The combination of the functionalities of feedback circuits is determinant for the number and size of attractors of molecular networks

ABSTRACT Molecular regulation was initially assumed to follow both a unidirectional and a hierarchical organization forming pathways. Regulatory processes, however, form highly interlinked networks with non-hierarchical and non-unidirectional structures that contain statistically overrepresented circuits (motifs). Here, we analyze the behavior of pathways containing non-hierarchical and non-unidirectional interactions that create motifs. In comparison with unidirectional and hierarchical pathways, our pathways have a high diversity of behaviors, characterized by the size and number of attractors. Motifs have been studied individually showing that feedback circuit motifs regulate the number and size of attractors. It is less clear what happens in molecular networks that usually contain multiple feedbacks. Here, we find that the way feedback circuits couple to each other (i.e., the combination of the functionalities of feedback circuits) regulate both the precise number and size of the attractors. We show that the different sets of expected results of epistasis analysis (a method to infer regulatory interactions) are produced by many non-hierarchical and non-unidirectional structures. Thus, these structures cannot be correctly inferred by epistasis analysis. Finally, we show that the structures producing the epistasis results have remarkably similar sets of combinations of functionalities, that combined with other network properties could greatly improve epistasis analysis.