Termination sequence between an inducible promoter and ubiquitous chromatin opening element (UCOE) reduces gene expression leakage and silencing

Abstract Inducible gene expression circuits offer precise control over target gene activation, making them essential tools for direct reprogramming, where cells are guided to differentiate into specific cell types. However, stable circuit function and consistent expression of key inducible proteins are crucial for effective reprogramming, and DNA methylation-induced silencing hinders this stability. To address this issue, A2-ubiquitous chromatin opening elements (A2UCOE) have gained attention for their ability to maintain open chromatin and prevent methylation, thereby stabilizing gene expression. In this study, we aimed to forward program iPSCs into thymic epithelial cells (TECs) using a compact, all-in-one gene circuit composed of a doxycycline-inducible Tet-On system, 863-bp A2UCOE (0.9 UCOE), and FOXN1 , a master transcription factor for TEC differentiation. This compact construct enables site-specific genome integration and stable inducible expression within iPSCs for renewably generating TECs. While the 0.9 UCOE promoted stable expression of constitutively expressed genes, it also caused unintended FOXN1 gene leakage, leading to unprogrammed gradual differentiation of the iPSCs. We generated A2UCOE fragments of varying lengths and found that gene leakage persisted regardless of fragment size. We tested spacer sequences between the A2UCOE and the Tet-On promoter consisting of varying AT-nucleotide content (35, 50, and 65%) and the 65% AT-rich SV40 poly-A terminator sequence and found that only the SV40 poly-A mitigated this leakage, and surprisingly, enhanced desired anti-silencing effects. These findings highlight the benefits and potential risks of using A2UCOE in iPSCs, and provides insights into a regulatory DNA architecture optimal for compact forward programming circuits for controlled iPSC differentiation. Graphical abstract