Jason T Lambert, Linda Su-Feher, Karol Cichewicz, Tracy L Warren, Iva Zdilar et al.
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.
Jason T Lambert, Linda Su-Feher, Karol Cichewicz, Tracy L Warren, Iva Zdilar, Yurong Wang, Kenneth J Lim, Jessica L Haigh, Sarah J Morse, Cesar P Canales, Tyler W Stradleigh, Erika Castillo Palacios, Viktoria Haghani, Spencer D Moss, Hannah Parolini, Diana Quintero, Diwash Shrestha, Daniel Vogt, Leah C Byrne, Alex S Nord. Parallel functional testing identifies enhancers active in early postnatal mouse brain. 2021-10-04. https://doi.org/10.7554/eLife.69479
A 16-element mouse miniMPRA library containing putative enhancer candidates and negative controls was cloned into the 3′ UTR of an Hsp1a minimal-promoter EGFP reporter. The scAAV9(2YF) library was delivered to P0 mouse prefrontal cortex and assayed in P7 brain using four biological replicate RNA/DNA sequencing measurements.
The main STAR408 screen tested 408 approximately 900 bp human candidate regulatory sequences from GWAS, LD, FBDHS, and PutEnh design groups in P7 mouse forebrain after P0 scAAV delivery. Four biological replicate DNA and RNA libraries were used to quantify enhancer activity by normalized RNA/DNA ratios and a GC-adjusted residual model; the packaged table retains the authors' 308 QC-passing amplicons.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 11 files (ZIP)elife-69479_article.pdfsource_notes.txtsupplementary_file_1_miniMPRA_counts_prop_act.csvsupplementary_file_2_STAR408_coordinates.csvsupplementary_file_3_STAR408_counts.csvsupplementary_file_4_STAR408_activity_qc.csvsupplementary_file_5_epigenomic_intersections.csvsupplementary_file_6_epigenomic_predictors.csvsupplementary_file_7_SNP_All_440.csvsupplementary_file_8_STAR408_GEO_Metadata.xlsxsupplementary_file_9_in_silico_PCR.csv