In a groundbreaking exploration of genome architecture, researchers have uncovered that chromatin loops, intricate DNA structures that fold the genome, are a foundational feature of the animal regulatory genome, stretching back to some of the earliest diverging animal lineages. The study focused on the ctenophore Mnemiopsis leidyi, a comb jelly species whose genomic organization sheds new light on how gene regulation has evolved over hundreds of millions of years.
Chromatin loops serve as pivotal points of interaction between promoters—the regions in DNA that initiate gene transcription—and enhancers, which amplify this gene expression, even over long genomic distances. This spatial organization is central to precise gene regulation, ensuring that genes are turned on and off at appropriate times and locations during development and in response to the environment. In M. leidyi, the physical genome architecture is richly dominated by thousands of these chromatin loops, a discovery that challenges previous assumptions about the evolutionary novelty of such structures.
The researchers identified over four thousand chromatin loops in the M. leidyi genome, with the majority bridging promoter and enhancer elements. Nearly two-thirds of these looping interactions link promoters directly to enhancers, highlighting a complex network of distal regulatory elements that coordinate gene expression. Additional loops connect enhancers to other enhancers, suggesting a multilayered regulatory topology that refines the genome’s functional output.
Detailed analyses revealed that nearly a thousand gene promoters participated in chromatin looping. Each promoter varied in its number of enhancer contacts, ranging from a single enhancer to as many as fifteen. Notably, these enhancers predominantly reside within intronic regions—the non-coding segments within genes—and intergenic spaces, which lie between genes. This distribution indicates a broad regulatory landscape wherein enhancers may influence multiple genes in proximity.
Crucially, the study employed chromatin immunoprecipitation sequencing (ChIP-seq) targeting the cohesin complex subunit SMC1, a known architectural protein involved in loop extrusion, to pinpoint loop anchor sites. Cohesin enrichment at these loci corroborated the existence of bona fide chromatin loops, underscoring their structural and functional relevance.
To ascertain whether these chromatin loop features are shared beyond M. leidyi, the team extended their chromatin contact profiling to another ctenophore, Hormiphora californensis, which diverged from M. leidyi approximately 180 million years ago. Despite the lower resolution of this dataset, strong chromatin loops were still detected, indicating that such looping is a conserved trait within ctenophores. Moreover, genes that participated in chromatin loops displayed higher expression levels in both species, implying that looping correlates with active transcriptional regulation.
Digging deeper into the molecular underpinnings, the researchers sought DNA sequence motifs enriched at loop anchors. A striking discovery was a GC-rich motif present in over 75% of loop anchors in both species. This motif was prevalent at promoters and enhancers engaged in looping interactions and even appeared in a subset of promoters without detected loops, suggesting it plays a broader role in genome regulation.
Interestingly, this GC-rich motif contains two CpG dinucleotides, known substrates for DNA methylation, an epigenetic modification that can regulate gene expression. Using long-read Nanopore sequencing data, the team observed an overall low methylation level in M. leidyi, consistent with prior reports. However, within loop anchor regions, the GC-rich motifs exhibited markedly reduced DNA methylation compared to the same motifs located outside loop anchors. This observation suggests that methylation status of these motifs modulates loop formation, possibly by influencing protein binding affinities at these critical genomic sites.
Further supporting the involvement of DNA-binding proteins in loop formation, ATAC-seq footprinting revealed protected regions at the identified motifs, indicative of protein-DNA interactions. To identify candidate architectural proteins that might specifically recognize these motifs, the team profiled the chromatin-bound proteome of M. leidyi. Among the most abundant zinc finger C2H2 domain-containing proteins—domains often linked to DNA binding and chromatin organization—two novel factors emerged: CTEP1 and CTEP2.
These proteins, unique to ctenophores, bound robustly to the GC-rich motif in DNA affinity purification sequencing (DAP-seq) assays, demonstrating strong preference for unmethylated sites. Their binding was inhibited at methylated motifs, reinforcing the hypothesis that DNA methylation dynamically regulates the recruitment of these architectural proteins to loop anchors. Together, CTEP1 and CTEP2 appear to be crucial mediators of chromatin looping in M. leidyi, orchestrating promoter-enhancer interactions in a methylation-sensitive manner.
Evolutionary analyses illuminated that chromatin loop anchor sequences are highly conserved across multiple ctenophore species, including Bolinopsis microptera, Pleurobrachia bachei, and H. californensis. These conserved regions are more preserved than other intronic or intergenic genomic intervals, underscoring their functional importance. Conversely, promoters of genes involved in distal contacts showed greater sequence divergence and higher transposable element content, indicative of dynamic regulatory modifications over evolutionary time.
The syntenic conservation—that is, the preservation of gene order—within enhancer-promoter loop regions was also elevated across ctenophore genomes compared to random genomic regions. This suggests that genome architecture exerts constraints on gene positioning, maintaining regulatory landscapes necessary for proper gene expression. Such organization likely facilitates coordinated gene regulation, reinforcing the idea that chromatin loops are integral to genome function rather than incidental features.
Collectively, these findings reveal that chromatin looping is not a recent evolutionary development exclusive to vertebrates or bilaterians but rather an ancestral characteristic embedded deeply in the animal kingdom, detectable even in early-diverging lineages like ctenophores. The ctenophore-specific architectural proteins CTEP1 and CTEP2 exemplify how lineage-specific factors can evolve to fulfill fundamental genomic functions while preserving underlying regulatory principles.
This research reshapes our understanding of genome regulation’s evolutionary roots and highlights the intricate interplay between DNA sequence motifs, epigenetic modifications, and protein factors in shaping the three-dimensional genome. As the physical folding of the genome emerges as a universal regulatory feature, future studies will undoubtedly explore how these ancient mechanisms influence development, adaptation, and complexity across the animal tree of life.
Subject of Research: Chromatin Loop Architecture and Genome Regulation in Ctenophores
Article Title: Chromatin loops are an ancestral hallmark of the animal regulatory genome
Article References:
Kim, I.V., Navarrete, C., Grau-Bové, X. et al. Chromatin loops are an ancestral hallmark of the animal regulatory genome. Nature (2025). https://doi.org/10.1038/s41586-025-08960-w
Image Credits: AI Generated
