The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Important note on gene symbol ambiguity: The symbol "ROF1" is used in Arabidopsis thaliana to designate an FKBP-type immunophilin (AtFKBP62), which is entirely unrelated to the yeast protein discussed here. In Saccharomyces cerevisiae (strain ATCC 204508 / S288c), ROF1 (Regulator of Fluffy 1) corresponds to the systematic open reading frame YHR177W (UniProt: P38867). This protein was initially annotated as uncharacterized but has since been functionally characterized as a WOPR-domain transcription factor belonging to the fungal-specific Gti1/Pac2 family (luo2024therolesof pages 2-4, luo2024therolesof pages 1-2, arita2021agenome‐scaleyeast pages 8-10).
The key characteristics of ROF1/YHR177W are summarized in the following table:
| Feature | Summary |
|---|---|
| Gene Name | ROF1 (Regulator of Fluffy 1); also referred to as YHR177W in the yeast systematic nomenclature; identified as the Pac2 ortholog in S. cerevisiae (luo2024therolesof pages 2-4, luo2024therolesof pages 1-2) |
| Systematic Name | YHR177W (luo2024therolesof pages 2-4, luo2024therolesof pages 1-2) |
| UniProt ID | P38867 (user-provided target identity) |
| Organism | Saccharomyces cerevisiae (strain ATCC 204508 / S288c), baker's yeast (luo2024therolesof pages 2-4) |
| Protein Family | Gti1/Pac2 family; fungal-specific WOPR-domain transcription factor family (luo2024therolesof pages 1-2, luo2024therolesof pages 4-5) |
| Domain(s) | Conserved N-terminal Gti1/Pac2 (WOPR) domain corresponding to PF09729/IPR018608; WOPR domain comprises WOPRa and WOPRb globular subdomains required for DNA binding (luo2024therolesof pages 1-2, tollot2016thewoprprotein pages 2-4, luo2024therolesof pages 4-5) |
| DNA Binding Motif | Promoters repressed by Rof1 are enriched for a WOPR-like motif: [A/T]TTAAACTTT (arita2021agenome‐scaleyeast pages 8-10) |
| Primary Function | Sequence-specific transcriptional repressor that regulates gene expression programs linked to colony morphology/biofilm-related traits and broader transcription-factor networks; it is not an enzyme or transporter, but a fungal transcriptional regulator (arita2021agenome‐scaleyeast pages 8-10, arita2021agenome‐scaleyeast pages 10-12) |
| Paralog | MIT1, the S. cerevisiae Gti1 ortholog and Wor1 homolog; Rof1 and Mit1 share overlapping DNA-binding regions and regulate many of the same genes (luo2024therolesof pages 2-4) |
| Subcellular Localization | Most likely nuclear, based on family-level evidence that Gti1/Pac2 proteins are nuclear-localizing transcriptional regulators with NLS features; direct YHR177W-specific localization evidence was not found in the gathered literature, so this is an inference rather than a direct experimental localization claim here (luo2024therolesof pages 6-7, luo2024therolesof pages 4-5) |
| Key Phenotypes (deletion and overexpression) | Deletion: YHR177W deletion alone had no clear effect on morphological switching/vegetative growth, but deletion suppresses the hog1Δ hyper-filamentous/complex colony/invasive growth phenotype in diploids; YHR177W is also described as required for biofilm formation in review-level synthesis (luo2024therolesof pages 7-7, luo2024therolesof pages 2-4, furukawa2011efficientconstructionof pages 5-6). Overexpression: reduces expression of a subset of target genes, causes fitness defects, prevents fluffy colony morphology, and reduces colony structure complexity (luo2024therolesof pages 4-5, arita2021agenome‐scaleyeast pages 8-10, arita2021agenome‐scaleyeast pages 10-12) |
| Regulon (key repressed genes) | Rof1-repressed genes include transcription factors MOT3, CUP9, PHD1, YAP6, NRG1, GAT2, TBS1, ACA1, GCN4, CIN5, and MIT1; repressed gene sets are enriched for water transport, siderophore transmembrane transport, and transcription factor binding functions (arita2021agenome‐scaleyeast pages 8-10) |
| Genetic Interactions | 264 synthetic dosage lethality interactions were identified upon ROF1 overexpression; strongest interactions involve the cell wall integrity (CWI) pathway (SLT2, BCK1, PKC1) and cell wall biosynthesis genes (GFA1, QRI1), plus chromatin/transcription regulators including SWR1, INO80, Rpd3L, NuA4, and COMPASS components (arita2021agenome‐scaleyeast pages 10-12) |
| Signaling Pathway Connections | Connected most clearly to morphogenesis/filamentation regulatory networks and genetically to the cell wall integrity (PKC1-BCK1-SLT2/Mpk1) MAPK pathway; YHR177W also emerges in hog1Δ suppressor screens, linking it indirectly to HOG-dependent control of filamentous growth. At the family level, Gti1/Pac2 proteins are associated with PKA and MAPK regulatory logic in fungi (arita2021agenome‐scaleyeast pages 10-12, luo2024therolesof pages 2-4, luo2024therolesof pages 6-7, furukawa2011efficientconstructionof pages 5-6) |
Table: This table summarizes the key molecular, functional, and phenotypic characteristics of the yeast gene ROF1/YHR177W from the gathered evidence. It is useful as a compact reference for identity verification, inferred function, regulatory targets, and pathway connections.
ROF1 encodes a sequence-specific DNA-binding transcriptional repressor. When Rof1 expression is induced (using the YETI inducible expression system), it rapidly reduces the expression of a defined subset of target genes (arita2021agenome‐scaleyeast pages 8-10). This repressive activity is mediated through its WOPR DNA-binding domain, which recognizes promoter sequences enriched for the motif [A/T]TTAAACTTT (arita2021agenome‐scaleyeast pages 8-10). Rof1 is not an enzyme or transporter; rather, it functions as a transcriptional regulator that modulates gene expression programs related to colony morphology, biofilm formation, and broader transcription factor networks.
The Rof1 regulon, identified by transcriptomic analysis following rapid induction, includes genes functionally enriched for water transport, siderophore transmembrane transport, and transcription factor binding activity (arita2021agenome‐scaleyeast pages 8-10). Notably, Rof1 represses the expression of numerous transcription factor genes, including MOT3, CUP9, PHD1, YAP6, NRG1, GAT2, TBS1, ACA1, GCN4, CIN5, and MIT1 (its own paralog) (arita2021agenome‐scaleyeast pages 8-10). This highly interconnected nature—a transcription factor that represses other transcription factors—positions Rof1 as a potential upstream modulator of multiple regulatory circuits.
ROF1 belongs to the Gti1/Pac2 protein family, also known as the WOPR (Wor1, Pac2, Ryp1) transcription factor family, which is fungal-specific (luo2024therolesof pages 1-2). The defining structural feature is a conserved N-terminal Gti1/Pac2 domain (Pfam PF09729 / InterPro IPR018608). This domain consists of two globular subdomains, WOPRa and WOPRb, separated by a linker region of variable length and sequence (tollot2016thewoprprotein pages 2-4, luo2024therolesof pages 4-5). Both subdomains are required for DNA binding activity—neither alone is sufficient (tollot2016thewoprprotein pages 2-4). The N-terminal Gti1/Pac2 domain also contains a conserved protein kinase A (Pka1) phosphorylation site, suggesting regulation by PKA-dependent signaling (luo2024therolesof pages 2-4, luo2024therolesof pages 1-2).
Structural modeling has confirmed that key amino acids critical for WOPR motif binding are highly conserved across WOPR family members. Crystal structures of the WOPR domain from Wor1 in Candida albicans and YHR177W from S. cerevisiae have been used as structural templates for comparative modeling (duttke2022decodingtranscriptionregulatory pages 7-8), indicating that the structural basis for DNA binding is conserved across the family.
The C-terminal region of Rof1, in contrast to the conserved N-terminal domain, is divergent and variable among Gti1/Pac2 family members, and this divergence may contribute to the functional differences between the two paralogs in S. cerevisiae (luo2024therolesof pages 5-6).
S. cerevisiae possesses two WOPR-family paralogs: Mit1 (the Gti1/Wor1 ortholog) and ROF1/YHR177W (the Pac2 ortholog) (luo2024therolesof pages 2-4). These two proteins share substantial functional overlap: over 50% of YHR177W-bound intergenic regions overlap with Mit1-bound regions, indicating shared DNA-binding specificities and co-regulation of many of the same target genes (luo2024therolesof pages 2-4). Despite this overlap, the two paralogs have distinct phenotypic consequences. Mit1 is a central regulator of morphological transitions—its absence prevents diploid pseudohyphal growth and haploid invasive growth—whereas deletion of YHR177W alone has no clear effect on morphological switching or vegetative growth (luo2024therolesof pages 2-4, luo2024therolesof pages 7-7). Instead, YHR177W is required for biofilm formation and complex colony structure (luo2024therolesof pages 2-4).
Intriguingly, deletion of MIT1 substantially increases YHR177W expression levels, suggesting a regulatory feedback loop between the two paralogs (luo2024therolesof pages 2-4). Both Rof1 and Mit1 are integrated into a broader morphogenesis transcriptional network that includes Phd1, Sok2, Mga1, Flo8, Tec1, and Ste12 (luo2024therolesof pages 2-4). The Rof1 overexpression regulon also includes Mit1 itself as a repressed target, further underscoring the interconnectedness of these regulatory circuits (arita2021agenome‐scaleyeast pages 8-10).
Although direct experimental localization data specific to YHR177W/Rof1 (e.g., from GFP fusion imaging) were not identified in the primary literature gathered here, the protein is predicted to localize to the nucleus. This inference is strongly supported by multiple lines of evidence: (i) Gti1/Pac2 family proteins across fungal species contain nuclear localization signals (NLS) and have been characterized as nuclear-localizing transcriptional regulators (luo2024therolesof pages 2-4, luo2024therolesof pages 6-7); (ii) Rof1 possesses a sequence-specific DNA-binding domain and functions as a transcription factor, which necessitates nuclear localization; and (iii) the Saccharomyces Genome Database annotates Rof1 localization as nuclear. These converging lines of evidence strongly support a nuclear site of action.
Rof1 plays a defined role in the complex colony morphology and filamentous growth programs of S. cerevisiae. In a genetic screen for suppressors of the hyper-filamentous phenotype caused by deletion of the Hog1 MAPK in diploid Σ1278b strains, YHR177W was identified as one of 30 genes whose insertion/deletion significantly suppressed enhanced morphological developments, including hyper-filamentous growth, agar invasion, and complex colony morphology (luo2024therolesof pages 2-4, furukawa2011efficientconstructionof pages 5-6). This screen categorized YHR177W as being involved in control of cell cycle or cell division, although the precise mechanistic basis for suppression was not elucidated (furukawa2011efficientconstructionof pages 5-6).
Overexpression of ROF1, conversely, reduces the complexity of colony structure and prevents the formation of "fluffy" colony morphology, a phenotype associated with biofilm-like growth (luo2024therolesof pages 4-5, arita2021agenome‐scaleyeast pages 10-12). This is consistent with Rof1's role as a transcriptional repressor of genes involved in morphological complexity.
A genome-wide synthetic dosage lethality (SDL) screen using the YETI system identified 264 genes with synthetic genetic interactions upon ROF1 overexpression (arita2021agenome‐scaleyeast pages 10-12). The strongest interactions were with components of the cell wall integrity (CWI) signaling pathway, specifically SLT2 (the MAPK), BCK1 (MAPKKK), and PKC1 (protein kinase C), as well as cell wall biosynthesis genes GFA1 and QRI1 (arita2021agenome‐scaleyeast pages 10-12). This genetic connection to the CWI pathway suggests that Rof1 overexpression imposes cell wall stress or reduces cell wall integrity, such that cells become dependent on the CWI pathway for survival.
Additionally, ROF1 displayed synthetic interactions with genes encoding components of multiple chromatin remodeling and transcriptional regulation complexes, including the SWR1 complex, INO80 complex, Rpd3L histone deacetylase complex, NuA4 histone acetyltransferase complex, and the COMPASS (Set1) histone methyltransferase complex (arita2021agenome‐scaleyeast pages 10-12). These interactions are consistent with Rof1's function as a transcriptional repressor that may require proper chromatin architecture for its activity or whose repressive effects create a cellular dependency on chromatin-mediated gene regulation.
The Gti1/Pac2 family is a well-conserved, fungal-specific transcription factor family found throughout the fungal kingdom (luo2024therolesof pages 1-2, luo2024therolesof pages 7-8). Most fungal genomes encode two paralogous WOPR proteins—one Gti1-type (e.g., Wor1 in Candida albicans, Ryp1 in Histoplasma capsulatum, Sge1 in Fusarium species, Mit1 in S. cerevisiae) and one Pac2-type (e.g., ROF1/YHR177W in S. cerevisiae, Pac2 in Schizosaccharomyces pombe) (luo2024therolesof pages 2-4, luo2024therolesof pages 5-6). The Gti1 orthologs have been more extensively studied and are generally considered master regulators of morphological switching and virulence in pathogenic fungi, while the Pac2 orthologs tend to play more minor or modulatory roles (luo2024therolesof pages 5-6, luo2024therolesof pages 7-8).
In S. pombe, the founding member Pac2 controls the onset of sexual development through a pathway independent of the cAMP cascade, specifically in response to nitrogen starvation (luo2024therolesof pages 2-4, luo2024therolesof pages 1-2). Gti1 in S. pombe functions as a downstream target of the Wis1-Sty1 MAPK (HOG) pathway and promotes gluconate uptake during glucose starvation (luo2024therolesof pages 2-4, luo2024therolesof pages 6-7). This family-level context—regulation by MAPK and PKA pathways and involvement in nutrient-responsive developmental decisions—provides a framework for understanding Rof1's role in S. cerevisiae as a regulator positioned at the intersection of nutrient sensing and morphological development.
ROF1/YHR177W encodes a fungal-specific WOPR-domain transcription factor that functions primarily as a transcriptional repressor in S. cerevisiae. It binds DNA through its conserved WOPRa/WOPRb domain to the motif [A/T]TTAAACTTT and represses a regulon of genes including multiple transcription factors, as well as genes involved in water transport and siderophore transport. Rof1 is the Pac2 ortholog in the Gti1/Pac2 family, paralogous to Mit1 (the Gti1/Wor1 ortholog), and the two share >50% of their DNA-binding targets. While deletion of Rof1 alone does not abolish morphological transitions, it is required for biofilm formation and was identified as a positive regulator of the hyper-filamentous growth phenotype in hog1Δ diploids. Its overexpression suppresses fluffy colony morphology and creates a genetic dependency on the cell wall integrity MAPK pathway. Rof1 is predicted to function in the nucleus as a DNA-binding transcriptional regulator, consistent with its family-level properties including conserved nuclear localization signals and its demonstrated capacity for sequence-specific DNA binding.
References
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