Engineering CREB‐activated promoters for adenosine‐induced gene expression

• DOI: 10.1002/biot.202300446
• Publication Date: 2024-02-26T02:05:47Z
• Authors: John Robert Cox; Andrea Fox; Conor Lenahan; Liza Pivnik; Matthew Manion; John Blazeck
• Keywords: 0303 health sciences; 03 medical and health sciences

Accumulation of the ribonucleoside, adenosine (ADO), triggers a cAMP response element binding protein (CREB)-mediated signaling pathway to suppress function immune cells in tumors. Here, we describe collection CREB-activated promoters that allow for strong and tunable ADO-induced gene expression human cells. By optimizing number CREB transcription factor sites altering core promoter region CREB-based hybrid promoters, created synthetic constructs drive higher levels than strong, endogenous mammalian presence ADO. These are induced up 47-fold by ADO, with minimal their "off" state. We further determine our activated other compounds act as analogs, combinatorial addition ADO these has synergistic impact on expression. Surprisingly, also detail how background degradation caused common cell culture media additive, fetal bovine serum (FBS), confounds experiments designed dose-responsiveness. show only after long-term heat deactivation FBS can enable induction at physiologically relevant Finally, demonstrate strength is enhanced incorporating sites.