Recombinant Ashbya gossypii Mediator of RNA polymerase II transcription subunit 9 (CSE2)

What is Ashbya gossypii and why is it relevant for studying the Mediator complex?

Ashbya gossypii is a filamentous ascomycete belonging to the yeast family of Saccharomycetaceae that has gained prominence in both industrial and research settings. This organism generates abundant amounts of riboflavin at the end of its growth phase and has been explored as a host system for recombinant protein production . A. gossypii is particularly valuable for studying eukaryotic transcriptional machinery, including the Mediator complex, due to several advantages:

• Its genome has been fully sequenced, providing a comparative genomic model that helped confirm the Whole Genome Duplication in the Saccharomyces lineage

• It possesses a highly efficient homologous recombination system that facilitates genetic manipulation

• The organism has a versatile research toolkit including different marker genes, regulatable promoters, Cre-lox based marker removal, and recent additions of CRISPR/Cas9 and CRISPR-Cpf1 for multiplex genome editing

• It shares significant genetic conservation with Saccharomyces cerevisiae but displays distinct multicellular filamentous growth, offering insights into how the Mediator complex may function in different cellular contexts

How does A. gossypii CSE2 compare structurally and functionally to its homologs in other fungi?

While detailed structural comparisons are not provided in the search results, we can analyze the evolutionary context:

The genomic context is particularly interesting as comparative genomics studies within the genus Eremothecium revealed chromosome number reductions from eight to seven chromosomes in A. gossypii and A. aceri, and from eight to six chromosomes in E. coryli, while E. cymbalariae maintains the ancestral eight chromosomes . These genomic rearrangements may have influenced the regulatory networks involving CSE2, potentially affecting its function in transcriptional regulation.

What are the recommended protocols for recombinant expression of A. gossypii CSE2?

For recombinant expression of A. gossypii CSE2, researchers should consider a multi-step approach:

Expression System Selection:

• For structural studies: E. coli BL21(DE3) with a T7 promoter-based vector

• For functional studies: Yeast expression systems (S. cerevisiae or native A. gossypii)

• For protein-protein interaction studies: Baculovirus-insect cell system

Optimization Parameters:

• Codon optimization based on A. gossypii preferred codon usage

• Addition of purification tags (His, GST, or MBP) that minimize interference with protein function

• Expression temperature (typically 16-25°C for complex proteins to ensure proper folding)

• Induction conditions (IPTG concentration or galactose percentage)

Expression Verification:

• Western blot analysis using specific antibodies such as CSB-PA751740XA01DOT (anti-CSE2 polyclonal antibody)

• SDS-PAGE for protein size confirmation

When working with A. gossypii as the expression host, researchers should note that the organism has distinct protein secretion characteristics, with most secreted proteins having isoelectric points between 4 and 6, and molecular weights above 25 kDa . This may inform purification strategies if secretion-based production is attempted.

What purification methods are most effective for isolating recombinant A. gossypii CSE2?

Based on protein characteristics and research objectives, the following purification workflow is recommended:

Primary Capture:

• Immobilized Metal Affinity Chromatography (IMAC) for His-tagged CSE2

• Glutathione-affinity chromatography for GST-tagged constructs

• Amylose resin for MBP-tagged proteins

Secondary Purification:

• Ion Exchange Chromatography (IEX) - Given that many A. gossypii proteins have isoelectric points between 4-6 , a cation exchange column at pH below the protein's pI would be effective

• Size Exclusion Chromatography (SEC) to separate monomeric CSE2 from aggregates or other contaminants

Advanced Methods (for structural studies):

• Affinity purification using engineered binding partners

• On-column refolding if inclusion bodies form

• Tag removal using specific proteases (TEV, PreScission, etc.)

Quality Control Metrics:

• Purity assessment: SDS-PAGE (>95% purity) and Western blot using anti-CSE2 antibody

• Activity assessment: DNA-binding assays, transcriptional assays

• Integrity verification: Mass spectrometry

For researchers seeking antibody reagents, the commercially available polyclonal CSE2 antibody (CSB-PA751740XA01DOT) raised against recombinant A. gossypii CSE2 protein can be used for detection methods including ELISA and Western blotting .

How can I design experimental controls when studying CSE2 function in A. gossypii?

Designing proper controls is critical for rigorous investigation of CSE2 function:

Genetic Controls:

• Complete CSE2 knockout strain (if viable)

• Conditional CSE2 knockdown using regulatable promoters

• Point mutation variants affecting specific domains

• Complementation with S. cerevisiae homologs to assess functional conservation

• Tag-only expression constructs to control for tag effects

Experimental Controls:

• Wild-type A. gossypii (strain ATCC 10895/CBS 109.51/FGSC 9923/NRRL Y-1056)

• Strains expressing unrelated proteins of similar size/characteristics

• Time-course sampling to establish baseline expression patterns

Molecular Controls for Interaction Studies:

• Known interactors of the Mediator complex as positive controls

• Non-interacting proteins as negative controls

• Competition assays with purified recombinant proteins

Transcriptional Analysis Controls:
When assessing transcriptional effects of CSE2 manipulation, include controls similar to those used in previous A. gossypii transcriptional studies, which identified differential expression of genes during developmental shifts from growth to sporulation .

How does CSE2 contribute to transcriptional regulation during A. gossypii's developmental transition from growth to sporulation?

The developmental shift from growth to sporulation in A. gossypii affects approximately 860 genes (560 upregulated, 300 downregulated) out of the ~5000 total genes . While CSE2's specific contribution is not directly addressed in the search results, its role as a Mediator complex component suggests involvement in this substantial transcriptional reprogramming.

Potential Regulatory Mechanisms:

• CSE2 may interact with key transcription factors that regulate sporulation genes

• It could facilitate chromatin remodeling required for developmental transitions

• CSE2 might participate in repression complexes that silence growth-related genes during sporulation

Key Regulatory Networks:
The core sporulation regulatory network in A. gossypii involves components conserved with S. cerevisiae, including IME1, IME2, IME4, and KAR4 . These factors likely interact with the Mediator complex, potentially through CSE2, to coordinate gene expression during sporulation.

Experimental Approach:
To investigate CSE2's role in this process, researchers could:

• Perform ChIP-seq to identify CSE2 binding sites during different developmental stages

• Analyze transcriptomes of CSE2 mutants during sporulation

• Conduct epistasis analysis between CSE2 and known sporulation regulators like IME1, IME2, and SOK2

• Examine protein-protein interactions between CSE2 and sporulation-specific transcription factors

The intrinsic negative regulators of sporulation identified in A. gossypii, including the α2 repressor and STE12, may interact functionally with CSE2 to fine-tune transcriptional responses .

What is the relationship between CSE2 function and protein secretion stress responses in A. gossypii?

A. gossypii has been explored as a host system for recombinant protein production , making the relationship between transcriptional regulation (involving CSE2) and secretion stress particularly relevant.

Transcriptional Response to Secretion Stress:
Studies of A. gossypii cells secreting recombinant Endoglucanase I (EGI) identified 21 differentially expressed genes correlated with recombinant protein secretion . The pattern suggested:

• Down-regulation of translation

• Up-regulation of ion and amino acid transmembrane transport

Potential CSE2 Involvement:
As a Mediator component, CSE2 could participate in:

• Sensing secretion stress and relaying signals to the transcriptional machinery

• Recruiting transcription factors that regulate stress response genes

• Modulating expression of genes involved in protein folding, transport, and quality control

Relevant Gene Expression Data:
This table shows genes differentially expressed during recombinant protein secretion in A. gossypii that may involve CSE2-mediated regulation:

This pattern suggests CSE2 may be involved in coordinating the downregulation of translation machinery during secretion stress .

How might CSE2 function differ between multinucleate and mononucleate fungal systems?

A. gossypii presents a unique model for studying transcriptional regulation in a multinucleate context, as its hyphae contain multiple nuclei that undergo asynchronous divisions within a shared cytoplasm .

Multinucleate Challenges for Transcriptional Regulation:

• Coordinating gene expression across multiple nuclei in a common cytoplasm

• Potentially different chromatin states and transcriptional activities between nuclei

• Nuclear-specific responses to local environmental signals

Hypothesized CSE2 Adaptations:

• Specialized interactions with nuclear transport machinery

• Mechanisms to ensure proper stoichiometry of Mediator components

• Altered kinetics of assembly/disassembly to accommodate multinucleate context

Comparative Framework:

Research Approach:
To investigate CSE2's multinucleate-specific functions, researchers could:

• Use nucleus-specific labeling combined with CSE2 visualization

• Compare ChIP-seq profiles across different nuclear populations within the same hypha

• Develop mathematical models of transcriptional regulation in multinucleate systems

What bioinformatic approaches are recommended for analyzing ChIP-seq data of CSE2 in A. gossypii?

Recommended Workflow for CSE2 ChIP-seq Analysis:

• Quality Control and Preprocessing:

  • Assess read quality using FastQC

  • Trim adapters and low-quality bases with Trimmomatic or similar tools

  • Map to A. gossypii reference genome (preferably strain ATCC 10895/CBS 109.51/FGSC 9923/NRRL Y-1056)

  • Remove PCR duplicates

• Peak Calling Considerations:

  • Use narrow peak callers (e.g., MACS2) as CSE2 likely has defined binding sites

  • Consider the multinucleate nature of A. gossypii when interpreting peak distributions

  • Employ appropriate controls: input DNA and non-specific antibody pulldowns

• Advanced Analysis:

  • Motif discovery to identify sequence preferences for CSE2 binding

  • Peak annotation relative to A. gossypii gene models

  • Integration with RNA-seq data to correlate binding with expression outcomes

  • Comparative analysis with S. cerevisiae CSE2 binding patterns

• Multinucleate-Specific Considerations:

  • Develop metrics to assess binding heterogeneity across nuclei

  • Consider spatial clustering of peaks that might reflect nuclear territories

  • Integrate with nuclear positioning data if available

Validation Approaches:

• qPCR validation of selected binding sites

• Reporter assays to confirm functional significance

• Genetic manipulation of binding sites using CRISPR/Cas9 system available for A. gossypii

How should researchers interpret conflicting data between transcriptomic and proteomic analyses of CSE2-dependent processes?

When confronted with discrepancies between transcriptomic and proteomic data in CSE2 studies, consider these systematic analysis approaches:

Sources of Discrepancy:

• Post-transcriptional regulation (particularly relevant in multinucleate systems)

• Protein stability differences

• Technical limitations in detection methods

• Temporal delays between transcription and translation

Analytical Framework:

Resolution Strategies:

• Perform time-course experiments to capture dynamics

• Utilize ribosome profiling to assess translation efficiency

• Examine protein half-life and degradation pathways

• Consider compartmentalization effects in the multinucleate system

• Apply integrated computational approaches that normalize and reconcile multi-omics data

Contextual Considerations:
In A. gossypii, protein secretion studies have shown distinct patterns in the secretome compared to intracellular proteins . This compartmentalization adds another layer of complexity when interpreting transcriptomic versus proteomic data.

What statistical approaches should be used to analyze CSE2 impact on global gene expression in A. gossypii?

Given the complexity of A. gossypii biology and CSE2's likely broad impact as a Mediator component, robust statistical approaches are essential:

Recommended Statistical Methods:

• Differential Expression Analysis:

  • LIMMA (Linear Models for Microarray Data) - previously used for A. gossypii transcriptomic analysis

  • DESeq2 or EdgeR for RNA-seq count data

  • Consider time-course analysis packages (e.g., maSigPro) for developmental transitions

• Correlation-Based Approaches:

  • Previous studies in A. gossypii successfully used correlation analysis to detect subtle expression changes that weren't captured by standard differential expression tests

  • Gene Set Enrichment Analysis (GSEA) to identify pathways and processes affected

• Multinucleate-Specific Considerations:

  • Develop statistical frameworks that account for potential heterogeneity between nuclei

  • Consider spatial statistics if nuclear position data is available

Multiple Testing Correction:

• Apply FDR correction methods (Benjamini-Hochberg) with appropriate thresholds

• Previous A. gossypii studies used FDR of 4.4% for correlation-based analyses

Biological Validation:

• Targeted qPCR of representative genes from each expression cluster

• Reporter assays for key regulated promoters

• Genetic epistasis tests with other transcriptional regulators

For integrating CSE2 studies with existing knowledge of A. gossypii biology, consider that previous work identified 560 genes upregulated and 300 genes downregulated during the developmental shift from growth to sporulation . The CSE2 contribution to this regulatory network would be a valuable research direction.

How can researchers address inconsistent expression of recombinant CSE2 in heterologous systems?

When facing challenges with recombinant CSE2 expression, consider these systematic troubleshooting approaches:

Expression Optimization Strategies:

• Codon Optimization:

  • Analyze A. gossypii codon usage bias and optimize for the expression host

  • Consider synonymous codon substitutions that preserve amino acid sequence but enhance expression

• Vector and Construct Design:

  • Try different promoter strengths (T7, tac, AOX1, GAL1)

  • Experiment with various fusion tags (His, GST, MBP, SUMO)

  • Test different vector backbones with varying copy numbers

  • Include translation enhancers (e.g., Shine-Dalgarno sequences, Kozak consensus)

• Host Strain Selection:

  • For E. coli: BL21(DE3), C41(DE3), SHuffle for disulfide bond formation

  • For yeast: Protease-deficient strains to minimize degradation

• Culture Conditions:

  • Temperature modulation (lower to 16-20°C during induction)

  • Induction timing and duration optimization

  • Media supplementation with cofactors or stabilizing agents

Troubleshooting Decision Tree:

Advanced Approaches:

• Co-expression with interacting partners from the Mediator complex

• Cell-free expression systems for toxic proteins

• Refolding protocols for inclusion body recovery

• Directed evolution of expression hosts for improved yield

What approaches can resolve specific antibody cross-reactivity issues when studying CSE2 in A. gossypii?

Antibody specificity is crucial for accurate CSE2 detection and functional studies. When facing cross-reactivity issues:

Antibody Optimization Strategies:

• Epitope Refinement:

  • Design peptide antigens unique to A. gossypii CSE2

  • Target regions with minimal conservation to related proteins

  • Consider recombinant fragments rather than full-length protein

• Validation Protocols:

  • Use CSE2 knockout/knockdown strains as negative controls

  • Perform peptide competition assays

  • Validate with orthogonal detection methods (e.g., MS/MS)

• Purification Approaches:

  • Affinity purification against recombinant CSE2

  • Negative selection against cross-reactive proteins

  • Immunodepletion of cross-reactive antibodies

• Technical Modifications:

  • Optimize blocking conditions (BSA, milk, specialized blockers)

  • Adjust antibody concentration and incubation parameters

  • Modify stringency of washing steps

Available Reagents:
The CSE2 antibody (CSB-PA751740XA01DOT) is a rabbit polyclonal antibody raised against recombinant A. gossypii CSE2 protein that has been affinity-purified and is recommended for ELISA and Western blot applications .

Alternative Detection Strategies:

• Tagged recombinant CSE2 expression with commercial tag antibodies

• Proximity labeling approaches (BioID, APEX)

• Mass spectrometry-based identification

• CRISPR-based endogenous tagging of CSE2

How can researchers differentiate between direct and indirect transcriptional effects of CSE2 in A. gossypii?

Distinguishing direct from indirect effects is a common challenge in transcriptional regulator studies:

Experimental Strategies:

• Temporal Resolution:

  • Perform high-resolution time course experiments after CSE2 perturbation

  • Early-response genes are more likely to be direct targets

  • Apply mathematical modeling to infer regulatory cascade dynamics

• Orthogonal Validation:

  • ChIP-seq to identify direct binding sites of CSE2

  • Reporter assays with wild-type and mutated binding sites

  • Inducible degron-tagged CSE2 for rapid depletion studies

• Genetic Approaches:

  • Epistasis analysis with known transcription factors

  • Targeted mutagenesis of predicted binding sites

  • Synthetic genetic array analysis to map genetic interactions

• Network Analysis:

  • Apply Bayesian network inference to model direct relationships

  • Use machine learning approaches to classify direct vs. indirect targets

  • Integrate multiple data types (ChIP-seq, RNA-seq, proteomic) for higher confidence predictions

• Potential asynchronous responses across different nuclei

• Nuclear-specific transcriptional territories

• Diffusion of transcription factors between nuclear zones

Previous studies of transcriptional regulation during sporulation in A. gossypii identified key regulators like IME1, IME2, and SOK2 . Researchers could investigate interactions between these factors and CSE2 to build a more comprehensive understanding of the regulatory network.

What emerging technologies could advance our understanding of CSE2 function in multinucleate systems like A. gossypii?

Cutting-Edge Methodologies for CSE2 Research:

• Spatial Transcriptomics Approaches:

  • Single-nucleus RNA-seq to profile individual nuclei within the same hypha

  • Multiplexed FISH to visualize transcripts with spatial resolution

  • Spatial proteomics to map protein distribution relative to nuclear positions

• Advanced Genomic Engineering:

  • CRISPR-based screening in A. gossypii to identify genetic interactions with CSE2

  • Base editing for precise single nucleotide modifications

  • Prime editing for targeted sequence replacements without double-strand breaks

• Structural Biology Innovations:

  • Cryo-EM of the A. gossypii Mediator complex with CSE2

  • Integrative structural modeling combining various experimental data types

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic interactions

• Single-Molecule Approaches:

  • Live-cell single-molecule tracking of labeled CSE2 in multinucleate hyphae

  • Super-resolution microscopy to visualize transcription factories

  • Single-molecule footprinting to map protein-DNA interactions at high resolution

• Computational Advances:

  • Agent-based modeling of transcriptional regulation in multinucleate systems

  • Deep learning approaches to predict CSE2 binding and functional outcomes

  • Multi-scale modeling integrating molecular dynamics with cellular-level simulations

A. gossypii's extensive genetic toolbox, including CRISPR/Cas9 and CRISPR-Cpf1 systems for multiplex genome editing , positions it well for applying these emerging technologies.

How might understanding CSE2 function contribute to biotechnological applications of A. gossypii?

A. gossypii is already utilized for industrial riboflavin production and has potential as a recombinant protein production platform . CSE2's role in transcriptional regulation suggests several biotechnological applications:

Potential Applications:

• Engineered Protein Production:

  • Modulating CSE2 to enhance secretory pathway capacity

  • Engineering CSE2-dependent promoters for controlled expression

  • Creating synthetic transcriptional circuits incorporating CSE2 function

• Metabolic Engineering:

  • Transcriptional reprogramming for improved riboflavin production

  • Targeting CSE2-regulated pathways for novel metabolite production

  • Developing stress-resistant strains through CSE2-mediated transcriptional adaptation

• Synthetic Biology Platforms:

  • Utilizing the multinucleate nature for compartmentalized reactions

  • Developing nucleus-specific gene expression systems

  • Creating synthetic developmental switches based on CSE2 regulatory networks

Relevant Findings:
Studies of protein secretion in A. gossypii have shown distinct protein profiles in different media conditions, with proteins having isoelectric points between 4 and 6, and molecular weights above 25 kDa . Understanding CSE2's role in regulating secretory pathway genes could help optimize these processes for biotechnological applications.

What can comparative studies between A. gossypii and related fungi reveal about the evolution of CSE2 function?

Comparative evolutionary analysis of CSE2 across related fungi offers insights into functional adaptation and conservation:

Evolutionary Research Approaches:

• Phylogenetic Analysis:

  • Construct CSE2 phylogenies across fungal lineages

  • Map functional domains and their conservation/divergence

  • Identify signatures of selection on specific residues or regions

• Functional Complementation:

  • Test cross-species complementation of CSE2 mutants

  • Identify species-specific interaction partners

  • Map functional divergence through domain swapping experiments

• Comparative Genomics:

  • Analyze synteny and genome context around CSE2 loci

  • Examine correlation between CSE2 sequence and genome architecture changes

  • Study CSE2 in the context of chromosome number reductions observed in the Eremothecium genus

Evolutionary Context:
The genus Eremothecium shows interesting genome evolution patterns, with chromosome number reductions from the ancestral eight chromosomes to seven in A. gossypii and A. aceri, and to six in E. coryli . This provides a natural experiment to study how CSE2 function may have adapted during these genomic reorganizations.

Comparative Framework:

This comparative approach could reveal how transcriptional regulation has evolved during the transition between growth forms and genomic reorganizations.