Multi-layered characterization of ~700,000 conserved noncoding elements within the human genome

Conserved noncoding elements (CNEs) have been extensively studied for their roles as regulatory elements, particularly enhancers. However, the advent of technologies like ChIP-seq and ATAC-seq has shifted research focus away from comparative genomics. Here, we leveraged data from large-scale projects like ENCODE to address the resulting gap in the comprehensive functional characterization of CNEs. We first derived a set of [~]700,000 CNEs in the human genome from a 470-way mammalian alignment. Phylogenetic inference identified [~]670,000 conserved elements within primates and [~]240,000 conserved elements across mammals. Our functional genomic analysis revealed that, irrespective of their level of conservation, approximately one third of CNEs exhibit concurrent chromatin accessibility and H3K27 acetylation in at least one of 19 examined tissues and cell lines and thus, are likely to act as cis-regulatory elements. Extrapolating these data to additional tissues and cell lines suggested that [~]40% of the CNE repertoire possesses cis-regulatory potential. Moreover, we found that the 3D organization of CNEs is non-random; specifically, CNEs are preferentially located toward the centers of topologically associating domains. CNE co-activation networks derived from chromatin accessibility and active histone marks revealed that evolutionary constraints acting on CNEs functioning as cis-regulatory elements reflect not only their isolated individual role, but their topological context. To summarize, we have generated a novel catalog of CNEs annotated with empirical cis-regulatory evidence. While evolutionary constraint and regulatory function are clearly linked, a comprehensive understanding of their interplay remains elusive. This resource provides a foundation for exploring this relationship systematically. Significance statementPrevious studies have investigated conserved noncoding element (CNE) evolution, epigenomic landscapes, and 3D genome organization separately, yet a systematic framework integrating these dimensions has been lacking. Here, we identified [~]700,000 CNEs, including [~]240,000 CNEs deeply conserved across mammals, and show that a large fraction display enhancer-associated epigenomic signatures and are preferentially enriched within TAD centers, highlighting their regulatory relevance. By generating and analyzing this CNE catalog within an integrated evolutionary, epigenomic, and 3D genomic context, our study bridges a critical gap and provides a comprehensive resource to better understand regulatory architecture and its potential contribution to disease-associated variation.