In the vast landscape of human DNA, a hidden regulatory layer is rewriting the textbook of genetic control.
We often think of our DNA as a blueprint, with genes as precise instructions for building and maintaining our bodies. Yet, only about 2% of our genome actually codes for proteins. The remainder, once dismissed as "junk DNA," is now known to be a complex control network. Among its most crucial components are enhancers—distant regulatory elements that act like powerful switches to turn genes on. Recently, scientists discovered that these switches themselves produce molecules called enhancer RNAs (eRNAs). Once considered mere byproducts, eRNAs are now emerging as pivotal players in human health and disease, influencing everything from our eye color to our susceptibility to cancer.
To understand the significance of enhancer RNAs, we must first grasp the basics of genetic regulation. Enhancers are short regions of DNA that can boost the activity of specific genes, often located far away on the chromosome chain. They act like orchestra conductors, coordinating when and where genes should be active without coding for any protein themselves.
Enhancers are regulatory DNA elements that control when and where genes are expressed, often located far from the genes they regulate.
When activated, enhancers transcribe enhancer RNAs (eRNAs) that function as regulatory signals rather than protein templates.
When an enhancer is activated, it doesn't just silently influence gene expression; it becomes a site of active transcription, producing a type of non-coding RNA called enhancer RNA (eRNA). Unlike messenger RNA (mRNA), which carries instructions for protein assembly, eRNA is not a template for proteins. Instead, it functions as a regulatory signal in its own right 12.
Research has shown that eRNAs play multiple critical roles in gene regulation. They can act as scaffolds that maintain the stability of the transcription complex, ensuring that enhancers and promoters physically interact correctly. They also serve as markers for actively engaged enhancers, providing a clear signpost of which regulatory elements are currently directing the genetic symphony 2.
Their discovery has opened new avenues for understanding how complex gene expression patterns are established and maintained across different tissues and developmental stages.
Before the creation of the Human enhancer RNA Atlas (HeRA), the expression landscape of eRNAs across normal human tissues remained largely unexplored. While individual studies had identified specific eRNAs with important functions, no comprehensive map existed to guide researchers through this complex regulatory network 1.
The HeRA project emerged to fill this critical knowledge gap. Launched in 2021 by a team at The University of Texas Health Science Center at Houston, HeRA represents a monumental effort to characterize eRNAs across 54 human tissues using data from the Genotype-Tissue Expression (GTEx) project 17. By analyzing a staggering 9,577 samples, the team identified 45,411 detectable eRNAs, creating the first systematic atlas of its kind 127.
Unprecedented scale for eRNA characterization
Comprehensive coverage of human anatomy
Vast expansion of known regulatory elements
Creating HeRA required a sophisticated methodological approach that integrated multiple massive datasets:
Researchers collected enhancer annotations from three major consortia: the ENCODE Project, the FANTOM Project, and the Roadmap Epigenomics Project 27. By integrating these resources and applying rigorous filtering—excluding regions that overlapped with known coding or non-coding genes—they established a comprehensive set of potential eRNA-producing regions.
Using RNA-seq data from GTEx, the team mapped sequencing reads to the human genome (hg19) and calculated eRNA expression levels using the Reads Per Million (RPM) method. Only eRNAs with RPM ≥ 1 were considered detectable, ensuring a focus on biologically relevant molecules 2.
The team investigated connections between eRNA expression and various traits—including gender, race, age, height, weight, and body mass index—using appropriate statistical tests with stringent significance thresholds 2.
Perhaps most importantly, HeRA constructed co-expression networks to identify putative regulators of eRNAs (such as transcription factors) and potential target genes, providing insights into the complex web of genetic regulation 12.
The construction of HeRA represents a landmark achievement in genomics. Let's examine the experimental approach that made this possible and the remarkable findings that emerged.
The team gathered enhancer annotations from three major epigenetic databases—ENCODE, FANTOM, and Roadmap Epigenomics—and integrated them into a unified set of potential eRNA regions, which were lifted over to the hg19 genome build for consistency 2.
Using 9,577 RNA-seq samples from the GTEx project across 54 human tissues, researchers mapped sequencing reads to the reference genome using HISAT2, a sophisticated alignment tool 2.
For each identified enhancer region, they calculated expression levels using SAMtools and normalized these values using the Reads Per Million (RPM) method to enable cross-sample comparison 2.
The team correlated eRNA expression with six key traits: gender, race, age, height, weight, and BMI. They employed appropriate statistical tests for each trait type—t-tests for gender, ANOVA for race, and Spearman correlation for continuous variables like age—with false discovery rate (FDR) correction for multiple testing 2.
Using co-expression patterns across tissues, the researchers identified millions of potential relationships between eRNAs and transcription factors, as well as between eRNAs and target genes within a 1 MB window, excluding those located within intronic regions of potential target genes 2.
Where possible, the team validated eRNA-transcription factor pairs using transcription factor binding site (TFBS) data from ENCODE ChIP-seq experiments, providing additional evidence for the regulatory relationships 2.
The analysis yielded several transformative discoveries about eRNAs and their roles in human biology:
HeRA identified tens of thousands of significant associations between eRNA expression and human traits. These connections suggest that eRNAs may contribute to shaping complex human characteristics and disease susceptibilities related to gender, race, and age 12.
The co-expression analysis revealed millions of potential regulatory relationships, mapping out a complex network of how eRNAs are controlled and how they in turn influence target genes. This network provides a rich resource for hypothesis generation about gene regulation mechanisms 12.
The atlas demonstrated that eRNA expression follows distinct patterns across different tissues, reflecting their roles in tissue-specific gene regulation programs. This tissue specificity helps explain how the same genome can produce dramatically different cell types and functions 1.
Perhaps the most compelling aspect of HeRA's findings lies in the connections between eRNAs and human disease. The atlas provides evidence that eRNAs are not just incidental molecules but play active roles in shaping human health outcomes.
| eRNA Name | Related Condition | Proposed Mechanism |
|---|---|---|
| OLMALINC | Body weight/Body fat regulation | Regulates SCD1 gene involved in triglyceride metabolism |
| Not specified | Autism spectrum disorders | Affects target gene expression in human brain |
| NET1e | Breast cancer tumorigenesis | Regulates NET1 oncogene expression |
| HPSEe | Cancer invasion and metastasis | Controls heparanase (HPSE) expression |
For instance, the database includes examples such as OLMALINC, an eRNA associated with body weight through its regulation of stearoyl-coenzyme A desaturase, a gene involved in serum triglyceride metabolism 2. Another eRNA has been linked to autism spectrum disorders through its effect on target gene expression in the human brain 2.
In cancer biology, eRNAs like NET1e regulate the expression of the oncogene NET1 in breast cancer to promote tumorigenesis, while HPSEe controls the expression of heparanase (HPSE) to facilitate cancer invasion and metastasis 2. These disease connections transform eRNAs from mere curiosities into potential therapeutic targets for future treatments.
For researchers venturing into the world of enhancer RNAs, HeRA provides an invaluable starting point. The web portal (https://hanlab.uth.edu/HeRA/) offers four main modules to explore eRNAs: Expression, associated Traits, potential Regulators, and potential Target genes across human tissues 4.
Beyond HeRA itself, the field relies on several crucial resources and methodologies that formed the foundation of this atlas:
| Resource/Method | Function | Role in eRNA Research |
|---|---|---|
| GTEx Project | Tissue-specific gene expression data | Provided the foundational RNA-seq data across 54 human tissues |
| ENCODE, FANTOM, Roadmap | Epigenomic annotation | Supplied enhancer region annotations for eRNA identification |
| HISAT2 | Sequence alignment | Mapped RNA-seq reads to the reference genome (hg19) |
| SAMtools | Sequence data processing | Quantified eRNA expression levels from aligned reads |
| ChIP-seq Data | Protein-DNA interaction mapping | Validated transcription factor binding at eRNA loci |
The creation of the Human enhancer RNA Atlas represents a paradigm shift in how we understand genetic regulation. By mapping the expression landscape of eRNAs across human tissues, HeRA has illuminated a hidden layer of our genome that influences everything from basic physiology to complex disease.
This resource, which characterizes 45,411 eRNAs from 9,577 samples across 54 human tissues, provides an unprecedented window into the complex regulatory networks that make us human 17. It enables researchers to explore trait-related eRNAs, identify potential regulators, and discover target genes through an intuitive web interface.
As we continue to explore this uncharted territory, each eRNA represents not just a scientific curiosity but a potential key to understanding human diversity, disease susceptibility, and the very mechanisms that coordinate the intricate dance of gene expression. The hidden switches of life, once invisible, are now coming into clear view—and they promise to revolutionize medicine in the decades to come.