A biofilm is an organized, resilient group of microbes where individual cells acquire properties, such as drug resistance, that are distinct from those observed in suspension cultures. Here we describe and analyze the transcriptional network controlling biofilm formation in the pathogenic yeast Candida albicans, whose biofilms are a major source of medical device-associated infections. We have combined genetic screens, genome-wide approaches, and two in vivo animal models to describe a master circuit controlling biofilm formation, composed of six transcription regulators that form a tightly woven network with ~1000 target genes. Evolutionary analysis indicates that the biofilm network has rapidly evolved: genes in the biofilm circuit are significantly weighted towards genes that arose relatively recently with ancient genes being underrepresented. This circuit provides a framework for understanding many aspects of biofilm formation by C. albicans in a mammalian host. It also provides insights into how complex cell behaviors can arise from the evolution of transcription circuits.

Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects of biology. In this work, we identify a transcriptional regulator from the human fungal pathogen Candida albicans that binds DNA specifically but has no detectable homology with any previously described DNA-or RNA-binding protein. This protein, named White-Opaque Regulator 3 (Wor3), regulates white-opaque switching, the ability of C. albicans to switch between two heritable cell types. We demonstrate that ectopic overexpression of WOR3 results in mass conversion of white cells to opaque cells and that deletion of WOR3 affects the stability of opaque cells at physiological temperatures. Genome-wide chromatin immunoprecipitation of Wor3 and gene expression profiling of a wor3 deletion mutant strain indicate that Wor3 is highly integrated into the previously described circuit regulating white-opaque switching and that it controls a subset of the opaque transcriptional program. We show by biochemical, genetic, and microfluidic experiments that Wor3 binds directly to DNA in a sequence-specific manner, and we identify the set of cis-regulatory sequences recognized by Wor3. Bioinformatic analyses indicate that the Wor3 family arose more recently in evolutionary time than most previously described DNA-binding domains; it is restricted to a small number of fungi that include the major fungal pathogens of humans. These observations show that new families of sequence-specific DNA-binding proteins may be restricted to small clades and suggest that current annotations-which rely on deep conservation-underestimate the fraction of genes coding for transcriptional regulators.transcriptional regulation | transcription factor | transcription networks | epigenetic switch

When transcription regulatory networks are compared among distantly related eukaryotes, a number of striking similarities are observed: a larger-than-expected number of genes, extensive overlapping connections, and an apparently high degree of functional redundancy. It is often assumed that the complexity of these networks represents optimized solutions, precisely sculpted by natural selection; their common features are often asserted to be adaptive. Here, we discuss support for an alternative hypothesis: the common structural features of transcription networks arise from evolutionary trajectories of “least resistance,” that is, the relative ease by which certain types of network structures are formed during their evolution.

Summary We examine how different transcriptional network structures can evolve from an ancestral network. By characterizing how the ancestral mode of gene regulation for genes specific to a-type cells in yeast species evolved from an activating paradigm to a repressing one, we show that regulatory protein modularity, conversion of one cis-regulatory sequence to another, distribution of binding energy among protein-protein and protein-DNA interactions, and exploitation of ancestral network features all contribute to the evolution of a novel regulatory mode. The formation of this derived mode of regulation did not disrupt the ancestral mode and thereby created a hybrid regulatory state where both means of transcription regulation (ancestral and derived) contribute to the conserved expression pattern of the network. Finally, we show how this hybrid regulatory state has resolved in different ways in different lineages to generate the diversity of regulatory network structures observed in modern species.

Epistasis—the non-additive interactions between different genetic loci—constrains evolutionary pathways, blocking some and permitting others1–8. For biological networks such as transcription circuits, the nature of these constraints and their consequences are largely unknown. Here we describe the evolutionary pathways of a transcription network that controls the response to mating pheromone in yeasts9. A component of this network, the transcription regulator Ste12, has evolved two different modes of binding to a set of its target genes. In one group of species, Ste12 binds to specific DNA binding sites, while in another lineage it occupies DNA indirectly, relying on a second transcription regulator to recognize DNA. We show, through the construction of various possible evolutionary intermediates, that evolution of the direct mode of DNA binding was not directly accessible to the ancestor. Instead, it was contingent on a lineage-specific change to an overlapping transcription network with a different function, the specification of cell type. These results show that analyzing and predicting the evolution of cis-regulatory regions requires an understanding of their positions in overlapping networks, as this placement constrains the available evolutionary pathways.