Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.

Harnessing bioluminescence for drug discovery and epigenetic research

“…A second 'nondisruptive' approach aims to couple the expression of the luciferase gene with the GOI, while preserving its function. This can be achieved using either an internal ribosomal entry site (IRES), as demonstrated for Vegfr3 (Olmeda et al, 2017) and Utrn (Vuorinen et al, 2021), or using a 2A self-cleaving peptide sequence (T2A site) (Van de Pette et al, 2017;Son et al, 2021;Pham et al, 2022;Van de Pette et al, 2022;Gleneadie et al, 2023). During translation, T2A cleavage results in expression of endogenous protein and a separate luciferase reporter.…”

“…This is particularly beneficial for multi-reporter models where a GOI is coupled to more than one reporter gene. Dual reporter models have been developed for Cidea (Son et al, 2021), Utrn (Gleneadie et al, 2023), Cdkn1c (Van de Pette et al, 2017), Dlk1 (Van de Pette et al, 2022), Fgf21 (Pham et al, 2022) and Dpd (Smith et al, 2022) (Table 1) where the reporters were separated from each other and from the endogenous gene by T2A sites. These models combine the high sensitivity and real-time dynamics of bioluminescence for in vitro screening and in vivo imaging with the cellular resolution of lacZ or fluorescent reporters (Day et al, 1998;Contag et al, 2000).…”

“…These models combine the high sensitivity and real-time dynamics of bioluminescence for in vitro screening and in vivo imaging with the cellular resolution of lacZ or fluorescent reporters (Day et al, 1998;Contag et al, 2000). The everexpanding diversity of luciferin/luciferase systems provides the opportunity to visualise expression of more than one gene of interest in the same animal using luciferase proteins which either require different substrates for activation (Jones et al, 2017) or produce spectrally distinct luminescence signal (Mezzanotte et al, 2011;Gleneadie et al, 2023;McMorrow et al, 2023) (exemplified in Figure 1A, right-hand panel, red and green).…”

“…BLI offers a powerful tool for preclinical research. Reporters can be used to visualise progression of a tumour (O'Farrell et al, 2013) or pathogen (Chang et al, 2014;Vande Velde et al, 2014;Daniel et al, 2015), or can represent expression of a disease-relevant gene, including epigenetically-silenced imprinted (Van de Pette et al, 2017; Van de Pette et al, 2022;Dimond et al, 2023) or X-linked genes (Tinsley et al, 2011;Sripathy et al, 2017;Lee et al, 2020;Grimm and Lee, 2022;Gleneadie et al, 2023). A key benefit of using bioluminescent reporters for drug screening is that one genetic model can be used for each stage of preclinical research, from in vitro drug screening to in vivo testing of candidate molecules (as illustrated in Figure 1B).…”

“…Initial reporters contained luciferase under the control of the Utrn promoter (Moorwood et al, 2011;Tinsley et al, 2011;Guiraud et al, 2015b) or the UTRs . To better represent the full genomic context of endogenous Utrn, we recently developed a Utrn reporter mouse with luciferase and lacZ inserted directly into the 3' UTR of endogenous Utrn, linked by T2A sites (Gleneadie et al, 2023) (Table 1). A screen of chromatin modifying drugs using a myoblast cell line derived from these mice determined that inhibition of EZH2, in combination with ERK inhibition, causes increased Utrn expression, outlining a potential route for supplementing Dmd deficiency with Utrn expression (Figure 1B, right) (Gleneadie et al, 2023).…”