Article An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase

Most tumor cells reactivate telomerase to ensure unlimited proliferation, whereas the expression of human telomerase reverse transcriptase (hTERT) is tightly regu-lated and rate-limiting for telomerase activity maintenance. Several general tran-scription factors (TFs) have been found in regulating hTERTtranscription; however, a systematic study is lacking. Here we performed an inducible CRISPR/Cas9 KO screen using an hTERTcore promoter-driven reporter. We identified numerous pos-itive regulators including an E3 ligase DTX2. In telomerase-positive cancer cells, DTX2 depletion downregulated hTERT transcription and telomerase activity, contributing to progressive telomere shortening, growth arrest, and increased apoptosis. Utilizing BioID, we characterized multiple TFs as DTX2 proximal pro-teins, among which NFIC functioned corporately with DTX2 in promoting hTERT transcription. Further analysis demonstrated that DTX2 mediated K63-linked ubiq-uitination of NFIC, which facilitated NFIC binding to the hTERT promoter and enhanced hTERT expression. These findings highlight a new hTERT regulatory pathway that may be exploited for potential cancer therapeutics.

Automated summary

This study identified multiple positive regulators, including the E3 ligase DTX2, through an inducible CRISPR/Cas9 KO screen. Depletion of DTX2 in telomerase-positive cancer cells resulted in downregulation of hTERT transcription and telomerase activity, leading to progressive telomere shortening, growth arrest, and increased apoptosis. BioID analysis revealed that DTX2 interacts with multiple transcription factors, with NFIC functioning cooperatively with DTX2 to promote hTERT transcription. Further investigation demonstrated that DTX2 mediates K63-linked ubiquitination of NFIC, which enhances NFIC binding to the hTERT promoter and increases hTERT expression.