MYH9 binds to lncRNA PTCSC2 and regulates FOXE1 in the 9q22 cancer risk locus

Yanqiang Wanga,b, Huiling Hea,b, Wei Lia,b, John Phayc, Rulong Shend, Lianbo Yue,f, Baris Hanciogluf, and Albert de la Chapellea,b,1

aHuman Cancer Genetics Program, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210; bDepartment of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210; cDepartment of Surgery, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210; dDepartment of Pathology, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210; eCenter for Biostatistics, The Ohio State University, Columbus, OH 43210; and fDepartment of Biomedical Informatics, The Ohio State University, Columbus, OH 43210

Contributed by Albert de la Chapelle, December 7, 2016 (sent for review August 30, 2016; reviewed by Bryan Haugen and Shioko Kimura) A locus on 9q22 harbors a SNP (rs965513) firmly expression of both PTCSC2 and FOXE1 (15). Three enhancer associated with risk of papillary thyroid carcinoma (PTC). The locus elements are located in a 33-kb linkage disequilibrium (LD) also comprises the forkhead box E1 (FOXE1) gene, which is impli- block within PTCSC2. Previous studies showed that PTCSC2 is cated in thyroid development, and a long noncoding RNA (lncRNA) transcribed in the opposite direction of FOXE1.Exon1andintron1 gene, papillary thyroid cancer susceptibility candidate 2 (PTCSC2). of PTCSC2 isoform C overlap with the FOXE1 promoter region How these might interact is not known. Here we report that PTCSC2 (15). Although the promoter region shared by PTCSC2 and FOXE1 binds myosin-9 (MYH9). In a bidirectional promoter shared by FOXE1 was found to be regulated via long-range looping interactions (18), and PTCSC2, MYH9 inhibits the promoter activity in both directions. the detailed mechanisms and their bearing on thyroid cancer need This inhibition can be reversed by PTCSC2, which acts as a suppres- to be explored. sor. RNA knockdown of FOXE1 in primary thyroid cells profoundly In the present study, we identified myosin-9 (MYH9) as an interferes with the pathway. We propose that the interaction lncRNA binding that targets the FOXE1 promoter re- between the lncRNA, its binding protein MYH9, and the coding gene gion through interactions with PTCSC2 and performs its regu- FOXE1 underlies the predisposition to PTC triggered by rs965513. latory function in thyroid cancer via downstream pathways. BIOCHEMISTRY These findings provide a better defined description of the com- lncRNA | MYH9 | transcriptional regulation | bidirectional promoter | plex mechanisms involved in the 9q22 thyroid cancer locus. thyroid cancer Results hyroid cancer is the most common endocrine malignancy. Identification of MYH9 as an LncRNA Binding Protein. To begin to TBased on the data from the National Cancer Institute for unravel the underlying mechanisms by identifying po- 2016, in total 64,300 new patients will be diagnosed with thyroid tentially interacting with PTCSC2, we performed biotin-labeled cancer, accounting for 3.8% of all cancer patients in the United RNA pull-down assays with protein lysate from normal thyroid States (https://seer.cancer.gov/statfacts/html/thyro.html). Thyroid tissue. Two PTCSC2 isoforms—isoform C (1,947 nt) and isoform cancer is classified into four main types: papillary, follicular, D (1,804 nt)—were used as baits by generating both sense and medullary, and anaplastic. Papillary thyroid carcinoma (PTC) ac- antisense (negative control) RNA probes for each of them (Fig. S1). – counts for 85 90% of all thyroid cancers (1). In efforts to explain Asaresult,astrand-specificbinding protein was discovered between the genomic background to PTC, genome-wide association studies (GWASs) have disclosed an SNP marker, rs965513, in 9q22.33 Significance strongly associated with PTC in European populations [odds ratio − (OR) = 1.75; P = 1.7 × 10 27] (2, 3). The association has been observed in other populations as well (4–8). Additionally, rs965513 Papillary thyroid carcinoma (PTC) is the most common endocrine has been reported to be associated with levels of thyroid-related cancer and displays strong heritability. So far, the most significant hormones, , goiter, and other abnormal thyroid known predisposing variant is rs965513 in 9q22. Although a long functions (2, 3, 9). SNP rs965513 resides in an intron of a recently noncoding RNA, papillary thyroid cancer susceptibility candidate 2 PTCSC2 detected long noncoding RNA (lncRNA), papillary thyroid cancer ( ), has been characterized in this locus, its mode of action in susceptibility candidate 2 (PTCSC2), which is located between two the carcinogenetic process is unknown. Here, we identify myosin- PTCSC2 coding , DNA damage recognition and repair factor (XPA) 9 (MYH9) as a binding protein of that regulates the bi- PTCSC2 and forkhead box E1 (FOXE1)(Fig. S1). directional promoter shared by and forkhead box E1 FOXE1 PTCSC2 FOXE1, also known as thyroid 2, is a single ( ). can rescue the promoter inhibition caused by exon coding gene belonging to the forkhead/winged helix-domain MYH9. The p53 pathway is profoundly affected by the inhibition FOXE1 PTCSC2 protein family (10). FOXE1 knockout mice were born alive but of . Our study discovers fundamental roles for , FOXE1 exhibited an ectopic or completely absent thyroid gland and severe MYH9, and in thyroid cancer and provides a description of cleft palate, accompanied by up-regulated TSH and down-regulated the regulatory mechanism. free thyroxine hormone levels (11). FOXE1 is essential for thyroid Author contributions: Y.W., H.H., and A.d.l.C. designed research; Y.W. performed research; gland development and the maintenance of thyroid differentiated W.L., J.P., and R.S. contributed new reagents/analytic tools; Y.W., L.Y., and B.H. analyzed status (12, 13). It also plays a role in the predisposition and devel- data; and Y.W., H.H., B.H., and A.d.l.C. wrote the paper. opment of thyroid cancer (14, 15). Moreover, somatic mutational Reviewers: B.H., University of Colorado School of Medicine; and S.K., National Cancer inactivation of FOXE1 has been found in PTC, suggesting FOXE1 Institute/National Institutes of Health. may contribute to tumorigenesis in a subset of thyroid cancers (16). The authors declare no conflict of interest. PTCSC2 was discovered in the region harboring rs965513 on Data deposition: The data reported in this paper have been deposited in the Gene Ex- 9q22 (15). Similar to some 25% of all known lncRNA genes (17), pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE83919). PTCSC2 has one unspliced isoform and several spliced isoforms, 1To whom correspondence should be addressed. Email: [email protected]. all of which display thyroid-specific expression. In PTC tumors, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the risk allele [A] of rs965513 is significantly associated with low 1073/pnas.1619917114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1619917114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 As PTCSC2 and FOXE1 are transcribed in opposite directions (15) and there is only 174 bp between their transcription start sites (TSSs), we postulated that they are transcribed by the same pro- moter with bidirectional activity. To test this hypothesis, a shorter (1,607 bp) and a longer (2,520 bp) fragment cloned from the FOXE1 promoter region were used for luciferase reporter assay in the BCPAP cell line with both forward (Promoter-S and Pro- moter-L) and inverted (Promoter-Inv-S and Promoter-Inv-L) orientation, as indicated (Fig. 3A). Although the inverted promoters showed lower activity compared with their corresponding forward ones, the fragment with both orientations exhibited a significantly higher level of promoter activity in comparison with the no pro- moter control. Moreover, cotransfection with the MYH9 expression vector showed that MYH9 can significantly inhibit the luciferase activity in both the forward and inverted promoter fragments. That MYH9 functions as a suppressor was disclosed in both the shorter and longer promoter fragments (Fig. 3 B and C). Thus, MYH9 had an impact on the FOXE1 gene, but its role in the presence of lncRNA PTCSC2 was not known. We transiently cotransfected the PTCSC2 expression vector in the BCPAP cell line Fig. 1. Identification of MYH9 as a PTCSC2 binding protein. (A) RNA pull- in the presence or absence of the MYH9 expression construct. down experiment with nontumorous thyroid tissue extract. Biotin pull-down Because there was no significant difference in promoter activity assays followed by SDS/PAGE separation were used to isolate the binding protein of PTCSC2 isoform C. Antisense RNA of PTCSC2 isoform C was used as between the two fragments of different sizes, only the longer pro- the negative control. The arrows indicate the binding protein bands ∼226 moter sets were used. Interestingly, an increase in promoter lucif- KD and 42 KD in size, respectively. (B) Information for the identification of erase activity was observed with cotransfection of the PTCSC2 MYH9 by MS. The full length of MYH9 protein is 1,960 amino acids, as shown expression vector, irrespective of MYH9 overexpression. The ef- in the lower diagram. Blue boxes indicate the peptides identified by MS fect was significant on the forward Promoter-L, whereas it was not analysis. (C) qRT-PCR detection of the indicated RNAs retrieved by MYH9 significant on the inverted Promoter-Inv-L, suggesting a rescuing antibody (RIP assay) in the BCPAP cell line with stable PTCSC2 isoform C effect on the inhibition caused by MYH9 (Fig. 4 A and B). overexpression. IgG was used as a negative control. The relative fold change PTCSC2 < ’ To further validate the roles of MYH9 and in regu- was normalized to the IgG control. **P 0.01. Student s t test. lating the endogenous FOXE1 promoter, a transient transfection assay with MYH9 and PTCSC2 expression vectors was per- 150 KD and 250 KD in size (Fig. 1A and Fig. S2). This protein formed in BCPAP and TPC1 cells. The expression of the en- FOXE1 was identified as MYH9, being 226 KD in size by mass spec- dogenous gene showed a significant decrease in the trometry (MS) (Fig. 1B). Another protein band that was noticed presence of MYH9 overexpression, which is consistent with the between 37 KD and 50 KD in size was identified as beta-actin role of MYH9 as an inhibitor in our luciferase assay. This in- (ACTB) (Fig. 1A and Figs. S2 and S3). As MYH9 is an ACTB hibitory effect becomes nonsignificant in the presence of PTCSC2, indicating that FOXE1 is regulated by PTCSC2 and binding partner participating in important cellular processes MYH9 differentially (Fig. 4 C and D). When we examined the (19), ACTB was not studied further here. PTCSC2 transcript levels and protein levels of MYH9 and The interaction between PTCSC2-isoform C and MYH9 was FOXE1 in 6 thyroid cancer cell lines and in nontumorous thyroid confirmed by RNA immunoprecipitation (RIP) assay in BCPAP tissue, we did not notice a correlation among the expression cells with PTCSC2-isoform C stable overexpression (Fig. 1C). PTCSC2 levels of these three genes (Fig. S4). was readily enriched by MYH9 antibody compared with SNP rs1867277 is in partial LD with rs965513 and is reported as IgG-negative controls (>sixfold), and this enrichment was sig- FOXE1 P < a functional variant in the regulation of expression and nificantly higher than in the 18S RNA controls ( 0.01), confers thyroid cancer susceptibility (14). As rs1867277 is located demonstrating the specificity of the assay. in the MYH9 interacting region R3 (Fig. 2A), we tested the reg- ulatory effects of MYH9 and PTCSC2 on the FOXE1 promoter MYH9 Binds to the FOXE1 Promoter Region. To determine whether with either the wild-type allele (G) or the thyroid cancer risk allele the PTCSC2 binding protein MYH9 is involved in the transcrip- FOXE1 (A). BCPAP cells were transiently transfected with either MYH9 tional expression of the gene, four overlapping regions or both MYH9 and PTCSC2 constructs using promoters with (R1, R2, R3, and R4) that cover the transcription factor enriched ′ opposite orientations. The promoter containing the A allele region (from ENCODE ChIP sequencing data) in the 5 UTR of exhibited significantly lower activity than the promoter with the FOXE1 were chosen for ChIP analysis using MYH9 antibody (Fig. G allele, and a similar difference was found in both forward and A 2 ). Of note, the previously reported thyroid cancer risk-associ- inverted promoters (Fig. S5). ated SNP rs1867277 (14) is located in the R3 genomic region. In the KTC1 cell line, significant enrichment was found in the R1 and FOXE1 Is Involved in the P53 Pathway in Human Primary Thyroid Cells. R3 regions (Fig. 2B). The enrichment was confirmed in non- FOXE1 is an important transcriptional regulator in the physio- tumorous thyroid tissue (Fig. 2C), suggesting the DNA binding logical functions of the thyroid as well as in thyroid cancer. activity of MYH9 is in the FOXE1 promoter, perhaps more spe- Lower expression level of FOXE1 is associated with PTC risk in cifically in regions R1 and R3. the presence of the risk allele of SNP rs965513 (15). To identify genes involved in FOXE1-mediated signaling pathways and MYH9 Functions as a Suppressor in the Promoter Region of PTCSC2 transcriptional regulation in thyroid, knockdown of FOXE1 was and FOXE1 with Bidirectional Activity. To investigate the role of performed with siRNAs in nontumorous human primary thyroid MYH9 binding to the FOXE1 promoter region, luciferase assays cells (20). Nontumorous human primary thyroid cells from the were performed in cells transiently transfected with FOXE1 same patient sample transfected with scrambled siRNAs were promoter or empty constructs (without promoter), together with used as the control. Knockdown of FOXE1 resulted in numerous MYH9 expression or empty expression vectors. changes in determined by RNA deep sequencing

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1619917114 Wang et al. Downloaded by guest on September 30, 2021 (fold change > 1.5, P < 0.001). Of the dysregulated genes (total is dependent on their expression. Many lncRNAs show tissue-spe- N = 107; up-regulated n = 80), 89.7% were coding genes (Table cific expression patterns. Several thyroid tissue- and cancer-associ- S1). Biological functional analysis using Ingenuity Pathway Anal- ated lncRNAs were recently discovered (23). Only a few lncRNAs ysis (IPA) software showed that cancer was in the top category related to thyroid and thyroid cancer have been well characterized, of “Diseases and Disorders” with the second lowest P value (Fig. including PTCSC2, PTCSC3,andNAMA (15, 24, 25), but even in S6). The top five categories of “Molecular and Cellular Func- these cases, the relevant molecular mechanisms are not known. It is tions”—namely, cellular movement, cell death and survival, cel- of special interest that these lncRNAs are involved in the genetic lular development, cellular growth and proliferation, and cell predisposition to thyroid cancer (germ-line involvement). Therefore, signaling—suggested its possible relevance to pathways related to their impact on nontumorous thyroid tissue needs to be explored, cell apoptosis, migration, or invasion (Table 1). These effects have which we here have endeavored to accomplish. We identified been widely reported as the major effects of the well-known p53 MYH9 as a noncoding RNA binding partner, which had not been signaling pathway (21, 22). Of the top 25 dysregulated genes, the reported before. thrombospondin 1 (THBS1, also known as TSP-1) and insulin-like MYH9 protein is a member of the nonmuscle myosin II growth factor binding protein 3 (IGFBP3) genes were found to be (NMII) group, which belongs to the myosin II subfamily (26). As up-regulated in FOXE1 knockdown cells (Fig. 5A and Table S1). a subunit of myosin II heavy chains, MYH9 takes part in the This inverse relationship was subsequently confirmed by quanti- generation of cell polarity, cell migration, cell–cell adhesion tative RT-PCR (qRT-PCR) in primary thyroid cells (Fig. 5B)and processes, and maintaining cytoskeleton structure by binding to also in the BCPAP and TPC1 cell lines (Fig. S7). We also analyzed actin filaments (19). Recently, additional functions of MYH9 gene expression data from 59 pairs of tumor/nontumor PTC have been reported. For example, MYH9 affects the expression samples available in the TCGA data portal (https://tcga-data.nci. of PAX5 by interacting with Thy28 (27), and it can activate nih.gov/docs/publications/tcga). In this analysis, the expression AKT through RAC1 and PAK1 (28), suggesting a role in gene − of FOXE1 was significantly (P = 2.02 × 10 05) higher in non- regulation. In 2014, a role for MYH9 as a tumor suppressor tumorous tissue, whereas the expression of both THBS1 and gene was discovered in squamous cell carcinomas (SCCs). This − IGFBP3 are significantly higher in tumorous tissue (P = 1.73 × 10 report implicated MYH9 in tumor development by regulating − 02 and P = 4.19 × 10 05, respectively) (Table S2). This is consistent posttranscriptional p53 stabilization, but the underlying mech- with our data and further supports the putative role of FOXE1 as a anism is still unknown (29). Our results are consistent with this suppressor of THBS1 and IGFBP3 in thyroid. Both THBS1 and finding and provide further evidence for a regulatory model by IGFBP3 are important members of the p53 pathway involved in which a specific noncoding RNA, PTCSC2, and MYH9 work BIOCHEMISTRY apoptosis, inhibition of the IGF1/mTOR pathway, and inhibition together by regulating FOXE1 promoter activity. Knockdown of of angiogenesis and metastasis, suggesting interactions between FOXE1 in primary human thyroid cells revealed that FOXE1 can FOXE1 and the p53 pathway in thyroid cells (Fig. 5C). regulate events in the p53 pathways. To further assess the function of FOXE1 in thyroid cells, we As a common occurrence, bidirectional transcription of two performed cell viability and apoptosis assays in FOXE1 knock- protein coding genes has been discovered and studied during down BCPAP cells. The down-regulation of FOXE1 led to re- the past decades (30, 31). More recently, many noncoding duced cell viability in CellTiter-Glo assay (Fig. 5D). Consistently, RNAs that are transcribed in the proximity of coding genes and apoptosis assay with flow cytometric analysis indicated that in- driven by the same bidirectional promoter have been reported creases of apoptotic cells at both early and late apoptosis stages (32, 33). Some noncoding transcripts exert repression of their were found in the FOXE1 knockdown cells (Fig. 5E). nearby coding genes by competing for the same polymerases and accessory factors or by transcriptional interference or his- Discussion tone modification (34–36). A number of highly expressed an- LncRNAs are important regulators of tissue physiology and disease tisense transcripts derived from bidirectional TSSs are able to processes including cancer. The regulatory effectiveness of lncRNAs enhance the expression of the corresponding protein coding gene

Fig. 2. Validation of the MYH9 binding site in the FOXE1 promoter region by ChIP assay. (A) Schematic view of a 900-bp transcription factor enrichment region in the FOXE1 promoter region. Genomic co- ordinate, chr9:100,615,431–100,616,330 (GRC37/hg19). DNase I hypersensitive sites, CpG islands, and ENCODE data were obtained from the UCSC Genome Browser. Red bars indicate the tested regions in ChIP assays. (B) ChIP assay using KTC1 cell line. The results represent four independent experiments, each in duplicates. Results are shown as means ± SD. *P < 0.05. Student’s t test. (C) ChIP assay using nontumorous thyroid tissue sample. The relative abundance was normalized to the input control.

Wang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 we consider thyroid primary cell culture to be a superior model for pathway and network analysis. In this study, we use human thyroid primary cell cultures that were recently established (20). Two important members of the p53 pathway, THBS1 and IGFBP3, have been reported to be involved in various cancer types. The regulation of tumor growth and metastasis by THBS1 has been widely described (42). THBS1 is regulated by BRAFV600E in thyroid cancer cells. The silencing of THBS1 results in decreased cell proliferation, adhesion, migration, and invasion (43). Moreover, THBS1 silencing can also cause changes in the levels of integrin receptors and anaplastic thyroid cancer cell morphology (44). THBS1 promotes human follicular thyroid carcinoma cell invasion mainly through up-regulation of urokinase plasminogen activator (PLAU) (45). This is consistent with our RNA-seq results, as PLAU is also one of the top 25 dysregulated genes in FOXE1 knockdown primary thyroid cells (Fig. 5A and Table S1). IGFBP3 inhibits tumor growth by promoting apoptosis and inhibiting cell proliferation in some cancer types, but in other circumstances, it increases cell survival and stimulates proliferation (46). Our data further em- phasize the roles played by these two genes (THBS1 and IGFBP3) in thyroid cancer. In conclusion, our study addresses the mechanisms and pro- vides a biological characterization of the widely reported GWAS locus 9q22 in thyroid cancer. We propose MYH9 as a binding protein of thyroid-specific noncoding RNA that may underlie the predisposition to thyroid cancer.

Fig. 3. Effect of MYH9 on FOXE1 promoter with bidirectional activity. (A)Sche- matic view of the vectors used in luciferase assays. Red boxes indicate the UTR regions of FOXE1; the yellow box indicates the FOXE1 coding sequences; blue boxes indicate the exons of PTCSC2; the thick gray line indicates the intron of PTCSC2; the thin black line indicates the intergenic genomic region. All of the position numbers are labeled relative to the FOXE1 TSS as +1. Arrows represent the fragment direction in the constructs according to the position in the ge- nome. Dual reporter luciferase assays in BCPAP cells cotransfected with con- structs containing a short promoter fragment (B) or long promoter fragment (C) having either forward or inverted orientation, with or without MYH9 over- expression vector. Values are presented as fold of the no promoter control treated with empty expression vector. Results are shown as means ± SD of four independent experiments, each in four replicates. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test.

in a tissue-specific manner (37). The PTCSC2–FOXE1 pair in our study further supports this regulatory model, which can be directed by the same inhibitor. Considering the complexity of the 9q22 re- Fig. 4. Effect of PTCSC2 on FOXE1 bidirectional promoter. Dual reporter gion including nearby CpG islands (38), it is reasonable to assume luciferase assay using the long promoter constructs with forward (A)or that other transcription factors and some epigenetic modifications inverted (B) orientations cotransfected with PTCSC2, MYH9, or empty ex- might be involved in the regulation of this bidirectional promoter, in pression vectors. The values at the bottom of each column represent the PTCSC2 portion of the total expression vectors. Results are shown as means ± SD of addition to MYH9 and . It will be of interest to learn < ’ whether other regulatory factors have any impact on MYH9, or four independent experiments, each in four replicates. *P 0.05. Student s t test. (C) qPCR detection of endogenous FOXE1 in the BCPAP cell line and (D) vice versa. the TPC1 cell line transiently transfected with PTCSC2, MYH9, or empty ex- Cell line models were used in most previous reports on pression vectors as indicated (n = 3). All values were normalized with the FOXE1 in the thyroid (39, 40). Because several thyroid markers values of the corresponding empty vector-transfected groups. Results are including lncRNAs lose their expression in most cell lines (41), shown as means ± SD. *P < 0.05; NS, not significant. Student’s t test.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1619917114 Wang et al. Downloaded by guest on September 30, 2021 Fig. 5. Gene expression profile of FOXE1 knock- down in thyroid primary cells indicating its in- volvement in the p53 pathway. (A) Gene expression differences of the top 25 genes caused by FOXE1 knockdown in primary thyroid cells on day 5 of cul- ture. The expression is plotted with heat-map color scale using relative expression fold change (FOXE1 knockdown culture vs. control culture) (fold change > 1.5, P < 0.001). Exp. 1, Exp. 2, and Exp. 3 represent three independent primary culture experiments de- rived from different patient samples. (B) Confirma- tion by qRT-PCR of two up-regulated genes, IGFBP3 (Left)andTHBS1 (Right), in FOXE1 knockdown pri- mary cells. si-Control represents the primary cell sample treated with scrambled siRNA control. Results are shown as means ± SD of three independent ex- periments, each in three replicates. All values were normalized with the values of the corresponding negative control siRNA-treated groups. *P < 0.05; ***P < 0.001. Student’s t test. (C) Part of the p53 signaling pathway including typical gene members and their functions from Kyoto Encyclopedia of Genes and Genomes (KEGG). THBS1 is labeled in a

purple ellipse, and IGFBP3 is labeled in two red el- BIOCHEMISTRY lipses. (D) Cell viability changes of BCPAP cell line at three time points (24, 48, and 72 h) with FOXE1 knockdown. For each time point, experiments were performed in four replicates of three biological rep- licates. All values were normalized with the values of the corresponding negative control siRNA-treated groups. (E) Example plots of cell apoptosis assays in BCPAP cell line treated with FOXE1 siRNA or negative control siRNA. Cells stained with only annexin V were evaluated as being in early apoptosis stage (lower right quadrant); cells stained with both annexin V and PI (propidium iodide) were evaluated as being in late apoptosis stage (upper right quadrant).

Materials and Methods (Sigma-Aldrich) following the instructions. Briefly, 5 μg antibody of rabbit anti-MYH9 (Santa Cruz, sc-98978) or rabbit IgG (Sigma, I5006-1MG) was The study was approved by the Institutional Review Board at The Ohio State used to incubate with the cell lysate in 1 mL RIP wash buffer with Protease University (OSU), and all subjects gave written informed consent before Inhibitor Mixture (Roche) and also RNase Inhibitor overnight at 4 °C after participation. ligation to Maganetic Beads. The RIP products were harvested by washing with 1 mL washing buffer up to five times. Finally, the washed product Cell Lines and Thyroid Tissue Samples. The human thyroid carcinoma cell lines μ used in this study were incubated in antibiotic-free RPMI medium 1640 (KTC1, resuspended in 200 L buffer was subjected to the purification step using BCPAP, C643, SW1736) or DMEM (TPC1, FTC133) supplemented with 10% (vol/vol) TRIzol reagent (Life Technologies). The quantification of target RNA in the final RIP products was determined by real-time qRT-PCR using the FBS (Gibco) at 37 °C in humidified air with 5% (vol/vol) CO2.Thyroid non- tumorous samples were snap-frozen in liquid nitrogen and kept at –80 °C Taqman method. after being obtained from patients with PTC during surgery. All cases were histologically diagnosed as PTC; clinical information on these samples will be ChIP Assay. A detailed description of the chromatin immunoprecipitation (ChIP) made available on request. assays to determine the degree of MYH9 enrichment on the KTC1 cell line and frozen thyroid tissue samples can be found in SI Materials and Methods.The In Vitro RNA Probe Synthesis and RNA Pull-Down. In vitro RNA synthesis primer sequences are provided in Table S3. procedures using MEGAscript T7 Transcription Kit for target probe and MEGAscript SP6 Transcription Kit for antisense control probe (Life Technol- ogies) were performed according to the manufacturers’ instructions. The Table 1. Molecular and cellular functions of the dysregulated whole-protein lysate from the human nontumorous thyroid sample was genes in FOXE1 knockdown cells by IPA analysis extracted using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Function P value Molecules Scientific). Protein concentration was determined by using the Bio-Rad − − Protein Assay Dye Reagent Concentrate (Bio-Rad) kit. RNA pull-down assay Cellular movement 1.30 × 10 03–7.65 × 10 10 42 was performed using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Cell death and survival 1.27 × 10−03–8.74 × 10−10 46 − − Fisher Scientific) according to the standard instructions. A detailed de- Cellular development 1.22 × 10 03–1.20 × 10 08 53 − − scription can be found in SI Materials and Methods. Cellular growth and 1.13 × 10 03–1.20 × 10 08 47

6 proliferation RIP Assay. In total, 2 × 10 BCPAP cells with PTCSC2 isoform C overexpression − − Cell signaling 9.58 × 10 04–6.75 × 10 08 19 were used for RIP lysis preparation using Harsh Lysis Buffer of Imprint RIP Kit

Wang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Transfection and Dual Luciferase Reporter Assay. For the luciferase reporter (Promega). The numbers of viable cells were represented by the luminescent assay, BCPAP cells were transiently transfected with reporter plasmid using signals, which were obtained by using GloMax 96 Microplate Luminometer Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the (Promega). Experiments were performed according to the manufacturer’sin- manufacturer’s instructions. Briefly, cells were seeded in a 24-well plate at 0.7 × structions. Apoptotic cell death in the BCPAP cell line transfected with FOXE1 5 10 cells per well and cultured overnight. The following day, 250 ng lucif- siRNA or negative control siRNA after 72 h was measured using the FITC erase reporter plasmids and 250 ng effector plasmids were cotransfected Annexin V Apoptosis Detection Kit (BD Biosciences) by flow cytometry along with 1.25 ng Renilla plasmid pRL-TK (Promega) as a control for each according to the manufacturer’s instructions. Three biological replicates μ well. Cells were lysed with 100 L passive lysis buffer (Promega) for luciferase were performed. activity analysis using the Dual-Luciferase Reporter Assay System (Promega) 24 h after transfection. A 20-μL aliquot of cell lysate was then assayed for ACKNOWLEDGMENTS. We thank Jerneja Tomsic and Sandya Liyanarachchi luciferase activity using GloMax 96 Microplate Luminometer (Promega). for helpful discussions about data analysis, Ann-Kathrin Eisfeld for help with knockdown experiments, Jan Lockman and Barbara Fersch for administrative Cell Viability Assay and Cell Apoptosis Assay. Cell viability in the BCPAP cell line help, and the OSU Comprehensive Cancer Center (OSUCCC) Proteomics transfected with FOXE1 siRNA or negative control siRNA at different time Shared Resource for help with MS. This work was supported by National points (24, 48, and 72 h) was analyzed by using the CellTiter-Glo assay Cancer Institute Grants P30CA16058 and P50CA168505.

1. Davies L, Welch HG (2006) Increasing incidence of thyroid cancer in the United States, 27. Fujita T, Kitaura F, Fujii H (2015) A critical role of the Thy28-MYH9 axis in B cell-specific 1973-2002. JAMA 295(18):2164–2167. expression of the Pax5 gene in chicken B cells. PLoS One 10(1):e0116579. 2. Gudmundsson J, et al. (2009) Common variants on 9q22.33 and 14q13.3 predispose to 28. Zhao B, et al. (2015) The non-muscle-myosin-II heavy chain Myh9 mediates colitis- thyroid cancer in European populations. Nat Genet 41(4):460–464. induced epithelium injury by restricting Lgr5+ stem cells. Nat Commun 6:7166. 3. Gudmundsson J, et al. (2012) Discovery of common variants associated with low TSH 29. Schramek D, et al. (2014) Direct in vivo RNAi screen unveils myosin IIa as a tumor levels and thyroid cancer risk. Nat Genet 44(3):319–322. suppressor of squamous cell carcinomas. Science 343(6168):309–313. 4. Ai L, et al. (2014) Associations between rs965513/rs944289 and papillary thyroid car- 30. Beck CF, Warren RA (1988) Divergent promoters, a common form of gene organi- cinoma risk: A meta-analysis. Endocrine 47(2):428–434. zation. Microbiol Rev 52(3):318–326. 5. Takahashi M, et al. (2010) The FOXE1 locus is a major genetic determinant for radi- 31. Trinklein ND, et al. (2004) An abundance of bidirectional promoters in the human ation-related thyroid carcinoma in Chernobyl. Hum Mol Genet 19(12):2516–2523. genome. Genome Res 14(1):62–66. 6. Matsuse M, et al. (2011) The FOXE1 and NKX2-1 loci are associated with susceptibility to 32. Seila AC, et al. (2008) Divergent transcription from active promoters. Science papillary thyroid carcinoma in the Japanese population. J Med Genet 48(9):645–648. 322(5909):1849–1851. 7. Zhu H, Xi Q, Liu L, Wang J, Gu M (2014) Quantitative assessment of common genetic 33. Pickrell JK, et al. (2010) Understanding mechanisms underlying human gene expres- variants on FOXE1 and differentiated thyroid cancer risk. PLoS One 9(1):e87332. sion variation with RNA sequencing. Nature 464(7289):768–772. 8. Wang YL, et al. (2013) Confirmation of papillary thyroid cancer susceptibility loci 34. Neil H, et al. (2009) Widespread bidirectional promoters are the major source of identified by genome-wide association studies of 14q13, 9q22, 2q35 cryptic transcripts in yeast. Nature 457(7232):1038–1042. and 8p12 in a Chinese population. J Med Genet 50(10):689–695. 35. Berretta J, Pinskaya M, Morillon A (2008) A cryptic unstable transcript mediates 9. Denny JC, et al. (2011) Variants near FOXE1 are associated with hypothyroidism and transcriptional trans-silencing of the Ty1 retrotransposon in S. cerevisiae. Genes Dev other thyroid conditions: Using electronic medical records for genome- and phenome- 22(5):615–626. wide studies. Am J Hum Genet 89(4):529–542. 36. Camblong J, Iglesias N, Fickentscher C, Dieppois G, Stutz F (2007) Antisense RNA 10. Zannini M, et al. (1997) TTF-2, a new forkhead protein, shows a temporal expression stabilization induces transcriptional gene silencing via histone deacetylation in in the developing thyroid which is consistent with a role in controlling the onset of S. cerevisiae. Cell 131(4):706–717. differentiation. EMBO J 16(11):3185–3197. 37. Uesaka M, et al. (2014) Bidirectional promoters are the major source of gene acti- 11. De Felice M, et al. (1998) A mouse model for hereditary thyroid dysgenesis and cleft vation-associated non-coding RNAs in mammals. BMC Genomics 15:35. palate. Nat Genet 19(4):395–398. 38. Abu-Khudir R, et al. (2014) Role for tissue-dependent methylation differences in the 12. Fernández LP, López-Márquez A, Santisteban P (2015) Thyroid transcription factors in expression of FOXE1 in nontumoral thyroid glands. J Clin Endocrinol Metab 99(6): development, differentiation and disease. Nat Rev Endocrinol 11(1):29–42. E1120–E1129. 13. De Felice M, Di Lauro R (2011) Minireview: Intrinsic and extrinsic factors in thyroid 39. Santisteban P, Acebrón A, Polycarpou-Schwarz M, Di Lauro R (1992) Insulin and in- gland development: An update. Endocrinology 152(8):2948–2956. sulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the 14. Landa I, et al. (2009) The variant rs1867277 in FOXE1 gene confers thyroid cancer thyroglobulin promoter. Mol Endocrinol 6(8):1310–1317. susceptibility through the recruitment of USF1/USF2 transcription factors. PLoS Genet 40. Venza I, et al. (2011) MSX1 and TGF-beta3 are novel target genes functionally reg- 5(9):e1000637. ulated by FOXE1. Hum Mol Genet 20(5):1016–1025. 15. He H, et al. (2015) Genetic predisposition to papillary thyroid carcinoma: Involvement of 41. Pilli T, Prasad KV, Jayarama S, Pacini F, Prabhakar BS (2009) Potential utility and FOXE1, TSHR, and a novel lincRNA gene, PTCSC2. J Clin Endocrinol Metab 100(1):E164–E172. limitations of thyroid cancer cell lines as models for studying thyroid cancer. Thyroid 16. Mond M, et al. (2015) Somatic mutations of FOXE1 in papillary thyroid cancer. Thyroid 19(12):1333–1342. 25(8):904–910. 42. Sid B, et al. (2004) Thrombospondin 1: A multifunctional protein implicated in the 17. Derrien T, et al. (2012) The GENCODE v7 catalog of human long noncoding RNAs: Analysis regulation of tumor growth. Crit Rev Oncol Hematol 49(3):245–258. of their gene structure, evolution, and expression. Genome Res 22(9):1775–1789. 43. Nucera C, et al. (2010) B-Raf(V600E) and thrombospondin-1 promote thyroid cancer 18. He H, et al. (2015) Multiple functional variants in long-range enhancer elements progression. Proc Natl Acad Sci USA 107(23):10649–10654. contribute to the risk of SNP rs965513 in thyroid cancer. Proc Natl Acad Sci USA 44. Duquette M, Sadow PM, Lawler J, Nucera C (2013) Thrombospondin-1 silencing down- 112(19):6128–6133. regulates integrin expression levels in human anaplastic thyroid cancer cells with 19. Conti MA, Adelstein RS (2008) Nonmuscle myosin II moves in new directions. J Cell Sci BRAF(V600E): New insights in the host tissue adaptation and homeostasis of tumor 121(Pt 1):11–18. microenvironment. Front Endocrinol (Lausanne) 4:189. 20. Wang Y, et al. (2016) Primary cell culture systems for human thyroid studies. Thyroid 45. Sid B, et al. (2008) Thrombospondin-1 enhances human thyroid carcinoma cell in- 26(8):1131–1140. vasion through urokinase activity. Int J Biochem Cell Biol 40(9):1890–1900. 21. Hwang CI, et al. (2011) Wild-type p53 controls cell motility and invasion by dual 46. Baxter RC (2014) IGF binding proteins in cancer: Mechanistic and clinical insights. Nat regulation of MET expression. Proc Natl Acad Sci USA 108(34):14240–14245. Rev Cancer 14(5):329–341. 22. Fridman JS, Lowe SW (2003) Control of apoptosis by p53. Oncogene 22(56): 47. Dulyaninova NG, House RP, Betapudi V, Bresnick AR (2007) Myosin-IIA heavy-chain 9030–9040. phosphorylation regulates the motility of MDA-MB-231 carcinoma cells. Mol Biol Cell 23. Iyer MK, et al. (2015) The landscape of long noncoding RNAs in the human tran- 18(8):3144–3155. scriptome. Nat Genet 47(3):199–208. 48. Kim D, et al. (2013) TopHat2: Accurate alignment of transcriptomes in the presence of 24. Jendrzejewski J, et al. (2012) The polymorphism rs944289 predisposes to papillary insertions, deletions and gene fusions. Genome Biol 14(4):R36. thyroid carcinoma through a large intergenic noncoding RNA gene of tumor sup- 49. Liao Y, Smyth GK, Shi W (2014) featureCounts: An efficient general purpose program pressor type. Proc Natl Acad Sci USA 109(22):8646–8651. for assigning sequence reads to genomic features. Bioinformatics 30(7):923–930. 25. Yoon H, et al. (2007) Identification of a novel noncoding RNA gene, NAMA, that is 50. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dis- downregulated in papillary thyroid carcinoma with BRAF mutation and associated persion for RNA-seq data with DESeq2. Genome Biol 15(12):550. with growth arrest. Int J Cancer 121(4):767–775. 51. Gordon A, Glazko G, Qiu X, Yakovlev A (2007) Control of the mean number of false 26. Sellers JR (2000) Myosins: A diverse superfamily. Biochim Biophys Acta 1496(1):3–22. discoveries, Bonferroni and stability of multiple testing. Ann Appl Stat 1(1):179–190.

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