An in vivo systemic massively parallel platform for deciphering animal tissue-specific regulatory function

Area of Science:

• Genomics
• Neuroscience
• Molecular Biology

Background:

• Non-protein coding enhancer sequences are critical for cell identity and function, especially in neural tissues.
• Understanding gene regulatory mechanisms in the brain is vital for deciphering neurodevelopment and neurological disorders.
• Challenges exist in studying non-coding regulatory elements in vivo, particularly in animal models.

Purpose of the Study:

• To introduce and validate a systemic Massively Parallel Reporter Assay (sysMPRA) for in vivo enhancer analysis in animals.
• To overcome limitations of traditional MPRA methods in animal studies, such as viral delivery and transduction efficiency.
• To assess the utility of sysMPRA in identifying tissue-specific enhancer function and regulatory element impacts.

Main Methods:

• Development of a systemic MPRA (sysMPRA) utilizing intravenous adeno-associated virus (AAV) delivery.
• Application of sysMPRA in mice to transduce MPRA libraries into diverse tissues.
• Analysis of enhancer activity, tissue specificity, and effects of genetic variations and transcription factor binding sites.

Main Results:

• Successful transduction of MPRA libraries into various mouse tissues using sysMPRA.
• Efficient identification of tissue-specific enhancer activity, consistent with machine learning predictions.
• Uncovered regulatory effects of MEF2C transcription factor binding site disruption, SNPs, and Alzheimer's disease-associated genetic variations.

Conclusions:

• SysMPRA is a robust method for delivering MPRA libraries systemically in vivo.
• This approach enables simultaneous determination of transcriptional functions for hundreds of enhancers across multiple tissues.
• SysMPRA facilitates the study of non-coding regulatory elements and their role in health and disease.