Aaron D. Hernday

The Evolution of Combinatorial Gene Regulation in Fungi

It is widely suspected that gene regulatory networks are highly plastic. The rapid turnover of transcription factor binding sites has been predicted on theoretical grounds and has been experimentally demonstrated in closely related species. We combined experimental approaches with comparative genomics to focus on the role of combinatorial control in the evolution of a large transcriptional circuit in the fungal lineage. Our study centers on Mcm1, a transcriptional regulator that, in combination with five cofactors, binds roughly 4% of the genes in Saccharomyces cerevisiae and regulates processes ranging from the cell-cycle to mating. In Kluyveromyces lactis and Candida albicans, two other hemiascomycetes, we find that the Mcm1 combinatorial circuits are substantially different. This massive rewiring of the Mcm1 circuitry has involved both substantial gain and loss of targets in ancient combinatorial circuits as well as the formation of new combinatorial interactions. We have dissected the gains and losses on the global level into subsets of functionally and temporally related changes. One particularly dramatic change is the acquisition of Mcm1 binding sites in close proximity to Rap1 binding sites at 70 ribosomal protein genes in the K. lactis lineage. Another intriguing and very recent gain occurs in the C. albicans lineage, where Mcm1 is found to bind in combination with the regulator Wor1 at many genes that function in processes associated with adaptation to the human host, including the white-opaque epigenetic switch. The large turnover of Mcm1 binding sites and the evolution of new Mcm1–cofactor interactions illuminate in sharp detail the rapid evolution of combinatorial transcription networks.

Biofilm Matrix Regulation by Candida albicans Zap1

The zinc-responsive transcription factor Zap1 has a striking role in fungal biofilm formation and is reported to regulate matrix formation.

The transcriptomes of two heritable cell types illuminate the circuit governing their differentiation.

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes.

White-Opaque Switching in Natural MTLa/α Isolates of Candida albicans: Evolutionary Implications for Roles in Host Adaptation, Pathogenesis, and Sex

All Mating Type Locus strain types of Candida albicans show white-opaque switching competency, not just MTL homozygotes, which allows them to adapt better to environmental changes.

Structure of the transcriptional network controlling white-opaque switching in Candida albicans

The human fungal pathogen Candida albicans can switch between two phenotypic cell types, termed ‘white’ and ‘opaque’. Both cell types are heritable for many generations, and the switch between the two types occurs epigenetically, that is, without a change in the primary DNA sequence of the genome. Previous work identified six key transcriptional regulators important for white‐opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, and Ahr1. In this work, we describe the structure of the transcriptional network that specifies the white and opaque cell types and governs the ability to switch between them. In particular, we use a combination of genome‐wide chromatin immunoprecipitation, gene expression profiling, and microfluidics‐based DNA binding experiments to determine the direct and indirect regulatory interactions that form the switch network. The six regulators are arranged together in a complex, interlocking network with many seemingly redundant and overlapping connections. We propose that the structure (or topology) of this network is responsible for the epigenetic maintenance of the white and opaque states, the switching between them, and the specialized properties of each state.

Candida albicans white and opaque cells undergo distinct programs of filamentous growth.

The ability to switch between yeast and filamentous forms is central to Candida albicans biology. The yeast-hyphal transition is implicated in adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis. A second form of morphological plasticity in C. albicans involves epigenetic switching between white and opaque forms, and these two states exhibit marked differences in their ability to undergo filamentation. In particular, filamentous growth in white cells occurs in response to a number of environmental conditions, including serum, high temperature, neutral pH, and nutrient starvation, whereas none of these stimuli induce opaque filamentation. Significantly, however, we demonstrate that opaque cells can undergo efficient filamentation but do so in response to distinct environmental cues from those that elicit filamentous growth in white cells. Growth of opaque cells in several environments, including low phosphate medium and sorbitol medium, induced extensive filamentous growth, while white cells did not form filaments under these conditions. Furthermore, while white cell filamentation is often enhanced at elevated temperatures such as 37°C, opaque cell filamentation was optimal at 25°C and was inhibited by higher temperatures. Genetic dissection of the opaque filamentation pathway revealed overlapping regulation with the filamentous program in white cells, including key roles for the transcription factors EFG1, UME6, NRG1 and RFG1. Gene expression profiles of filamentous white and opaque cells were also compared and revealed only limited overlap between these programs, although UME6 was induced in both white and opaque cells consistent with its role as master regulator of filamentation. Taken together, these studies establish that a program of filamentation exists in opaque cells. Furthermore, this program regulates a distinct set of genes and is under different environmental controls from those operating in white cells.

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

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.

Genetics and molecular biology in Candida albicans.

Candida albicans is an opportunistic fungal pathogen of humans. Although a normal part of our gastrointestinal flora, C. albicans has the ability to colonize nearly every human tissue and organ, causing serious, invasive infections. In this chapter we describe current methodologies used in molecular genetic studies of this organism. These techniques include rapid sequential gene disruption, DNA transformation, RNA isolation, epitope tagging, and chromatin immunoprecipitation. The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, have greatly facilitated research into this important pathogen. Candida albicans is a normal resident of the human gastrointestinal tract; it is also the most common fungal pathogen of humans, causing both mucosal and systemic infections, particularly in immune compromised patients. C. albicans and Saccharomyces cerevisiae last shared a common ancestor more than 900 million years ago; in terms of conserved coding sequences, the two species are approximately as divergent as fish and humans. Although C. albicans and S. cerevisiae share certain core features, they also exhibit many significant differences. This is not surprising as C. albicans has the ability to survive in nearly every niche of a mammalian host, a property not shared by S. cerevisiae. Research into C. albicans is important in its own right, particularly with regards to its ability to cause disease in humans; in addition, comparison with S. cerevisiae can reveal important insights into evolutionary processes. Many of the methodologies developed for use in S. cerevisiae have been adapted for C. albicans, and we describe some of the most common. Although alternative procedures are described in the literature, we have found those described below to be the most convenient. Because the C. albicans parasexual cycle is cumbersome to use in the laboratory, genetics in this organism has been based almost entirely on directed mutations. Because the organism is diploid, creating a deletion mutant requires two rounds of gene disruption. We describe a rapid method for creating sequential disruptions, one which can be scaled up to create large collections of C. albicans deletion mutants. We also describe a series of additional techniques including DNA transformation, mRNA isolation, epitope tagging, and chromatin immunoprecipitation (ChIP). The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, has greatly increased the quality and pace of research into this important pathogen.

Ssn6 Defines a New Level of Regulation of White-Opaque Switching in Candida albicans and Is Required For the Stochasticity of the Switch

ABSTRACT The human commensal and opportunistic pathogen Candida albicans can switch between two distinct, heritable cell types, named “white” and “opaque,” which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicans functional homolog of the Saccharomyces cerevisiae transcriptional corepressor Cyc8, as a new regulator of white-opaque switching. In a or α mating type strains, deletion of SSN6 results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6 deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators. IMPORTANCE The most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named “white” and “opaque,” is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans. The most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named “white” and “opaque,” is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans.

An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen. ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.