Journal article
Massively parallel cis-regulatory analysis in the mammalian central nervous system
Genome research, Vol.26(2), pp.238-255
02/01/2016
DOI: 10.1101/gr.193789.115
PMCID: PMC4728376
PMID: 26576614
Abstract
Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.
Details
- Title: Subtitle
- Massively parallel cis-regulatory analysis in the mammalian central nervous system
- Creators
- Susan Q. Shen - Washington University in St. LouisConnie A. Myers - Washington University in St. LouisAndrew E. O. Hughes - Washington Univ, Sch Med, Dept Pathol & Immunol, St Louis, MO 63110 USALeah C. Byrne - University of California, BerkeleyJohn G. Flannery - University of California, BerkeleyJoseph C. Corbo - Washington University in St. Louis
- Resource Type
- Journal article
- Publication Details
- Genome research, Vol.26(2), pp.238-255
- DOI
- 10.1101/gr.193789.115
- PMID
- 26576614
- PMCID
- PMC4728376
- NLM abbreviation
- Genome Res
- ISSN
- 1088-9051
- eISSN
- 1549-5469
- Publisher
- Cold Spring Harbor Lab Press, Publications Dept
- Number of pages
- 18
- Grant note
- HG006790 / National Institutes of Health (National Human Genome Research Institute); United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Human Genome Research Institute (NHGRI) 275579 / Simons Foundation Autism Research Initiative EY018826; EY022975; EY024958; 5T32EY013360 / National Institutes of Health (National Eye Institute); United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Eye Institute (NEI) Foundation Fighting Blindness
- Language
- English
- Date published
- 02/01/2016
- Academic Unit
- Psychiatry; Iowa Neuroscience Institute
- Record Identifier
- 9984618522202771
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