In silico design of context-responsive mammalian promoters with user-defined functionality

Abstract

Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation.

CHO genome mining for synthetic promoter design

“…In this context, synthetic promoters are an attractive alternative as they can be customdesigned to engineer recombinant gene transcription predictably, removing functionally illdefined and uncontrollable elements in expression vectors. Indeed, we have previously reported synthetic promoters that offer stable, precise control of recombinant transcriptional activity in CHO cells (Brown et al 2014(Brown et al , 2017. These genetic constructs were designed by (i) analysis of CHO cell transcription factor (TF) expression profiles, and (ii) determination of the transcription factor regulatory element (TFRE) compositions of commonly utilized viral promoters.…”

“…Accordingly, design rules for constructing synthetic elements with desired functionalities can be obtained by either (i) determination of host cell TF repertoires under appropriate experimental conditions, or (ii) analysis of TFRE compositions of endogenous promoters exhibiting suitable expression dynamics. Whilst synthetic promoters have been created for CHO cells by analysis of TF expression levels (Brown et al 2017), CHO genomic (i.e. endogenous TFRE) information has never been utilized, potentially limiting opportunities for synthetic promoter design.…”

“…This analysis identified 11 TFREs that significantly increased expression (>5-fold) over basal expression from the minimal core promoter (NF B, NF B-p65, GABP , DMP1, AhR/ARNT, USF1, STAT3, Sp1, ZBED1, Pax3 and EGR-1). Amongst these, only 3 TFREs -NF B, USF1 (E-box) and Sp1 (GC-box) have been identified for our previous viral-and TF-derived synthetic promoter constructs (where the former was the most active element in CHO cells; Brown et al 2014Brown et al , 2017.…”

Whole synthetic pathway engineering of recombinant protein production

“…A component with this functionality has reportedly been constructed for use in CHO cells. Synthetic promoter 100RPU.1 was specifically designed in silico, using transcription factor binding sites (TFBSs) with requisite functionalities, to exhibit maximal transcriptional activity and long-term expression stability without imposing off-target effects on CHO cell growth or viability (Brown, Gibson, Hatton, & James, 2017).…”

“…We hypothesized that the output from 100RPU.1 could be enhanced by either increasing the number of TFBSs in the proximal region (limited to 14 in the original design), or replacing the nonengineered minimal core region (taken from the hCMV-IE1 promoter). We therefore (a) constructed a hybrid promoter (100RPU_PH) containing the proximal regions from both 100RPU.1 and a second highly active synthetic part, 100RPU.2 (Brown et al, 2017), and (b) created elements (100RPU.1_SC2, 100RPU.1_SC3, and 100RPU.1_SC4) where the hCMV-IE1 core promoter was substituted by varying "super cores" that are specifically designed to maximize transcription initiation rates (Even et al, 2016;Juven-Gershon et al, 2006; see Supporting Information Figure S1).…”