We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o cells.
Cancer progression involves genetic and epigenetic changes that disrupt chromatin 3D organization, affecting enhancer-promoter interactions and promoting growth. Here, we provide an integrative approach, combining chromatin conformation, accessibility, and transcription analysis, validated by in silico and CRISPR-interference screens, to identify relevant 3D topologies in pediatric T cell leukemia (T-ALL and ETP-ALL). We characterize 3D hubs as regulatory centers for oncogenes and disease markers, linking them to biological processes like cell division, inflammation, and stress response.
The dynamics of three-dimensional (3D) genome organization are essential to transcriptional regulation. While enhancers regulate spatiotemporal gene expression, chromatin looping is a means for enhancer-promoter interactions yielding cell-type-specific gene expression. Further, non-canonical DNA secondary structures, such as G-quadruplexes (G4s), are related to increased gene expression.
The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale.
The orchestration of our genes heavily relies on coordinated communication between enhancers and promoters, yet the mechanisms behind this dynamic interplay during active transcription remain unclear. Here, we investigated enhancer-promoter (E-P) interactions in relation to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence-fluorescence in situ hybridization analyses. Our data reveal that the active state of specific genes is characterized by specific proximities between different genomic regions and the accumulation of transcriptional regulatory factors.
