Parallel functional testing identifies enhancers active in early postnatal mouse brain

Cis-regulatory elements such as enhancers 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 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 the self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identify 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 normal and pathogenic brain development. Overall design: 1) Massively Parallel Reporter Assays (MPRA) testing 408 ~900 bp human amplicons in AAV vector: 4 paired RNA and DNA replicates, 1 AAV plasmid library; 1 high-PCR-cycle technical replicate. Illumina PE150 reads. 2) Massively Parallel Reporter Assay with 16 ~900 bp mouse amplicons with human orthologs (mini_MPRA): 4 RNA replicates and 1 AAV plasmid library. Illumina PE75 reads.