## 3 Results
### Screening of 20 ETS in Cell Lines Shows Vvariable Effects on TERT Expression  
Taking the list of 20 ETS factors expressed in thyroid cell lines and tumors, we employed short-hairpin RNAs (shRNA) targeting each factor individually and generated knocked-down stable cell lines (Figure 2A and Table 1). Initially, four thyroid cancer cell lines were employed: TCO1, SW1736, and C643, which all harbor _TERT_ c-124C\(>\)T mutation, and Cal62, which was used as a control for a wild-type _TERT_ promoter. In these cell lines, stable knocking down of specific ETS factors had a variable effect on _TERT_ expression when compared with scrambled shRNA cells (Figure 2B, left panel, and Supplementary Figure S1). We reasoned that ETS factors that are specifically recruited to the de novo ETS site formed by the -124C\(>\)T mutation would modify _TERT_ expression in TCO1, SW1736, and C643 cells, while Cal62 cells would remain unchanged. However, this pattern was not observed. Indeed, multiple factors exerted an effect on TERT transcription in a manner that was not always evidently TPM-mediated. Indeed, ELK1, ELK3, ELK4, ELF3, ELF4, ERF, and ETV1 knockdown resulted in significant reductions in _TERT_ expression in at least two out of three _TERT_-mutant cell lines. Of those, ELK1, ELK3, ELF3, and ER knockdown also induced at least 45% reductions in TERT expression in the Cal62 cell line (TERT wildtype). To further study the effects of these factors in an extended panel of cell lines, we selected the aforementioned seven ETS proteins, along with EHF and ETS2, which showed marginally  
Figure 1: General classification and expression patterns of the ETS family of transcription factors in thyroid cancer specimens. ETS factors are divided in four (I–IV) classes (**top row**) based on their binding sequence preferences (**second row**). Consensus (“Cons”) sites are displayed, with core sequences shown in bold and the de novo ETS binding sites created by _TERT_ promoter mutations at \(-124\) and \(-146\) highlighted in red and blue font, respectively (both C\(>\)T transitions, shown in the reverse strand in the dotted line box). The third row lists the names of the 12 ETS families, based on the presence of other protein domains. Graphs show the normalized expression levels from published thyroid cancer datasets, including PTC from the TCGA study (**top panel**) [9], and our data on PDTC + ATC (**middle**) [8] and thyroid cancer cell lines (**bottom**) [24]. The seven ETS factors not expressed in thyroid cancers are indicated with red crosses. Abbreviations: ETS = E26 transformation specific; TCGA = The Cancer Genome Atlas; PTC = Papillary Thyroid Cancer; PDTC = Poorly Differentiated Thyroid Cancer; ATC = Anaplastic Thyroid Cancer. significant reductions in _TERT_ expression, and GABPA, which had been reported in other cancer lineages. Interestingly, in our experiments, GABPA knockdown produced more variable results and did not consistently result in a reduction in _TERT_ levels as previously described upon short-term disruption to GABPA expression. We subsequently screened the selected 10 ETS factors that showed considerable reductions in _TERT_ expression in three additional cell lines: BHT101, BCPAP, and T238 (Figure 2B, right panel). From these data, knockdown of ETV1, ELK1, ELF3, and ERF consistently resulted in downregulation of the _TERT_ expression across all or most cell lines appraised, with varying degrees of magnitude. In many cases where knockdown of an ETS factor resulted in a downregulation of _TERT_ expression, a correlating reduction in _TERT_ was observed in the Cal62 control cell line. Therefore, these observations may not represent factors that specifically regulate the mutant _TERT_ promoter. Of note, we confirmed the functional knockdown of ETS factors at the RNA level in these same specimens (Figure 2C and Supplementary Figure S1). In 85% of these stable cell lines, there was at least a 50% reduction in expression of the targeted ETS factor compared to the scrambled shRNA control.