A biologically informed framework for classifying and interpreting chromatin interactions is developed and dual-state regulatory elements connected to these dual-state regulatory elements are enriched for developmental and signaling pathways and exhibit increased expression specificity across cell types, consistent with specialized roles in context-dependent gene regulation.
Abstract
Gene regulation depends on coordinated interactions between promoters and distal cis-regulatory elements, yet understanding how these regulatory elements communicate remains a fundamental challenge in mammalian genomics. Chromatin interaction assays provide one approach for identifying potential regulatory relationships, but interpreting the biological significance of individual interactions remains difficult; chromatin interactions comprise multiple biologically distinct classes that are only partially captured by any single assay. Here, we integrate Hi-C, RNAPII ChIA-PET, and CTCF ChIA-PET with the ENCODE Registry of candidate cis-regulatory elements (cCREs) and complementary functional genomic datasets to develop an integrative framework for classifying and interpreting promoter-centric chromatin interactions. Using this framework, we identify a distinct class of candidate architectural promoter-enhancer interactions that are characterized by increased recurrence across cellular contexts, broader promoter connectivity, and reduced dependence on linear genomic proximity. We further show that many regulatory elements anchoring these interactions transition between enhancer and CTCF-only states while maintaining stable chromatin interactions. These dual-state regulatory elements also acquire context-specific transcription factor inputs within evolutionarily conserved architectural scaffolds, suggesting that stable chromatin architecture can be repeatedly repurposed for new regulatory functions. Genes connected to these dual-state regulatory elements are enriched for developmental and signaling pathways and exhibit increased expression specificity across cell types, consistent with specialized roles in context-dependent gene regulation. Together, our findings provide a biologically informed framework for classifying and interpreting chromatin interactions and support a model in which conserved chromatin architecture provides a stable foundation upon which new regulatory programs evolve.
Nucleosomes organize genomes and regulate DNA access, yet accumulating evidence suggests that their constituent histones may have functions beyond canonical chromatin regulation, but the breadth of such regulatory diversity remains unclear. Here, we used the six-residue loop 2 (L2) of H2A and H2A.Z to map, at single-residue resolution, how nucleosome-core variation reshapes cellular function. Genome-scale interaction mapping identified hundreds of regulatory connections spanning chromatin, as well as actin organization, endocytosis, and membrane trafficking. Interactions were residue-specific and differed between H2A and H2A.Z, revealing a regulatory landscape encoded by single residues. Transcriptome profiling showed limited expression changes and little overlap between differentially expressed genes and regulatory partners, indicating that non-chromatin connections are not readily explained by altered transcription. L2 substitutions also preferentially conferred benefits under cell wall and membrane stress. Thus, the nucleosome is linked to cellular-periphery functions beyond classical chromatin regulation, identifying histone variation as a source of phenotypic innovation.
Zachary H. Harvey, Benjamin Gundinger, Jian-Yi Kok et al.· bioRxiv· 0 citations
Developmental genes are frequently regulated by multiple enhancers distributed across large cis-regulatory regions. How these enhancers communicate with their target promoter and how their interactions are shaped by distinct developmental transcriptional environments remain incompletely understood. Here, we investigate the chromatin organization of the Drosophila shavenbaby locus, a developmental gene controlled by seven distal enhancers. Tissue-specific UMI-4C revealed extensive enhancer-promoter and enhancer-enhancer interactions, including in cell populations where individual enhancers are inactive. Quantitative three-dimensional DNA-FISH revealed compact enhancer-promoter hubs enriched in shavenbaby-expressing cells, yet also present in non-expressing cells and prior to expression. Perturbation of shavenbaby regulators, transcription factors, and architectural proteins revealed that multiple factors contribute to hub organization. Their relative contributions differed between epidermal populations, indicating that similar hubs can be supported by different combinations of regulators. Perturbations that reduced hub organization were frequently associated with reduced shavenbaby-dependent trichome formation. Together, our results identify a robust, multi-factorial enhancer-promoter hub that is shaped by distinct regulatory inputs across developmental contexts.
Sujay Naik, Srijani Roy, Ella Preger-Ben Noon· bioRxiv· 0 citations
ABSTRACT Transcription and three‐dimensional (3D) genome organization are closely coupled, but their precise relationship remains unresolved. Evidence from perturbation, imaging, and modeling studies suggests that transcription is not strictly required for the establishment of large‐scale genome features such as compartments and topologically associating domains (TADs). Instead, transcription seems to exert more prominent effects at finer spatial scales, where it influences enhancer–promoter interactions, local chromatin loops, and microcompartments. RNA molecules, polymerase‐driven supercoiling, and R‐loop formation may further modulate chromatin organization at the gene level. At the same time, many architectural proteins and transcriptional regulators operate with functional overlap, complicating efforts to disentangle cause and consequence. Together, these observations suggest that genome architecture provides a relatively stable framework for regulated gene expression, while transcription and associated factors refine chromatin organization in a context‐dependent manner. Understanding how these processes are integrated will require systematic, multi‐scale perturbations across different biological and biochemical systems.
Low-affinity transcription-factor (TF) motifs are an important element of the cis-regulatory code, yet they are notoriously difficult to map and mechanistically incompletely understood, limiting our ability to interpret non-coding variation in development, evolution, and disease. Here, we investigate their role in pioneering and leverage sequence-to-profile models of chromatin accessibility in mouse embryonic stem cells to reliably map and interpret low-affinity motifs across the genome. We find that low-affinity motifs have outsized effects by cooperating with nearby motifs through intra-nucleosomal soft syntax. By modeling nucleosome-mediated cooperativity with a kinetic model, we discover and validate that pioneer cooperativity makes a motif operate at higher pioneering ranges across changing TF concentrations, thereby raising the regulatory potential. These results show that low-affinity motifs can be accurately mapped, shape the properties of developmental enhancers, and likely play a widespread role in fine-tuning enhancers during evolution.
M. Weilert, Kaelan J. Brennan, Khyati Dalal et al.· Cell Genomics· 0 citations
In eukaryotes, transcription factors (TFs) must continuously compete with nucleosomes to access their binding sites, leading to cell-to-cell variability in chromatin accessibility at regulatory regions. Although critical to understand enhancer function in transcription, the mechanisms that define how frequently an enhancer is active in a cell population remain unclear. Here we used single-molecule footprinting to quantify the frequency at which chromatin is accessible at enhancers in response to TF perturbations and changes in their chromatin environment. We find that, individually, most TFs open chromatin in a small fraction of cells, and that cumulative TF binding controls enhancer activation frequency. Moreover, testing the functionality of hundreds of enhancers when inserted at an ectopic genomic location indicates that p300 activity is required for their full activation. Our data support a model in which enhancer activation frequency depends on the cumulative function of multiple TFs and is modulated by p300 activity. This study uses single-molecule footprinting to quantify chromatin accessibility at enhancers and promoters in mouse embryonic stem cells and to dissect the contributions of transcription factor binding and chromatin context.
Transcriptional regulation is governed by interactions between cis-regulatory elements (CREs) and trans-acting regulators in a context-specific manner. Although DNA and single-cell foundation models have enabled modeling regulatory biology at scale, most represent either sequence or cellular state alone, limiting their ability to capture context-dependent gene regulation. Here we present RegFM, a context-aware foundation model for human transcriptional regulation. RegFM treats transcriptional regulation as a dialogue between cis-regulatory sequences (e.g., CREs) and trans-acting regulators (e.g., transcription factors (TFs) and chromatin regulators (CRs)) by coupling long-range CRE representations with TFs and CRs activity. Trained on large-scale ENCODE and CELLxGENE transcriptomic profiles, RegFM learns gene-centered regulatory representations that generalize across unseen cellular contexts. In a wide range of tasks, including gene expression prediction, cis-regulatory element annotation, bivalent promoter and dosage-sensitivity classification, and perturbation-response prediction, RegFM consistently improves over existing methods. RegFM emerges as a scalable and interpretable framework for modeling human transcriptional regulation and provides insights into context-dependent gene regulatory programs.
Zijing Gao, Yining Sun, Hao-Chen Wang et al.· bioRxiv· 0 citations
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