Abstract
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.
Highlights
Cis-regulatory elements such as enhancers are critical drivers of spatiotemporal gene expression within the developing and mature brain[1]
After counterstaining brain sections transduced as described above with antibodies for Ctip[2], we found that EGFP+ cells driven by Dlx exhibited significantly lower frequencies of Ctip2+ nuclei compared to either CAG-driven mRuby3+ cells or EGFP+ cells driven by amplicon #161 (Figure 3B, C). These results demonstrate that our massively parallel reporter assays (MPRAs) could accurately reflect enhancer activity of particular candidate sequences in vivo. 354 Dissection of regulatory elements within the third intron of CACNA1C Our library included amplicons spanning across a psychiatric disorder-associated LD interval within the ~330 kb third intron of the gene CACNA1C, which encodes the α1 subunit of the L357 type voltage-gated calcium channel CaV1.2
We show that amplicons active in our MPRA were more likely to have enhancer signature across functional genomics datasets and that orthologous mouse sequences tested in an independent parallel reporter assay showed strong activity correlation
Summary
Cis-regulatory elements such as enhancers are critical drivers of spatiotemporal gene expression within the developing and mature brain[1]. 327 Confirmation of in vivo P7 cortex MPRA results for single candidate sequences To validate enhancer activity in the mouse brain at P7, we cloned individual amplicons for selected positive and negative hits from the MPRA into the same HspMinP-EGFP 3’UTR oriented reporter and generated scAAV9 for each construct. Amplicon #161 (FBDHS group), an enhancer candidate that overlaps both a DNaseI hypersensitive site in fetal human brain and a copy number variant region near the autism- and epilepsy-associated gene SCN2A, and that displayed strong activity in the screen (Figure 3–figure supplement 1A), showed consistent expression of EGFP in the mouse brain at P7 (Figure 3A, top row). In a follow-up experiment, we transduced scAAV9-HspMinP-EGFP-#3 and AAV9-CAG-mRuby[3] at P0 but waited to collect the brains until P28, at which time we observed that amplicon #3 drove EGFP expression in cortical neurons of adolescent mice (Figure 4C, Figure 4–figure supplement 2). 382 These results suggest that the AAV MPRA implementation was effective at screening putative regulatory sequences for in vivo activity in the brain, with reporter expression concordant between MPRA results and single candidate tests of EGFP expression in P7 mouse forebrain for five individually validated amplicons, and that these results could be extended to study activity in later development
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