Gut microbiota modulation of regulatory DNA elements revealed by massively parallel functional characterization

Area of Science:

• Genomics
• Molecular Biology
• Hepatology

Background:

• Cis-regulatory elements (CREs) are crucial for gene regulation in liver cells (hepatocytes).
• Current functional annotations of CREs often rely on in vitro models, which may not accurately reflect physiological conditions in vivo.
• Understanding in vivo regulation is essential for comprehending liver function and disease.

Purpose of the Study:

• To systematically profile and functionally annotate human liver-derived CREs under matched in vitro and in vivo conditions.
• To investigate the role of gut microbiota-derived signals in modulating hepatic CRE activity and gene expression.
• To identify transcription factors and pathways involved in in vivo regulation of hepatic CREs.

Main Methods:

• Massively parallel reporter assays (MPRAs) were used to profile 109,386 human liver-derived CREs.
• Hepatocytes were studied under both in vitro and in vivo conditions for comparative analysis.
• Chromatin marks (H3K27ac) and accessibility were analyzed, alongside transcription factor binding and microbial metabolite treatments.

Main Results:

• Identified in vivo-active functional CREs (fCREs) enriched for specific epigenetic marks and transcription factor binding.
• Demonstrated that gut microbiota signals modulate fCRE activity and target gene expression in vivo, partly through the KEAP1/NFE2L2 antioxidant pathway.
• Showed that microbial metabolites directly impact fCRE activity and that genetic variations within fCREs influence their response to microbial signals.

Conclusions:

• Gut microbiota significantly influences hepatic CRE activity and gene expression in vivo.
• Revealed condition-specific gene regulatory mechanisms in the liver, highlighting the importance of the in vivo environment.
• Established a link between microbial metabolites, hepatic fCREs, and the KEAP1/NFE2L2 pathway, offering insights into liver physiology and potential therapeutic targets.