A number of proteins are known to be associated with TAD formation including the protein CTCF and the protein complex cohesin. It is also unknown what components are required at TAD boundaries; however, in mammalian cells, it has been shown that these boundary regions have comparatively high levels of CTCF binding. In addition, some types of genes (such as transfer RNA genes and housekeeping genes) appear near TAD boundaries more often than would be expected by chance.
Computer simulations have shown that chromatin loop extrusion driven by cohesin motors can generate TADs. In the loop extrusion model, cohesin binds chromatin, pulls it in, and extrudes chromatin to progressively grow a loop. Chromatin on both sides of the cohesin complex is extruded until cohesin encounters a chromatin-bound CTCF protein, typically located at the boundary of a TAD. In this way, TAD boundaries can be brought together as the anchors of a chromatin loop. Indeed, in vitro, cohesin has been observed to processively extrude DNA loops in an ATP-dependent manner and stall at CTCF. However, some in vitro data indicates that the observed loops may be artifacts. Importantly, since cohesins can dynamically unbind from chromatin, this model suggests that TADs (and associated chromatin loops) are dynamic, transient structures, in agreement with in vivo observations.
Other mechanisms for TAD formation have been suggested. For example, some simulations suggest that transcription-generated supercoiling can relocalize cohesin to TAD boundaries or that passively diffusing cohesin “slip links” can generate TADs.