Three-dimensional (3D)-chromatin organization is critical for proper enhancer-promoter communication and, therefore, for a precise execution of the transcriptional programs governing cellular processes. The emergence of Chromosome Conformation Capture (3C) methods, in particular Hi-C, has allowed the investigation of chromatin interactions on a genome-wide scale, revealing the existence of overlapping molecular mechanisms that we are just starting to decipher. Therefore, disentangling Hi-C signal into these individual components is essential to provide meaningful biological data interpretation. Here, we discuss emerging views on the molecular forces shaping the genome in 3D, with a focus on their respective contributions and interdependence. We discuss Hi-C data at both population and single-cell levels, thus providing criteria to interpret genomic function in the 3D-nuclear space.
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