Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and drug discovery applications. Although the setting up and running of TSA reactions is a relatively simple process, the subsequent analysis of TSA data, especially in high-throughput format, requires substantial amount of effort if conducted manually. We therefore developed the Thermal Shift Assay–Curve Rapid and Automatic Fitting Tool (TSA-CRAFT), a freely available software that enable automatic analysis of TSA data of any throughput. TSA-CRAFT directly reads real-time PCR instrument data files and displays the analyzed results in a web browser. This software features streamlined data processing and Boltzmann equation fitting, which is demonstrated in this study to provide more accurate data analysis than the commonly used first-derivative method. TSA-CRAFT is freely available as a cross-operating system-compatible standalone tool (https://sourceforge.net/projects/tsa-craft/) and also as a freely accessible web server (http://tbtlab.org/tsacraft.html).

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA–binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.