Identification and characterization of a previously undescribed family of sequence-specific DNA-binding domains

Abstract

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

Transcription factor evolution in eukaryotes and the assembly of the regulatory toolkit in multicellular lineages

“…This contrasts with the relatively less complex TF repertoire of other multicellular lineages, such as Fungi, the brown algae E. siliculosus, or even the red alga Chondrus crispus (51). There exists the possibility that our analysis has missed as-yet-undescribed TFs that are unique to those lineages, as TFs that are restricted to small clades may be more prevalent than expected (52). In any case, fungi and brown and red algae did not expand their repertoire of paneukaryotic TFs (Homeoboxes, bHLH, and bZIPs) nor their protein domain architectures, in contrast to embryophytes or metazoans.…”

Ras Signaling Gets Fine-Tuned: Regulation of Multiple Pathogenic Traits of Candida albicans

“…When WOR1 is overexpressed in ras1, pde2, cyr1, tpk1, or tpk2 mutant backgrounds, the opaque state results, showing that Wor1 is the transcriptional effector downstream of the Ras signaling components that regulates the W-O switch (106). Other studies have identified additional transcription factors that control the W-O switch in concert with Wor1, namely, Czf1, Efg1, Wor2 (58, 110), the recently described Wor3 (56), and the mating-type proteins a1 and ␣2 (110) that create the genetic environment required for the W-O switch (Fig. 3E).…”

“…While a direct association with Ras signaling and Czf1, Wor2, or Wor3 has not been described, these transcription factors act in concert in a transcriptional regulatory-feedback loop with Ras-regulated Efg1 and Wor1. Similarly to the complex regulatory circuit that controls biofilm formation, Czf1, Wor1, Wor2, and Wor3 participate in an interlocking regulatory-feedback loop with Efg1 to control the W-O switch (56,110) and thus provide additional support for the idea of fine-tuning of Ras signaling by a complex, context-specific, transcriptional regulatory network that controls phenotypic switching in C. albicans. This transcriptional circuit further is regulated in a complex manner by the regulation of chromatin structure, as the phenotype of the hda1 mutant suggests that the expression of key switch regulators is impacted by Hda1-mediated histone positioning (96).…”

“…The role for signaling through the Ras/ cAMP/PKA pathway in virulence of C. albicans has been clearly established and thoroughly reviewed (51,52). Cutting-edge research on this fungal pathogen has revealed important new insights into the transcriptional regulatory networks that act in concert with Ras-regulated transcription factors to control cell adhesion (53), biofilm formation (54), morphogenesis (55), and W-O switching (56)(57)(58), processes in which Ras/cAMP/PKA signaling plays a central but not a sole role (Fig. 2).…”

Budding off: bringing functional genomics toCandida albicans

“…In the case of the white-opaque switch, a network of at least six transcription factors (AHR1, CZF1, EFG1, WOR1, WOR2 and WOR3) controls expression of 748 target genes [30, [75][76][77][78]. A detailed analysis of this circuit included ChIP-chip, global transcriptome analysis and mechanically induced trapping of molecular interactions (MITOMI) [75].…”