Anin vivomassively parallel platform for deciphering tissue-specific regulatory function

Abstract

Genetic studies are rapidly identifying non-protein-coding human disease-associated loci. Understanding the regulatory mechanisms underlying these loci remains a challenge because the causal variants and the tissues in which they act are often unclear. Massively parallel reporter assays (MPRAs) have the potential to link differences in genome sequence, including genetic variants, to tissue-specific regulatory function. Although MPRA and similar technologies have been widely adopted in cell culture, there have been several barriers to widespread use in animals. We overcome these challenges with a new whole-animal MPRA (WhAMPRA), where systemic intravenous AAV effectively transduces the plasmid MPRA library to mouse tissues. Our WhAMPRA approach revealed models of tissue-specific regulation that generally match machine learning model predictions. In addition, we measured the regulatory effects of disrupting MEF2C transcription factor binding sites and impacts of late onset Alzheimers disease-associated genetic variations. Overall, our WhAMPRA technology simultaneously determines the transcriptional functions of hundreds of enhancers in vivo across multiple tissues.

Targeted mutagenesis of specific genomic DNA sequences in animals for thein vivogeneration of variant libraries

“…The advent of MPRAs has revolutionized our ability to query biological phenomena, from dissecting gene regulatory regions to systematically studying protein function (Arnold et al, 2013; Brown et al, 2022; Chan et al, 2023; Inoue and Ahituv, 2015; Ireland et al, 2020; Jores et al, 2020; Kheradpour et al, 2013; Lagunas et al, 2023; Lalanne et al, 2024; Qi et al, 2020; Staller et al, 2022). Yet, these advances have been mostly relegated to cells in culture, where the transfection of massive libraries of DNA is feasible.…”

“…To uncover these rules it is therefore necessary to find the binding sites within an enhancer with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively Parallel Reporter Assays (MPRAs) have made it possible to perform this feat in the context of cells in culture and in some multicellular organisms (Arnold et al, 2013; Brown et al, 2022; Chan et al, 2023; Inoue and Ahituv, 2015; Ireland et al, 2020; Jores et al, 2020; Kheradpour et al, 2013; Lagunas et al, 2023; Lalanne et al, 2024; Qi et al, 2020). Here, a barcoded library of reporter genes, each driven by a randomly mutated regulatory region or genome fragments, is generated in vitro and transfected into cells.…”

“…Indeed, the incorporation of a large number of DNA variants is not possible in most multicellular organisms. As a result, with some notable exceptions in adult mouse brains, C. elegans , Ciona intestinalis and tobacco leaves (Brown et al, 2022; Chan et al, 2023; Farley et al, 2015; Jores et al, 2020; Lagunas et al, 2023; Stevenson et al, 2023), the field lacks a reliable pipeline to incorporate DNA diversity into animals and plants, and to deploy MPRAs to uncover the rules that dictate the development and physiology of multicellular organisms.…”

“…A second limitation of MPRAs-which applies to both cells in culture and multicellular organisms-is their reliance on reporter constructs: enhancers within the library are typically integrated into a plasmid that allows for the simultaneous measurement of enhancer sequence and reporter gene expression level (Arnold et al, 2013;Brown et al, 2022Brown et al, , 2022Chan et al, 2023Chan et al, , 2023Farley et al, 2015;Inoue and Ahituv, 2015;Ireland et al, 2020;Jores et al, 2020Jores et al, , 2020Kheradpour et al, 2013;Lagunas et al, 2023Lagunas et al, , 2023Lalanne et al, 2024;Qi et al, 2020). In most cases these reporter constructs remain episomal and lack chromatin such that they cannot capture information about the genomic context that might be at play such as histone modification landscape.…”

Rapid and Quantitative Functional Interrogation of Human Enhancer Variant Activity in Live Mice

“…Likewise, loss-of-function enhancer variants often result in loss of enhancer activity in one cell type, while in other cell types, its activity is unaffected (Bengani et al, 2021; Bhatia et al, 2015, 2021; Shin et al, 2023; Spieler et al, 2014). These cell-type-specific effects of enhancer variants are difficult to capture with high-throughput methods such as massively-parallel reporter assays (MPRAs) and CRISPR inhibitor/activator screens, both of which are primarily performed in vitro (Findlay, 2021; Inoue & Ahituv, 2015; Maricque et al, 2018) or in one tissue (Brown et al, 2022; Capauto et al, 2023; Deng et al, 2023; Lagunas et al, 2023; Patwardhan et al, 2012; White et al, 2013). Transgenic enhancer-reporter assays in mice enable visualization of enhancer activity in the whole animal and are a gold-standard for functionally testing when and where a human enhancer is active in vivo (Kothary et al, 1989; Pennacchio et al, 2006; Visel et al, 2006).…”