The human genome, often conceptualized as a linear chemical code, is physically arranged in a highly organized, three-dimensional structure within the nucleus of a cell. This intricate folding plays a fundamental role in controlling which genes are turned on or off. Promoter Capture Hi-C (PC Hi-C) is a specialized laboratory technique developed to map these complex spatial relationships on a genome-wide scale. The method identifies which distant pieces of DNA physically touch the regions that initiate gene expression, known as promoters. By revealing these physical contacts, PC Hi-C provides a high-resolution map of the genome’s wiring diagram, showing how distant regulatory elements interact with their target genes. This approach offers a powerful way to understand gene regulation and how its disruption can contribute to disease.
Understanding the 3D Genome Structure
The physical organization of DNA is a multi-layered hierarchy that directly influences cellular function and gene expression. At the largest scale, each chromosome occupies a distinct, non-overlapping space within the nucleus called a chromosome territory. Within these territories, the DNA folds into complex domains, creating physical proximity between sequences separated by vast distances along the linear code.
A major level of organization is the Topologically Associating Domain (TAD). A TAD is a segment of DNA that preferentially interacts with itself rather than with sequences outside its boundary. TADs act like segregated neighborhoods, typically spanning hundreds of thousands to a million base pairs. Regulatory elements within a TAD are constrained to interact primarily with the genes contained within the same domain.
Nested within these large domains are smaller, more dynamic structures known as chromatin loops. These loops bring specific regulatory sequences, such as enhancers, directly into contact with the promoter of a target gene, sometimes bridging hundreds of kilobases of intervening DNA. The formation and dissolution of these precise loops are the immediate physical mechanism controlling gene expression. Standard DNA sequencing provides only the linear arrangement of the code, which is insufficient to determine which distant enhancer regulates which specific promoter.
How Promoter Capture Hi-C Works
Promoter Capture Hi-C is an advanced variant of the general Hi-C method, which identifies all possible physical contacts within the genome. The standard Hi-C method begins by chemically fixing the DNA in the cell, locking physically interacting segments into place. The DNA is then cut into small pieces. Segments that were physically close are chemically ligated, or joined together, even if they were far apart in the linear sequence.
PC Hi-C drastically improves efficiency by introducing a targeted enrichment step using specialized “bait” sequences. These baits are short, synthetic RNA probes designed to be complementary to the sequence of nearly all known gene promoters in the genome. After the initial ligation step, these biotinylated RNA baits are introduced to the sample. They act like molecular magnets, specifically hybridizing only to the DNA fragments that contain a promoter sequence.
A magnetic purification step then pulls down only the promoter-containing fragments, along with any other DNA segments that were physically ligated to them. This selective capture focuses the subsequent high-throughput sequencing entirely on promoter-centric interactions. This achieves a substantial enrichment—often 35- to 100-fold—over traditional Hi-C, allowing researchers to map these specific contacts with much higher resolution and efficiency. The resulting sequenced fragments provide a direct, quantitative measure of how frequently any given promoter interacts with other regions of the genome.
Unlocking Gene Regulation and Disease Links
The high-resolution maps generated by PC Hi-C have clarified the relationship between enhancers and promoters. Before this technique, the targets of the estimated one million enhancers in the human genome were largely unknown, as linear distance was a poor predictor of regulatory control. PC Hi-C directly maps enhancer-promoter loops, revealing the true target gene for thousands of distant regulatory elements in a single experiment. This often shows that a gene is regulated by a far-away enhancer while bypassing several closer genes.
One significant application of PC Hi-C is interpreting the results of Genome-Wide Association Studies (GWAS). GWAS have identified thousands of genetic variations linked to complex diseases like diabetes, heart disease, and autoimmune disorders. The vast majority of these disease-associated variations, or Single Nucleotide Polymorphisms (SNPs), fall within non-coding regions of the genome, meaning they do not alter a protein sequence. Instead, these SNPs often reside in enhancers, where they alter the regulatory element’s function.
By integrating GWAS data with PC Hi-C interaction maps, researchers can trace a non-coding, disease-associated SNP to the specific promoter it physically contacts and regulates. For instance, a variant linked to body mass index (BMI) was found using PC Hi-C to reside in a regulatory element that loops to the promoter of the MAP2K5 gene, despite the gene being located far away on the linear sequence. This ability to link a non-coding risk variant to its actual target gene is transforming disease research by identifying the precise molecular pathway affected by a genetic change.
PC Hi-C has been used to generate atlases of long-range promoter interactions in various cell types. This offers a comprehensive view of how the 3D genome architecture dictates the expression patterns that characterize different cell identities and disease states.