Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.
All supplementary information, including links to raw and processed data, can be found at the Nord Lab Resources page (https://nordlab.faculty.ucdavis.edu/resources/). Software can be found at the Nord Lab Git Repository (https://github.com/NordNeurogenomicsLab/) and https://github.com/NordNeurogenomicsLab/Publications/tree/master/Lambert_eLIFE_2021. Sequencing data have been deposited in GEO under accession code GSE172058.
Parallel functional testing identifies enhancers active in early postnatal mouse brainNCBI Gene Expression Omnibus, GSE19373 GSE172058.
Roadmap Consolidated Peak DatasetGEO GSM530651, GSM595913, GSM595920, GSM595922, GSM595923, GSM595926, GSM595928, GSM806934, GSM806939, GSM621457, GSM706999, GSM806935, GSM621427, GSM707000, GSM806936, GSM621393, GSM707001, GSM806937, GSM621410, GSM707002, GSM806938.
- Jason T Lambert
- Linda Su-Feher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All procedures were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of California Animal Care and Use Committee (AUP #R200-0913BC). Surgery was performed under anesthesia, and all efforts were made to minimize suffering.
- Genevieve Konopka, University of Texas Southwestern Medical Center, United States
- Received: April 19, 2021
- Accepted: October 2, 2021
- Accepted Manuscript published: October 4, 2021 (version 1)
© 2021, Lambert et al.
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