Gut microbiota-derived signals modulate functional cis-regulatory element activity and target gene expression in the liver in vivo, in part via the KEAP1/NFE2L2 antioxidant pathway, revealing microbiota-dependent regulation of hepatic gene regulatory mechanisms.
Key Findings
Methods
A massively parallel reporter assay (MPRA) systematically profiled 109,386 human liver-derived cis-regulatory elements (CREs) under matched in vitro and in vivo conditions in hepatocytes.
The study used massively parallel reporter assays (MPRAs) to functionally characterize CREs at scale.
CREs were profiled under both in vitro and in vivo conditions to allow direct comparison.
The CREs tested were derived from human liver.
Hepatocytes were used as the cellular model system.
Results
In vivo-active functional CREs (fCREs) were enriched for H3K27ac histone marks and chromatin accessibility compared to inactive elements.
fCREs identified in vivo showed enrichment for H3K27ac, an active enhancer mark.
Chromatin accessibility was also enriched at in vivo-active fCREs.
These epigenomic features distinguished in vivo-active from inactive regulatory elements.
The enrichment patterns suggest that in vivo conditions better capture physiologically relevant regulatory states.
Results
In vivo-active fCREs were regulated by diverse transcription factors active in the human liver.
Multiple transcription factors were implicated in regulating the identified fCREs.
Transcription factor activity was characterized specifically in the context of human liver.
The diversity of transcription factors suggests broad regulatory complexity at these elements.
This regulation was observed under in vivo conditions, distinguishing it from in vitro findings.
Results
Gut microbiota-derived signals modulate fCRE activity and target gene expression in the liver in vivo.
The study demonstrated that gut microbiota signals influence hepatic CRE function in vivo.
Both fCRE activity and the expression of fCRE target genes were affected by microbial signals.
This represents a direct link between gut microbiota and hepatic gene regulatory element function.
The modulation was observed in vivo, underscoring the physiological relevance of the findings.
Results
The KEAP1/NFE2L2 antioxidant pathway is partially responsible for mediating gut microbiota modulation of hepatic fCRE activity.
The KEAP1/NFE2L2 (also known as NRF2) pathway was identified as a mechanistic link between microbial signals and CRE regulation.
Modulation of fCRE activity by gut microbiota occurred 'in part via the KEAP1/NFE2L2 antioxidant pathway.'
The antioxidant pathway is a well-known sensor of cellular redox status and can be activated by microbial metabolites.
This finding implicates oxidative stress signaling as a conduit for microbiota-liver communication at the gene regulatory level.
Results
Specific microbial metabolites directly altered the activity of selected fCREs.
Specific (not all) microbial metabolites were shown to directly modulate fCRE activity.
This demonstrates a metabolite-level mechanism by which gut bacteria influence hepatic gene regulation.
The finding suggests metabolite specificity in microbiota-CRE interactions rather than a general effect.
These effects were observed in the context of the broader MPRA functional characterization framework.
Results
Genetic variation within fCREs modified their responsiveness to microbial signals.
Variants located within fCREs altered how those elements responded to gut microbiota-derived signals.
This finding connects common genetic variation to condition-specific (microbiota-dependent) gene regulation.
The result suggests that individual genetic differences may influence the liver's transcriptional response to microbiota.
This interaction between genetic variants and microbial signals highlights a potential mechanism for gene-environment interactions in liver function.
Background
In vitro models poorly capture physiological CRE regulation compared to in vivo conditions.
Most existing functional annotations of CREs derive from in vitro models.
The study directly compared in vitro and in vivo conditions using matched experimental designs.
In vivo-active fCREs showed distinct epigenomic and transcription factor features not fully captured in vitro.
This discrepancy motivates the use of in vivo MPRA approaches for physiologically relevant regulatory annotation.
What This Means
This research suggests that the bacteria living in our gut can influence how genes are turned on and off in the liver by modulating specific DNA 'switches' called cis-regulatory elements (CREs). Using a powerful technique that tested over 109,000 human liver DNA sequences simultaneously — both in cell culture and in living organisms — the researchers found that the set of regulatory elements that are truly active in a living body is different from what is seen in laboratory cell cultures alone. The active regulatory elements in vivo were marked by specific chemical tags on DNA packaging proteins and were controlled by a variety of liver-specific proteins that bind DNA.
A key finding is that signals from gut microbes — the trillions of bacteria inhabiting the intestine — reach the liver and change the activity of these regulatory DNA elements, partly through a well-known cellular stress-response pathway called KEAP1/NFE2L2 (the antioxidant or NRF2 pathway). Specific molecules produced by gut bacteria (metabolites) were shown to directly switch certain regulatory elements on or off. Additionally, natural genetic differences between individuals (variants in the DNA) within these regulatory elements changed how strongly they responded to microbial signals, hinting at why people may differ in their liver gene regulation depending on their gut microbiome.
This research matters because it reveals a previously underappreciated layer of communication between gut bacteria and liver gene regulation, and demonstrates that studying gene regulation in living systems — rather than just in laboratory dishes — is essential for understanding how the liver actually works. The findings could have implications for understanding liver diseases and conditions where both gut microbiome composition and liver gene expression play important roles.
Zaratiana C, M Y, Lee Y, Ong A, Liu T, Low S, et al.. (2026). Gut microbiota modulation of regulatory DNA elements revealed by massively parallel functional characterization.. Molecular cell. https://doi.org/10.1016/j.molcel.2026.03.036