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MYH9 Binds to Lncrna Gene PTCSC2 and Regulates FOXE1 in the 9Q22 Thyroid Cancer Risk Locus

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MYH9 Binds to Lncrna Gene PTCSC2 and Regulates FOXE1 in the 9Q22 Thyroid Cancer Risk Locus MYH9 binds to lncRNA gene PTCSC2 and regulates FOXE1 in the 9q22 thyroid 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 chromosome 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 p53 pathway. We propose that the interaction lncRNA binding protein 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 proteins 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, hypothyroidism, 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 genes, 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 transcription factor 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.
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