LAUSR

Expression of recombinant genes in HEK293 cells is frequently utilized for production of recombinant proteins and viral vectors. These systems frequently employ the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. However, the mechanistic basis of CMV‐mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis‐regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c‐Rel, and NF‐κB. We also identified critical Sp1‐dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP‐Jκ binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV‐derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild‐type counterpart.