Recombinant Candida glabrata Transcription factor NRM1 (NRM1)

What is the primary function of NRM1 in Candida glabrata?

NRM1 (Negative Regulator of MBF targets 1) functions as an MBF-specific transcriptional repressor that acts at the transition from G1 to S phase of the cell cycle. It serves as a critical nexus between the cell cycle and transcriptional regulation, primarily constraining the expression of G1-S phase genes through a negative feedback mechanism. This role appears to be evolutionarily conserved among different yeast species, suggesting its fundamental importance in cell cycle regulation .

How does NRM1 interact with MBF complexes?

NRM1 binds to the MBF (MCB-Binding Factor) complex primarily through interaction with the Cdc10 component. Research using MuDPIT (MultiDimensional Protein Interaction Technology) analysis of affinity-purified proteins confirmed that NRM1 is a component of the MBF complex. Specifically, NRM1 interacts with Cdc10 but shows reduced binding in the absence of other components like Res2. The C-terminal region of Cdc10 appears essential for this interaction, as evidenced by the cdc10-C4 mutant (lacking the C-terminal 61 amino acids), which abrogates MBF binding to NRM1 .

What promoter regions does NRM1 typically bind to?

NRM1 binds to promoters containing MCB (Mlu1 Cell cycle Box) sequence motifs through its association with MBF. These promoters typically regulate genes involved in DNA replication and cell cycle progression at the G1-S transition. In experimental contexts, NRM1 has been demonstrated to bind to well-established MBF target promoters such as cdc22+. This binding is crucial for the repression of MBF-regulated promoters outside of the G1-S phase .

What are the recommended approaches for expressing recombinant C. glabrata NRM1?

For expressing recombinant C. glabrata NRM1, researchers should consider using vector systems optimized for fungal expression. Based on methodologies used for other C. glabrata transcription factors, a recommended approach involves:

• Amplifying the NRM1 gene from C. glabrata genomic DNA using PCR with primers containing appropriate restriction sites

• Cloning the gene into a suitable expression vector (e.g., pGREG576)

• Replacing weak promoters like GAL1 with stronger constitutive promoters such as PDC1

• Verifying the construct through DNA sequencing before transformation

This approach has been successful for other C. glabrata transcription factors, where the PDC1 promoter provided stronger constitutive expression compared to the GAL1 promoter, which allows only very low expression of downstream genes in C. glabrata .

How can researchers effectively assess NRM1 binding to target promoters?

To assess NRM1 binding to target promoters, researchers should employ chromatin immunoprecipitation (ChIP) assays. The methodology involves:

• Crosslinking proteins to DNA in vivo using formaldehyde

• Cell lysis and DNA fragmentation through sonication

• Immunoprecipitation with antibodies against tagged NRM1 (e.g., myc-tagged)

• PCR amplification of the precipitated DNA using primers specific for suspected target promoters

• Quantification of binding through qPCR

This approach allows for the detection of dynamic changes in NRM1 binding to promoters under different conditions, such as normal cell cycle progression versus replication stress. The technique has been successfully used to demonstrate that NRM1 binding to promoters is abolished in response to replication stress induced by hydroxyurea (HU) .

What protein purification methods are most effective for recombinant NRM1?

For purification of recombinant NRM1, researchers should consider the following approach:

• Express NRM1 with an affinity tag (e.g., His-tag or GST-tag) for easier purification

• Use fungal expression systems rather than bacterial systems to ensure proper post-translational modifications

• Employ affinity chromatography as the initial purification step

• Follow with size exclusion chromatography to improve purity

• Validate protein integrity through SDS-PAGE and Western blotting

When analyzing NRM1's phosphorylation state, researchers should include phosphatase treatment controls to confirm that slower migrating bands observed during gel electrophoresis are indeed due to phosphorylation, as demonstrated with NRM1 isolated from hydroxyurea-arrested cells .

How is NRM1 activity regulated during the cell cycle?

NRM1 activity is regulated through multiple mechanisms during the cell cycle:

• Transcriptional regulation: NRM1 is itself an MBF target gene, creating a negative feedback loop where NRM1 accumulates during G1-S and then represses its own expression along with other MBF targets

• Post-translational modifications: NRM1 undergoes phosphorylation in response to cellular conditions

• Protein-protein interactions: NRM1's interaction with the MBF complex is dynamic and can be modulated by other regulatory factors

This multilevel regulation ensures tight control of G1-S transcription, limiting it to the appropriate cell cycle phase under normal conditions, while allowing for rapid modulation in response to cellular stresses .

What is the relationship between NRM1 and DNA replication checkpoint activation?

NRM1 serves as a critical target of the DNA replication checkpoint pathway. When DNA replication is stressed or stalled:

• NRM1 becomes phosphorylated, appearing as slower migrating bands on SDS-PAGE

• This phosphorylated form of NRM1 dissociates from MBF-regulated promoters

• Dissociation leads to de-repression of MBF target genes outside of the normal G1-S window

• This response occurs with various replication stress agents, including hydroxyurea (HU), methyl methanesulfonate (MMS), and camptothecin

This mechanism allows cells to maintain expression of DNA replication and repair genes when DNA replication is compromised, providing an elegant linkage between checkpoint activation and transcriptional regulation. The phosphorylation of NRM1 appears to be the key molecular event that inactivates its repressor function in response to replication stress .

How can researchers investigate the phosphorylation sites on NRM1 that respond to replication stress?

To investigate NRM1 phosphorylation sites that respond to replication stress, researchers should employ the following methodology:

• Mass spectrometry analysis:

  • Express and purify recombinant NRM1 from cells treated with and without replication stress agents

  • Perform tryptic digestion of purified NRM1

  • Analyze peptides using LC-MS/MS with phosphopeptide enrichment strategies

  • Compare phosphopeptide profiles between conditions to identify differentially phosphorylated sites

• Site-directed mutagenesis:

  • Generate serine/threonine to alanine mutations at candidate phosphorylation sites

  • Express these mutants in cells and assess their response to replication stress

  • Evaluate whether mutations prevent the shift to slower migrating forms on SDS-PAGE

  • Determine if phospho-deficient mutants maintain promoter binding during replication stress

• Functional validation:

  • Perform ChIP assays with phospho-mutant versions to assess promoter binding

  • Analyze transcriptional profiles of cells expressing phospho-mutant NRM1 versions

  • Evaluate cell cycle progression and replication stress survival in cells expressing mutant versions

This systematic approach would help identify the specific residues that mediate checkpoint-dependent regulation of NRM1 function and provide insight into the molecular mechanism of this regulation .

What role might NRM1 play in antifungal resistance mechanisms in C. glabrata?

Although direct evidence linking NRM1 to antifungal resistance in C. glabrata is not established in the provided search results, we can propose a research framework based on knowledge of other transcription factors in C. glabrata:

• Gene expression analysis:

  • Compare NRM1 expression levels between antifungal-sensitive and resistant C. glabrata strains

  • Perform RNA-sequencing of NRM1 deletion mutants with and without antifungal exposure

  • Identify potential NRM1 target genes involved in drug resistance pathways

• Target gene investigation:

  • Determine if NRM1 regulates genes involved in membrane composition, drug efflux, or stress responses

  • Similar to CgMar1, which regulates genes related to lipid biosynthesis pathways and contributes to plasma membrane sphingolipid incorporation and membrane permeability

  • Assess whether NRM1 directly or indirectly influences expression of known resistance genes

• Stress response integration:

  • Investigate whether antifungal exposure triggers replication stress that might influence NRM1 phosphorylation and activity

  • Examine if NRM1 deletion affects susceptibility to various classes of antifungals

  • Determine if NRM1 overexpression confers resistance to specific antifungals

Given that other transcription factors like CgMar1 contribute to azole resistance through regulation of membrane composition and drug accumulation , NRM1 could potentially influence resistance through cell cycle-dependent expression of genes involved in similar processes.

How does C. glabrata NRM1 function compare to its orthologs in other Candida species and S. cerevisiae?

To investigate functional conservation and divergence of NRM1 across fungal species, researchers should:

• Perform phylogenetic analysis:

  • Align NRM1 protein sequences from C. glabrata, other Candida species, and S. cerevisiae

  • Identify conserved domains and species-specific sequence variations

  • Construct phylogenetic trees to understand evolutionary relationships

• Conduct complementation studies:

  • Express C. glabrata NRM1 in S. cerevisiae nrm1Δ strains and vice versa

  • Assess whether cross-species expression rescues phenotypes associated with NRM1 deletion

  • Evaluate if regulation mechanisms (e.g., phosphorylation in response to replication stress) are conserved

• Compare protein-protein interactions:

  • Use techniques like yeast two-hybrid or co-immunoprecipitation to identify interaction partners of NRM1 in different species

  • Determine if MBF component binding is conserved across species

  • Investigate whether regulatory mechanisms are similar across species

This comparative approach would illuminate how NRM1 function has evolved across fungal species and potentially identify species-specific adaptations relevant to pathogenicity or stress responses .

What are the differences in NRM1 target gene networks between model yeasts and pathogenic C. glabrata?

To characterize differences in NRM1 target gene networks, researchers should implement the following methodological approach:

• Genome-wide binding analysis:

  • Perform ChIP-seq with tagged NRM1 in C. glabrata and model yeasts like S. cerevisiae

  • Identify and compare binding sites across species

  • Analyze promoter motifs to determine if recognition sequences are conserved

• Transcriptome analysis:

  • Conduct RNA-seq in NRM1 deletion mutants across different species

  • Perform differential expression analysis to identify species-specific target genes

  • Compare expression changes during cell cycle progression and under replication stress

• Functional categorization:

  • Group target genes by biological function in each species

  • Identify pathogen-specific targets that might contribute to virulence or stress adaptation

  • Determine if C. glabrata NRM1 has acquired regulation of unique gene sets related to its pathogenic lifestyle

How might NRM1 influence C. glabrata virulence in infection models?

To investigate NRM1's potential role in virulence, researchers should consider:

• Infection model studies:

  • Generate C. glabrata strains with NRM1 deletion or overexpression

  • Evaluate virulence in infection models such as Galleria mellonella larvae, similar to approaches used for CgDtr1

  • Assess parameters including host survival rate, fungal proliferation in host tissues, and host immune response

• Survival in phagocytes:

  • Analyze NRM1 mutant survival within macrophages or neutrophils

  • Determine if NRM1 regulates genes that contribute to survival within phagocytes

  • Examine if NRM1 phosphorylation status changes during phagocytosis

• Stress resistance analysis:

  • Evaluate resistance of NRM1 mutants to stresses encountered during infection (oxidative stress, nutrient limitation, pH changes)

  • Similar to analysis of CgDtr1, which was found to confer resistance to oxidative and acetic acid stress

  • Assess if NRM1-regulated gene expression contributes to these stress responses

Based on findings with other transcription factors like CgDtr1, which influences C. glabrata virulence by protecting cells from stress factors within phagocytes , NRM1 might similarly coordinate cell cycle regulation with stress responses relevant to pathogenesis.

What experimental designs best elucidate NRM1's role during host-pathogen interactions?

To effectively study NRM1's role during host-pathogen interactions, researchers should implement:

• Time-course studies during infection:

  • Monitor NRM1 expression, phosphorylation status, and localization at different timepoints post-infection

  • Track binding to target promoters during different infection stages

  • Correlate these changes with host immune response and fungal proliferation metrics

• Conditional NRM1 expression systems:

  • Develop systems for inducible NRM1 expression or depletion during specific infection phases

  • Determine the consequences of altering NRM1 activity at different infection stages

  • Assess if NRM1 function becomes particularly important during specific host-pathogen interaction phases

• Ex vivo and in vitro models:

  • Use cell culture systems to mimic specific host environments (e.g., macrophages, epithelial cells)

  • Monitor NRM1 activity and cell cycle progression during host cell interactions

  • Examine if host factors directly influence NRM1 regulation

These experimental approaches would provide temporal and spatial information about NRM1 function during host-pathogen interactions, potentially identifying critical windows where NRM1 activity influences infection outcomes.

What are the main difficulties in expressing and purifying functional recombinant C. glabrata NRM1?

Researchers working with recombinant C. glabrata NRM1 may encounter several technical challenges:

• Expression system selection:

  • C. glabrata-specific promoters may behave differently than in model organisms

  • As observed with other C. glabrata proteins, the GAL1 promoter allows only very low expression of downstream genes in C. glabrata

  • Solution: Use strong constitutive promoters like PDC1 for reliable expression

• Post-translational modifications:

  • NRM1 undergoes phosphorylation that is critical to its function

  • Bacterial expression systems may not reproduce these modifications accurately

  • Solution: Express in eukaryotic systems that maintain proper phosphorylation patterns

• Protein solubility and stability:

  • Transcription factors often contain regions prone to aggregation

  • Solution: Consider expressing functional domains separately, use solubility tags, or optimize buffer conditions

• Maintaining native conformation:

  • Ensuring the recombinant protein maintains DNA-binding activity

  • Solution: Include functional assays (e.g., electrophoretic mobility shift assays) to confirm activity of purified protein

Each challenge requires specific methodological approaches to overcome, with attention to the particular properties of transcription factors and C. glabrata expression systems.

How can researchers effectively control for NRM1 phosphorylation states in experimental systems?

To effectively control for NRM1 phosphorylation states in experimental systems, researchers should:

• Develop phospho-specific antibodies:

  • Generate antibodies that specifically recognize phosphorylated forms of NRM1

  • Use these for Western blotting and immunoprecipitation to monitor phosphorylation status

  • Validate specificity using phosphatase treatments and phospho-null mutants

• Create phospho-mimetic and phospho-null mutants:

  • Replace key serine/threonine residues with aspartic acid/glutamic acid (phospho-mimetic) or alanine (phospho-null)

  • Express these in cells to simulate constitutively phosphorylated or unphosphorylated states

  • Use these mutants to dissect the functional consequences of phosphorylation

• Employ mass spectrometry-based quantification:

  • Develop targeted mass spectrometry methods to quantify specific phosphorylated peptides

  • Use stable isotope labeling approaches for precise quantification of phosphorylation stoichiometry

  • Track changes in phosphorylation in response to different stimuli

• Control experimental conditions rigorously:

  • Include phosphatase inhibitors during protein extraction to preserve phosphorylation states

  • Use cell synchronization methods to control for cell cycle-dependent phosphorylation

  • Include appropriate controls for replication stress induction (e.g., hydroxyurea treatment)

These approaches would enable researchers to precisely monitor and manipulate NRM1 phosphorylation states, facilitating investigation of how phosphorylation regulates its function.