Parallel functional testing identifies enhancers active in early postnatal mouse brain

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Enhancers are cis-regulatory elements crucial for gene expression in the brain.
• Massively parallel reporter assays (MPRAs) allow high-throughput screening of DNA sequences for enhancer activity.
• Understanding non-coding sequences' role in development and disease is vital.

Purpose of the Study:

• To adapt MPRA for in vivo screening of enhancer function in the early postnatal mouse brain.
• To identify and validate novel brain-specific enhancers.
• To investigate regulatory elements associated with neuropsychiatric disorders.

Main Methods:

• Adapted a self-transcribing regulatory element MPRA strategy.
• Delivered the MPRA system to the mouse brain using recombinant adeno-associated virus (rAAV).
• Performed paired screening and single enhancer in vivo functional testing.

Main Results:

• Successfully identified and validated putative enhancers driving reporter gene expression in the mouse forebrain.
• Discovered regulatory elements within an intronic CACNA1C linkage disequilibrium block.
• Demonstrated the utility of rAAV-mediated MPRA for in vivo enhancer characterization.

Conclusions:

• rAAV-delivered MPRA is a powerful tool for large-scale in vivo enhancer screening in the brain.
• Identified novel enhancers, including those linked to neuropsychiatric disorder risk genes like CACNA1C.
• This approach advances the understanding of enhancer function in brain development and disease.