CeGAL: revisiting a widespread fungal-specific TF family using an in silico error-aware approach to identify missing zinc cluster domains

Transcription factors (TF) regulate gene activity in eukaryotic cells by binding specific regions of genomic DNA. In fungi, the most abundant TF class contains a fungal-specific ‘GAL4-like’ Zn2C6 DNA binding domain (DBD), while the second class contains another fungal-specific domain, known as ‘fungal_trans’ or Middle Homology Domain (MHD), whose function remains largely uncharacterized. Remarkably, almost a third of MHD-containing TF in public sequence databases apparently lack DNA binding activity, since they are not predicted to contain a DBD. Here, we reassess the domain organization of these ‘MHD-only’ proteins using an in silico error-aware approach. Our large-scale analysis of ~17000 MHD-only TF sequences showed that the vast majority (>90%) result from gene annotation errors, thus contradicting previous findings that the MHD-only TF are widespread in fungi. We show that they are in fact exceptional cases, and that the Zn2C6-MHD domain pair represents the canonical domain signature defining a new TF family composed of two fungal-specific domains. We call this family CeGAL, after the most characterized members: Cep3, whose 3D structure has been determined and GAL4, an archetypal eukaryotic TF. This definition should improve the classification of the Zn2C6 TF and provide critical insights into fungal gene regulatory networks.IMPORTANCEIn fungi, extensive efforts focus on genome-wide characterization of potential Transcription Factors (TFs) and their targets genes to provide a better understanding of fungal processes and a rational for transcriptional manipulation. The second most abundant families of fungal-specific TFs, characterized by a Middle Homology Domain, are major regulators of primary and secondary metabolisms, multidrug resistance and virulence. Remarkably, one third of these TFs do not have a DNA Binding Domain (DBD-orphan) and thus are excluded from genome-wide studies. This particularity has been the subject of debate for many years. By computationally inspecting the close genomic environment of about 20,000 DBD-orphan TFs from a wide range of fungal species, we reveal that more than 90% contained sequences encoding a zinc-finger DBD. This analysis implies that the arrays of DBD containing TFs and their control DNA-sequences in target genes need to be reconsidered and expands the combinatorial regulation degree of the crucial fungal processes controlled by this TF family.