for Human Primary Thyroid Cell Culture and Nucleofection. Human the subsequent protein binding step in protein–RNA binding buffer primary thyroid cells were cultured in customized 6H medium as for 150 min at 4 °C with agitation. The final RNA–magnetic beads– previously described (20). Briefly, fresh nontumorous thyroid ∼ protein complex was washed three times with wash buffer, where- tissue samples (0.3 1.5 g) were obtained from patients with PTC after 12 μL elution buffer was added to retrieve the pull-down by surgery. The tissue was immediately dissected into fragments protein products. The retrieved proteins were resolved in gradient as small as possible using a sterile razor blade in a cell culture ’ gel electrophoresis followed by MS identification. hood. After one wash in Hanks Balanced Salt Solution (Life Technologies), the tissue fragments were transferred to 0.25% ChIP Assay. ChIP assays to determine the degree of MYH9 en- trypsin solution for an overnight digestion. On the second day, richment were performed using the Magna ChIP A/G Chromatin the fragments were digested with 1% trypsin (Life Technologies) Immunoprecipitation Kit (17-10085, EMD Millipore) on KTC1 and 0.35% collagenase 4 (Worthington Biochemical) solution cells according to the manufacturer’s instructions. Briefly, chro- for 90 min at 37 °C. The digested material was filtered through matin was cross-linked with 1% formaldehyde for 10 min at nylon mesh (100 μm, FALCON). After centrifugation at 1,000 × g room temperature. After sonication, chromatin was immuno- for 5 min, the supernatant was discarded and 1 mL red blood cell precipitated with rabbit anti-MYH9 antibody (sc-98978X, Santa lysing buffer (Sigma) was added for 2 min to eliminate the blood Cruz Biotechnology) or IgG (sc-2027X, Santa Cruz Biotechnology) cells. The cells were washed twice with Hanks’ solution and at 4 °C overnight. The protein/DNA complexes were eluted from centrifuged at 1,000 × g for 5 min. Finally, the cells were counted the magnetic beads after standard washing steps. The cross-links using a TC20 Automated Cell Counter (Bio-Rad) and seeded to were reversed by incubating at 62 °C for 2 h and 95 °C for 10 min. a density of 105∼106 cells per well on six-well plates. Final DNA products were purified by using QIAquick PCR Purifi- Human FOXE1 siRNA (ON-TARGET plus SMART pool) cation Kit (QIAGEN). Then, qPCR assays were performed by using and negative control siRNA (ON-TARGET plus control pool) the purified DNA as template with primers covering the transcrip- were purchased from Dharmacon. Human thyroid primary cells tion factor-enriched region of the FOXE1 promoter (Fig. 2A). were cultured in 6H medium for 5 d until they had reached ChIP assays on frozen thyroid tissue samples were performed as 80∼90% confluency. Then, primary cells were electroporated by follows: Briefly, ∼50 mg of frozen tissue was minced into small NucleofectorII device (Amaxa) with 75 pmol siRNA in 100 μLof pieces (∼2 mm) using sterile blades. Protein/DNA cross-linking Basic Nucleofector Medium for Primary Mammalian Epithelial was performed by incubating with formaldehyde at 1% concen- Cells (Lonza, VPI-1005) for each well of the six-well plates. Cells tration for 10 min at room temperature. After washing twice with were then resuspended in 6H medium, incubated for 24 h, and PBS, fixed tissues were homogenized. Cell pellets were resus- used for further analysis. Wang et al. www.pnas.org/cgi/content/short/1619917114 1of8 RNA-Seq Sample Preparation and Detection. The total RNA samples using TopHat2 (48). Raw read counts for each gene were for RNA-seq were extracted by TRIzol reagent (Invitrogen) and quantified by using featureCounts software (49) that uses the then treated by DNase-I (Ambion) to eliminate DNA contami- GENCODE v.22 Gene Transfer Format (GTF) file as a transcript nation. RNA concentration was determined by using Qubit 2.0 reference (GENCODE annotation). Genes with counts below 5 Fluorometer (Agilent Technologies) with an RNA HS Assay Kit. for at least two samples out of three within each group were The integrity of the RNA samples was assessed by BioAnalyzer filtered out. Then, the counts were normalized toward the (Agilent). All RNA integrity numbers (RINs) were greater than 8. common library size. The count data were assumed to follow a The purified RNAs with no visible sign of genomic DNA con- negative binomial distribution. To improve the estimate of tamination from the HS Nanochip tracings were used for total RNA library generation. overdispersion and to identify genes differentially expressed Furthermore, Illumina TruSeq Stranded Total RNA Sample between samples, R package DESeq2 (50) was used to estimate Prep Kit with Ribo-Zero Gold (catalog no. RS-122-2201) was the smoothed overdispersion parameters and to calculate P used to transform RNA into cDNA after removing rRNA and values with Wald test for group comparison under a generalized mitochondrial RNA. The RNA-seq libraries were prepared linear model. The P value cutoffs were determined by controlling according to the manufacturer’s protocol. Finally, 75 bp paired- the mean number of false positives (51). end sequencing was performed using the Illumina HiSeq 2500 system. Western Blotting. Western blotting was performed according to standard procedures. Antibodies used were MYH9 (Santa Cruz, Gene Abundance Estimate and Differential Gene Expression Analysis. sc-98978), FOXE1 (Abcam, ab134129), and beta-Actin (Santa RNA-seq reads were first mapped to the human genome hg19 Cruz, sc-47778). Fig. S1. A diagrammatic view of the 9q22 locus. The diagram shows the lead SNP rs965513 in GWAS, the two flanking coding genes (XPA and FOXE1), and the lncRNA PTCSC2 isoforms C and D. Red arrows indicate transcriptional orientations. Blue filled boxes represent exons. Wang et al. www.pnas.org/cgi/content/short/1619917114 2of8 Fig. S2. Identification of MYH9 as a PTCSC2 isoform D binding protein. (A) RNA pull-down experiment with nontumorous thyroid tissue extract. Biotin pull- down assays followed by SDS/PAGE separation were used to isolate the protein binding PTCSC2 isoform D. Antisense RNA of PTCSC2 isoform D was used as the negative control. The arrows indicate the binding protein bands ∼226 KD and 42 KD in size, respectively. (B) Information for the identification of MYH9 by MS. Fig. S3. The detailed information of MS analysis for ACTB protein identified in the RNA pull-down assay.