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

Ashley R Brown1,2, Grant A Fox1,2, Irene M Kaplow1,2,3

  • 1Ray and Stephanie Lane Department of Computational Biology, Carnegie Mellon University, Pittsburgh, PA, United States.

Frontiers in Genetics
|April 24, 2025
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Summary

Systemic MPRA (sysMPRA) enables efficient in vivo analysis of gene enhancers across multiple tissues. This method overcomes challenges in animal studies, revealing regulatory effects of genetic variations and transcription factor binding sites.

Keywords:
PHP.eBaavbrainenhancerin vivomachine learningtissue specifictranscriptional regulation

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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.