Supplementary Table 1. Differential gene expression data list of BLM6 and BLM14 cells (-146C>T vs. -146C) for the genes that were predicted to be off-targets of the guide RNA that has been used for the CRISPR/Cas9 genome engineering. Supplementary Table 2. Table showing list of interacting regions obtained from 4C analysis. 4C was performed in BLM6 and BLM14 cells. Differential interactions were obtained from 4C analysis. List shows the genomic coordinates (column 1-3) and their Refseq identities (column 4) of regions interacting with mutant Tert promoter. Column 5 shows whether the regions were tested by 3C-qPCR assay. Y = Yes, N=No. Column 6 shows whether the region was removed by CRISPR/Cas9 editing in the later part of the study. * mark is named as T-INT1 region. Supplementary Table 3. Primer sequences for ChIP-qPCR, 4C and 3C and RT-qPCR experiments.
ARTICLE ABSTRACT
Cancer-specific TERT promoter mutations (-146C>T and -124C>T) have been linked to reactivation of the epigenetically silenced telomerase reverse transcriptase gene (TERT). Understanding how these single-nucleotide alterations drive TERT reactivation is a fundamental unanswered question and is key for making successful therapeutics. We show that unlike wild-type promoters, recruitment of the transcription factor GABPA specifically to mutant TERT promoters mediates long-range chromatin interaction and enrichment of active histone marks, and hence drives TERT transcription. CRISPR-mediated reversal of mutant TERT promoters, or deletion of its long-range interacting chromatin, abrogates GABPA binding and long-range interactions, leading to depletion of active histone marks, loss of POL2 recruitment, and suppression of TERT transcription. In contrast, de novo introduction of a TERT promoter mutation enables GABPA binding and upregulation of TERT via long-range interactions, acquisition of active histone marks, and subsequent POL2 recruitment. This study provides a unifying mechanistic insight into activation of mutant TERT promoters across various human cancers.Significance: This study identifies a key mechanism by which cancer-specific mutant TERT promoters cause reactivation of TERT. Because the mechanism uncovered here is not utilized by promoters that drive TERT in normal cells, this mechanism could be exploited to make inhibitors which have the potential to block telomerase function and hence the progression of up to 90% of human cancers. Cancer Discov; 6(11); 1276–91. ©2016 AACR.See related commentary by Min and Shay, p. 1212.This article is highlighted in the In This Issue feature, p. 1197
