Recombinant Candida glabrata DNA-directed RNA polymerase II subunit RPB2 (RPB2), partial

What is the role of RNA polymerase II subunit RPB2 in Candida glabrata?

RNA polymerase II (RNAPII) is essential for transcription in eukaryotes, with RPB2 being its second-largest subunit. In C. glabrata, RPB2 (encoded by the gene CAGL0L04246g) forms part of the 12-subunit RNAPII complex that is critical for transcription of protein-coding genes. Research indicates that RPB2 contributes to:

• Core transcriptional machinery function

• Stress response during host-pathogen interactions

• Adaptation to environmental stressors

The RNAPII complex is a major limiting factor in transcription, with studies showing that a 50% reduction in nuclear amounts of RNAPII results in approximately 40% reduction in global RNAPII occupancy, while similar reductions in general transcription factors only reduced occupancy by 5-10% .

How can researchers express and purify recombinant C. glabrata RPB2 for experimental studies?

Expression of recombinant C. glabrata RPB2 can be achieved through several expression systems:

Standard purification protocol includes:

• Expression with appropriate tags (His-tag is common)

• Cell lysis under optimized conditions

• Affinity chromatography as primary purification step

• Quality control via SDS-PAGE (target purity ≥85%)

When designing constructs, consider using partial RPB2 constructs focusing on specific domains rather than the full-length protein, which can be challenging to express due to its large size.

What methods are most effective for studying RPB2 function in C. glabrata?

Several complementary approaches have proven effective:

• ChIP-seq against elongating RNAPII: Provides genome-wide mapping of transcriptional activity with high temporal resolution

  • Particularly powerful when combined with spike-in normalization for quantitative comparison between conditions

  • Can distinguish between initiated (S5-P) and elongating (S2-P) forms of RNAPII

• Genetic manipulation approaches:

  • Deletion mutants (where non-lethal)

  • Conditional depletion using systems like anchor-away approach

  • Flap loop deletions (e.g., Δ873-884 in human RNAPII RPB2)

• Expression systems for functional studies:

  • Anhydrotetracycline-inducible promoters for controlled overexpression

  • Heterozygous diploids with FRB-tagged alleles for rapid nuclear depletion

• Infection models for studying RPB2's role in pathogenesis:

  • Galleria mellonella larvae model

  • Neutropenic mouse model of disseminated candidiasis

  • Macrophage infection assays

How does C. glabrata RNAPII contribute to transcriptional dynamics during host-pathogen interactions?

RNAPII in C. glabrata orchestrates dynamic transcriptional responses during infection with remarkable temporal precision:

• Chronological activation pattern: Studies mapping genome-wide RNAPII occupancy reveal that genes of specialized pathways are activated at specific timepoints during macrophage infection

• Stress-responsive transcription: RNAPII mediates transcriptional programs that allow C. glabrata to withstand oxidative stress when engulfed by macrophages that generate reactive oxygen species

• Virulence regulation: Transcription factors like CgXbp1 interact with the RNAPII machinery to regulate virulence-related genes during infection. Deletion of CgXBP1 leads to accelerated transcriptional activation during macrophage infection, with 369 genes showing faster expression in the mutant compared to wild-type

Methodologically, researchers can study these dynamics by:

• Performing time-course ChIP-seq experiments targeting RNAPII during infection

• Using spike-in normalization for quantitative comparisons between timepoints

• Coupling with deletion mutants of transcriptional regulators to identify regulatory mechanisms

What structural elements of RPB2 are critical for C. glabrata's transcriptional activity?

Based on structural studies of RNAPII across species, several RPB2 domains appear critical:

Interestingly, studies in human RNAPII have shown that the flap loop (residues 873-884) that contacts transcription factor IIB (TFIIB) can be deleted without affecting global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation . Similar structural studies in C. glabrata RPB2 could reveal species-specific features that contribute to its pathogenicity.

How does RPB2 function intersect with histone modification in regulating C. glabrata responses to host defense peptides and antifungal drugs?

Research indicates a complex interplay between RNAPII function and chromatin regulation:

• Histone modification is a central mechanism by which C. glabrata withstands stress from host defense peptides and echinocandin antifungal drugs

• Transcriptional adapter protein Ada2, which interacts with the RNAPII machinery, is necessary for C. glabrata to tolerate a wide variety of stressors, providing a compelling explanation for its role in virulence

• Histone deacetylases like Rpd3 and Hos2 affect susceptibility to protamine and caspofungin, with the rpd3Δ mutant showing attenuated virulence in mice

This interplay can be studied through:

• ChIP-seq targeting both RNAPII and histone modifications

• Deletion mutants of chromatin regulators followed by RNAPII occupancy analysis

• Drug susceptibility assays combined with transcriptional profiling

How do cell size and growth conditions affect RPB2-dependent transcription in C. glabrata?

Studies in budding yeast have revealed important principles that likely apply to C. glabrata:

• Cell size scaling: Total amount of RNAPII loaded on the genome increases with cell size, with uniform increases in both initiated (S5-P) and elongating (S2-P) RNAPII populations

• Dynamic equilibrium: RNAPII binding appears to be driven by dynamic equilibrium kinetics rather than simple titration against the genome

• Limiting component: RNAPII itself is a major limiting component of the pre-initiation complex (PIC), with transient overexpression of all 12 RNAPII subunits sufficient to increase polymerase loading on the genome

Experimental approach to study this in C. glabrata:

• Generate populations of different cell sizes (e.g., using centrifugal elutriation and cell cycle arrest)

• Perform spike-in normalized ChIP-seq to quantify RNAPII occupancy

• Develop inducible expression systems for RNAPII subunits to test limiting component models

What is the relationship between RPB2 function and antifungal drug resistance in C. glabrata?

C. glabrata exhibits high intrinsic resistance to azole antifungals, with RNAPII playing a crucial role in the transcriptional responses that mediate this resistance:

• Transcription factor regulation: Novel transcription factors like CgXbp1 that work through the RNAPII machinery regulate not only virulence-related genes but also genes associated with drug resistance

• Stress response genes: RNAPII mediates transcription of oxidative stress response genes regulated by TFs CgSkn7, CgYap1, and CgMsn2/4, which may contribute to antifungal resistance

• DNA repair connection: Over half of all C. glabrata clinical isolates contain mutations in the mismatch repair gene MSH2, which leads to higher frequency of resistance emergence. These genetic adaptations may affect how RNAPII responds to drug exposure

Methodological approaches:

• Compare RNAPII occupancy profiles between drug-sensitive and resistant strains

• Perform ChIP-seq for both RNAPII and relevant transcription factors before and after drug exposure

• Create reporter constructs to monitor transcriptional responses to antifungal drugs

How has the RPB2 gene evolved within the Candida genus and related fungi?

The RPB2 gene shows important evolutionary patterns:

• Phylogenetic utility: RPB2 sequences have been used to reconstruct the phylogeny of various fungal genera, including Hordeum, revealing its evolutionary conservation and utility as a phylogenetic marker

• Genomic element insertions: In some fungi, miniature inverted-repeat terminal elements (MITEs) have been found to insert into the RPB2 gene, potentially affecting its evolution

• Geographical distribution: Indel length in some genomes corresponds well to geographical distribution, suggesting regional evolutionary pressures

Comparative analysis of RPB2 across Candida species and related fungi reveals:

• Regions of high conservation corresponding to functional domains

• Species-specific variations that may relate to host adaptation

• Evidence of selection pressure on specific domains that interact with transcription factors

How do regulatory networks involving RPB2-mediated transcription differ between C. glabrata and closely related species?

Despite C. glabrata being more closely related to Saccharomyces cerevisiae than to C. albicans, it has evolved distinct regulatory strategies:

• Oxidative stress response: In C. glabrata, all three factors (CgSkn7, CgYap1, and CgMsn2/4) are strongly induced in response to hydrogen peroxide, whereas only Yap1 is induced and Skn7 is repressed in S. cerevisiae

• Adaptive evolution: C. glabrata has evolved specific transcriptional responses for adaptation to a human commensal/opportunistic pathogen lifestyle

• Novel transcription factors: Factors like CgXbp1 have acquired specialized functions in C. glabrata for survival in macrophages and drug tolerance

The evolutionary relationship can be studied by:

• Comparative ChIP-seq targeting RNAPII across multiple species

• Analysis of transcription factor binding site conservation

• Functional complementation experiments between species

What methodological considerations are important when designing RNAPII ChIP-seq experiments in C. glabrata?

Effective ChIP-seq for studying RNAPII in C. glabrata requires careful attention to:

Experimental Design Considerations:

Advanced Analysis Approaches:

• Separate genes into bins based on promoter escape efficiency (TR values)

• Generate normalized aggregate RNAPII occupancy profiles

• Compare RNAPII distributions around transcription start sites and across gene bodies

• Integrate with other genomic data (e.g., histone modifications, transcription factor binding)

How can researchers distinguish between direct and indirect effects when studying RPB2 mutations in C. glabrata?

Distinguishing direct from indirect effects requires a multi-faceted approach:

• Rapid depletion systems:

  • Anchor-away approach to conditionally deplete targeted factors from the nucleus

  • Allows observation of immediate effects before compensatory responses occur

  • Heterozygous diploids with one FRB-tagged allele enable ~50% depletion upon rapamycin treatment

• Combined genomic approaches:

  • ChIP-seq for both RNAPII and transcription factors

  • RNA-seq to measure mature transcript levels

  • Integration of data to identify discrepancies between transcription and steady-state mRNA levels

• Structure-guided mutations:

  • Target specific domains based on structural information

  • Create allelic series with varying degrees of functional impairment

  • Compare phenotypic outcomes across mutation spectrum

• Complementation studies:

  • Reintroduce wild-type or mutant RPB2 into deletion background

  • Test domain-swapping between species to identify functional regions

  • Use inducible systems to control timing and level of expression

This integrated approach allows researchers to build causal models distinguishing primary effects of RPB2 mutations from downstream adaptive responses.

What are the best approaches for creating conditional RPB2 mutants in C. glabrata?

Creating conditional RPB2 mutants requires specialized approaches since RPB2 is essential:

• Tetracycline-inducible systems:

  • Engineer strains where all 12 RNAPII subunits can be simultaneously and conditionally overexpressed

  • Anhydrotetracycline-inducible promoter (TetPr) allows 2-3 fold overexpression in a defined time window

  • Can study effects of increased RNAPII concentration

• Anchor-away approach:

  • FRB-tag one allele in a heterozygous diploid to allow conditional nuclear depletion

  • Enables ~50% reduction in nuclear amounts upon rapamycin treatment

  • Allows study of partial loss-of-function

• Domain-specific mutations:

  • Target non-essential domains like the flap loop

  • Design deletions that remove specific interaction interfaces with transcription factors

  • Create point mutations in conserved residues

• Degron-based systems:

  • Fusion of destabilizing domains for rapid protein degradation

  • Temperature-sensitive alleles for conditional inactivation

  • Auxin-inducible degron for controlled protein depletion

Each approach has advantages for specific experimental questions, with the choice depending on whether transient or sustained effects are being studied.

How can virulence assays be optimized to study the role of RPB2 in C. glabrata pathogenesis?

Multiple virulence models have been established, each with specific advantages:

In vivo models:

In vitro models:

Optimized approaches combine:

What are the critical controls for studying RPB2-dependent transcription in C. glabrata?

Robust experimental design requires several key controls:

For ChIP-seq experiments:

• Spike-in controls: Mix C. glabrata and S. cerevisiae samples in different ratios before immunoprecipitation to confirm quantitative linearity

• Antibody controls: Include both RPB2-specific antibodies and antibodies against the RPB1 C-terminal domain (8WG16)

• Input samples: Always compare to input chromatin to control for DNA abundance biases

• Biological replicates: Minimum of 3 biological replicates to enable statistical analysis

• Quantile normalization: Apply between replicates and conditions to enable fair comparisons

For genetic manipulation studies:

• Complementation controls: Reintroduce wild-type RPB2 to confirm phenotype rescue

• Empty vector controls: Include in all transformations

• Growth rate normalization: Ensure similar growth rates when comparing strains

• Strain background verification: Confirm genetic background using markers

For drug susceptibility testing:

• Standard drug control strains: Include reference strains with known susceptibility

• Growth medium controls: Test media without drugs to establish baseline growth

• Time-course measurements: Monitor growth over time rather than endpoint only

• Concentration series: Use multiple drug concentrations to establish dose-response curves

How can researchers integrate RNAPII occupancy data with other genomic datasets to understand C. glabrata biology?

Integrative analysis provides deeper insights than any single data type:

• Multi-omics integration approaches:

  • Combine RNAPII ChIP-seq with mRNA-seq to distinguish transcription from RNA stability effects

  • Integrate with ChIP-seq for histone modifications to understand chromatin-mediated regulation

  • Incorporate transcription factor binding data to identify regulatory networks

• Analytical frameworks:

  • Calculate traveling ratio (signal within gene body/signal at promoter) to identify genes with different regulatory mechanisms

  • Generate normalized aggregate profiles around transcription start sites

  • Perform sliding window analysis using 500-bp windows every 125 bp from -3 kb to +3 kb relative to TSS

• Visualization and clustering:

  • Group genes by expression patterns during infection or drug treatment

  • Identify co-regulated gene modules that may share regulatory mechanisms

  • Map temporal dynamics to biological pathways

• Functional validation:

  • Target identified genes for deletion or overexpression

  • Test phenotypic consequences in infection models

  • Confirm direct regulation through targeted ChIP-qPCR

This integrated approach allows researchers to move beyond descriptive genomics to mechanistic understanding of C. glabrata biology.

How might single-cell approaches revolutionize our understanding of RPB2-dependent transcription in C. glabrata populations?

Single-cell technologies offer new perspectives on heterogeneity in fungal populations:

• Population heterogeneity: Single-cell approaches could reveal subpopulations with distinct transcriptional states during infection or drug exposure

• Bet-hedging strategies: May uncover how C. glabrata uses transcriptional diversity as a survival strategy

• Host-pathogen dynamics: Could track individual cell fates during macrophage infection

Emerging technologies applicable to C. glabrata include:

• Single-cell RNA-seq adapted for fungi

• CUT&Tag for profiling RNAPII occupancy in small cell numbers

• Live-cell imaging with tagged RNAPII to track dynamics in real-time

• Spatial transcriptomics to map fungal gene expression in tissue context

What potential does RPB2 hold as an antifungal drug target in C. glabrata?

RPB2 presents intriguing possibilities as a therapeutic target:

Research approaches to explore this potential:

• Structure-based drug design targeting fungal-specific regions

• Screens for compounds that disrupt specific RPB2 interactions

• Transcription-based screens to identify molecules that selectively inhibit fungal transcription