Recombinant Schizosaccharomyces pombe Uncharacterized protein C11C11.05 (SPBC11C11.05)

What is the genomic context of SPBC11C11.05 and how might this inform functional hypotheses?

SPBC11C11.05 is positioned in proximity to the gene SPBC11C11.06c, which is downstream of the PRO.08 tDNA loci in the S. pombe genome . This genomic neighborhood is particularly interesting because research has demonstrated interactions between RNA Polymerase II (Pol II) and Pol III transcription in this region.

Studies have shown that Pol II transcription initiated downstream of the PRO.08 tDNA is involved in chromatin remodeling that primes efficient Pol III transcription . The expression of SPBC11C11.06c has been shown to be independent of transcription factors Pcr1 and Atf1, which regulate stress responses in S. pombe . This genomic context suggests that SPBC11C11.05 might be involved in transcriptional regulation, chromatin remodeling, or related processes.

How can recombinant SPBC11C11.05 protein be properly stored and handled for research applications?

Recombinant SPBC11C11.05 is typically provided in a Tris-based buffer with 50% glycerol, specifically optimized for this protein's stability . For optimal results, researchers should adhere to the following storage and handling guidelines:

• Store the protein at -20°C for general storage and -80°C for extended preservation

• Avoid repeated freeze-thaw cycles as these can compromise protein integrity

• Maintain working aliquots at 4°C for up to one week only

• Include appropriate protease inhibitors when performing experimental manipulations

• Verify protein quality before experiments using methods such as SDS-PAGE or size exclusion chromatography

These storage conditions are essential for maintaining protein stability and functionality for downstream experimental applications.

What are the most effective approaches for studying the expression pattern of SPBC11C11.05?

To comprehensively characterize the expression pattern of SPBC11C11.05, researchers should employ multiple complementary techniques:

• Transcriptomic Analysis:

  • RNA-seq profiling across different growth conditions, cell cycle stages, and stress responses

  • Integration with existing S. pombe transcriptome datasets, particularly those identifying cell cycle-regulated genes

  • Quantitative RT-PCR validation of expression patterns under specific conditions

• Promoter Analysis:

  • Identification of potential regulatory motifs in the SPBC11C11.05 promoter

  • Comparison with known cell cycle-specific or stress-responsive promoter elements

  • Studies have identified four promoter motifs in S. pombe with strong association to cell cycle phase-specific expression

• Chromatin Immunoprecipitation (ChIP):

  • Analysis of transcription factor binding at the SPBC11C11.05 promoter

  • Techniques similar to those used for studying Sty1 MAP kinase recruitment to stress-induced genes

  • Integration with histone modification data to understand chromatin context

• Reporter Gene Systems:

  • Creation of SPBC11C11.05 promoter-reporter constructs

  • Analysis of expression patterns in different conditions and genetic backgrounds

  • Mutation of putative regulatory elements to identify essential promoter features

These approaches should be integrated to develop a comprehensive understanding of when, where, and how SPBC11C11.05 is expressed in the cell.

What strategies can be employed to study protein-protein interactions involving SPBC11C11.05?

Identifying protein interaction partners is crucial for understanding the functional context of SPBC11C11.05. Several complementary approaches should be considered:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Expression of epitope-tagged SPBC11C11.05 in S. pombe

  • Immunoprecipitation under native conditions to preserve protein complexes

  • Mass spectrometric identification of co-purifying proteins

  • This approach has been successful in identifying components of protein complexes in S. pombe, such as the ELL transcription elongation complex

• Proximity-Based Labeling:

  • Fusion of SPBC11C11.05 with enzymes like BirA* or APEX2

  • Biotinylation of proteins in close proximity in living cells

  • Identification of labeled proteins by streptavidin purification and mass spectrometry

  • This approach can capture transient interactions missed by conventional co-immunoprecipitation

• Yeast Two-Hybrid Screening:

  • Use of SPBC11C11.05 as bait to screen S. pombe cDNA libraries

  • Identification of direct binary interactions

  • Validation of positive hits using orthogonal methods

• Co-localization Studies:

  • Creation of fluorescently tagged SPBC11C11.05

  • Live-cell imaging to track subcellular localization

  • Co-localization with candidate interaction partners

  • Fluorescence resonance energy transfer (FRET) to confirm direct interactions

When analyzing protein interaction data, particular attention should be paid to connections with known transcription regulators, chromatin remodelers, or components of RNA polymerase complexes, given the genomic context of SPBC11C11.05 .

How can the function of SPBC11C11.05 be investigated through gene deletion or modification approaches?

Genetic manipulation provides powerful tools for investigating SPBC11C11.05 function. The following strategies should be considered:

• CRISPR-Cas9 Knockout Strategy:

  • Design of guide RNAs targeting the SPBC11C11.05 coding sequence

  • Integration of appropriate selection markers

  • Verification of knockout by PCR, sequencing, and protein detection methods

  • Phenotypic analysis under various conditions including normal growth, stress, and cell cycle perturbation

• Conditional Expression Systems:

  • Placement of SPBC11C11.05 under control of regulatable promoters (e.g., nmt1)

  • Enabling temporal control of gene expression

  • Particularly valuable if complete knockout proves lethal

  • Analysis of phenotypes during depletion and re-expression

• Domain Deletion and Mutation Analysis:

  • Creation of systematic truncations or point mutations

  • Functional complementation assays

  • Identification of essential domains or residues

  • This approach can reveal functional domains without prior structural knowledge

• Fusion Tag Strategies:

  • C- or N-terminal tagging with epitopes for detection and purification

  • Care must be taken to ensure tags don't disrupt function

  • Special consideration should be given to the impact of C-terminal tags, as structural data from related systems suggests they may impede conformational changes required for some protein functions

A comprehensive genetic analysis should include phenotypic characterization under conditions that test cell cycle progression, stress response, and transcriptional regulation, given the contextual information available about SPBC11C11.05 .

What evidence suggests potential roles for SPBC11C11.05 in transcriptional regulation?

Several lines of evidence suggest SPBC11C11.05 may function in transcriptional regulation processes:

• Genomic Context and RNA Polymerase Dynamics:

  • SPBC11C11.05 is located near the PRO.08 tDNA loci, where Pol II transcription influences Pol III activity

  • Studies have demonstrated that Pol II transcription in this region is required to prime and maintain nucleosome depletion at Pol III loci

  • Pol II transcription initiated from a CRE motif located approximately 550 bp downstream of the PRO.08 tDNA has been shown to influence chromatin accessibility

• Potential Connection to Transcription Elongation:

  • S. pombe contains a Pol II transcription elongation factor complex that includes Ell1, Eaf1, and Ebp1, which is functionally similar to the metazoan Super Elongation Complex (SEC)

  • This complex plays important roles in regulating genes involved in cell separation

  • SPBC11C11.05 could potentially interact with or function in parallel to this complex

• Chromatin Modification Connection:

  • Histone modifications, particularly H3 acetylation by HATs like Gcn5 and Mst2, are important for efficient Pol III transcription in S. pombe

  • CTD S2 phosphorylation of Pol II is required to maintain nucleosome-depleted regions at class III loci

  • SPBC11C11.05 might participate in these chromatin-modifying processes

To investigate these potential roles, researchers should consider ChIP-seq experiments to map SPBC11C11.05 binding sites, RNA-seq analysis in SPBC11C11.05 mutants, and genetic interaction studies with known transcription regulators.

How might SPBC11C11.05 be involved in cell cycle regulation or stress response pathways?

Based on contextual information and S. pombe biology, SPBC11C11.05 may function in cell cycle regulation or stress response:

• Cell Cycle Regulation:

  • S. pombe is a well-established model for cell cycle studies, with 747 genes identified as having cell cycle-regulated expression

  • Genome-wide studies have identified promoter motifs associated with cell cycle phase-specific expression

  • Analysis of SPBC11C11.05 expression throughout the cell cycle could reveal patterns suggestive of a cell cycle role

• Stress Response Pathways:

  • The stress-activated MAP kinase Sty1 and transcription factor Atf1 regulate stress-induced gene expression in S. pombe

  • Stress response often involves chromatin remodeling and transcriptional reprogramming

  • SPBC11C11.05 could function in stress adaptation, particularly if its expression or activity is modulated under stress conditions

• Growth Transition Regulation:

  • Pol II transcription is required for efficient recruitment of Pol III upon exit from stationary phase

  • This process is important for cells to efficiently reinitiate growth after periods of quiescence

  • If SPBC11C11.05 functions in this pathway, mutants might show defects in transitions between growth states

Experimental approaches to test these hypotheses should include:

• Analysis of SPBC11C11.05 expression and localization during cell cycle progression and under various stresses

• Phenotypic characterization of SPBC11C11.05 mutants during cell cycle arrest/release and stress exposure

• Genetic interaction studies with known cell cycle and stress response regulators

What computational methods can provide insights into SPBC11C11.05 function prior to extensive experimentation?

Computational approaches can generate valuable functional hypotheses for SPBC11C11.05:

• Sequence and Structural Analysis:

  • Identification of conserved domains or motifs using tools like InterPro, Pfam, and SMART

  • Secondary structure prediction using PSIPRED

  • 3D structure prediction with AlphaFold2 or similar tools

  • Analysis of potential functional sites based on structural features

• Comparative Genomics:

  • Identification of orthologs in related species

  • Analysis of evolutionary conservation patterns

  • Investigation of synteny and gene neighborhood conservation

  • S. pombe and S. cerevisiae comparative data can be particularly informative despite their evolutionary divergence

• Promoter and Regulatory Element Analysis:

  • Identification of transcription factor binding motifs in the SPBC11C11.05 promoter

  • Comparison with promoters of genes with known functions

  • Assessment of potential cell cycle-regulated promoter elements, as four such motifs have been identified in S. pombe

• Network-Based Function Prediction:

  • Integration with existing protein-protein interaction networks

  • Co-expression analysis across multiple conditions

  • Pathway enrichment analysis for genes with similar expression patterns

  • Guilt-by-association approaches to infer function from network neighbors

These computational approaches should be used to generate hypotheses that can be tested through targeted experimental approaches.

What are the considerations for analyzing potential post-translational modifications of SPBC11C11.05?

Post-translational modifications (PTMs) often play crucial roles in protein function and regulation. A systematic approach to studying PTMs in SPBC11C11.05 should include:

• Global PTM Profiling:

  • Mass spectrometry-based proteomics to identify PTMs

  • Enrichment strategies for specific modifications (e.g., phosphopeptide enrichment)

  • Comparison of PTM patterns under different conditions (cell cycle stages, stress)

  • Integration with known S. pombe PTM datasets

• Phosphorylation Analysis:

  • S. pombe extensively uses phosphorylation in cell cycle and stress regulation

  • The CTD of RNA Polymerase II is phosphorylated on S2 by Lsk1 (Cdk12), which affects chromatin structure at tRNA genes

  • Similar phosphorylation events might regulate SPBC11C11.05

  • Techniques like Phos-tag gels can separate phosphorylated protein forms

• Acetylation Investigation:

  • Histone acetyltransferases like Gcn5 and Mst2 play roles in S. pombe chromatin regulation

  • These enzymes can also acetylate non-histone proteins

  • The mutation of the SRI domain of Set2, which affects H3K36me3 deposition, abolishes the chromatin recruitment of Mst2

  • Similar mechanisms might regulate SPBC11C11.05

• Site-Directed Mutagenesis Validation:

  • Mutation of putative modification sites to non-modifiable residues

  • Creation of phosphomimetic mutations (e.g., S/T to D/E)

  • Phenotypic analysis of mutants to determine functional significance

  • In vitro modification assays to identify responsible enzymes

Understanding the PTM landscape of SPBC11C11.05 could provide crucial insights into its regulation within cellular pathways and its potential role in processes like transcriptional regulation or chromatin remodeling.

How can ChIP-seq be optimized for studying SPBC11C11.05 chromatin interactions?

Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for mapping protein-DNA interactions. For SPBC11C11.05, consider these optimization strategies:

• Antibody Selection and Validation:

  • Generate specific antibodies against SPBC11C11.05 or use epitope-tagged versions

  • Rigorously validate antibody specificity through Western blotting and immunoprecipitation

  • Include appropriate controls (pre-immune serum, IgG controls)

  • Validate using cells where SPBC11C11.05 is deleted or depleted

• Crosslinking Optimization:

  • Test different formaldehyde concentrations and incubation times

  • Consider dual crosslinking approaches (e.g., DSG followed by formaldehyde)

  • Optimize for chromatin fragmentation to achieve appropriate fragment sizes

  • Similar approaches to those used for Sty1 MAP kinase ChIP could be adapted

• ChIP Protocol Refinements:

  • Optimize sonication conditions for S. pombe chromatin

  • Test different buffer compositions for immunoprecipitation

  • Include spike-in controls for quantitative comparisons

  • Consider sequential ChIP for co-occupancy studies

• Data Analysis Considerations:

  • Compare SPBC11C11.05 binding sites with known Pol II and Pol III occupancy

  • Integrate with histone modification data, particularly H3 acetylation

  • Analyze enrichment at specific genomic features (promoters, tRNA genes)

  • Look for motifs enriched in binding sites

ChIP-seq analysis should pay particular attention to class III genes (tRNA genes) and their surrounding regions, given the potential role of SPBC11C11.05 in processes related to transcription by Pol II and Pol III .

What techniques can reveal the potential role of SPBC11C11.05 in nucleosome positioning or chromatin remodeling?

Given the genomic context suggesting potential involvement in chromatin-related processes, several techniques can be employed to investigate SPBC11C11.05's role in nucleosome dynamics:

• MNase-seq for Nucleosome Mapping:

  • Compare nucleosome positioning patterns between wild-type and SPBC11C11.05 mutant cells

  • Focus on regions around tRNA genes, where Pol II has been shown to maintain nucleosome-depleted regions (NDRs)

  • Analyze during different growth conditions, particularly exit from stationary phase

  • Examine changes in nucleosome occupancy at specific genomic features

• ATAC-seq for Chromatin Accessibility:

  • Assess global chromatin accessibility changes in SPBC11C11.05 mutants

  • Integrate with transcriptomic data to correlate accessibility with gene expression

  • Focus on class III loci, where accessibility is known to be regulated by Pol II transcription

  • Compare with data from mutants affecting CTD S2 phosphorylation of Pol II

• Histone Modification ChIP-seq:

  • Analyze histone H3 occupancy at class III loci, which increases when CTD S2 phosphorylation is inhibited

  • Examine histone acetylation patterns, particularly those mediated by Gcn5 and Mst2

  • Investigate H3K36 methylation, which is linked to transcription elongation

  • Compare patterns between wild-type and SPBC11C11.05 mutant cells

• In Vitro Nucleosome Assembly/Remodeling Assays:

  • Test whether recombinant SPBC11C11.05 affects nucleosome assembly or positioning in vitro

  • Examine potential interactions with known chromatin remodeling complexes

  • Assess the impact on RNA polymerase progression through chromatin templates

These approaches can reveal whether SPBC11C11.05 contributes to the maintenance of chromatin structure at specific loci, potentially connecting to the established role of Pol II in priming efficient Pol III transcription through chromatin remodeling .

How can multi-omics strategies be applied to comprehensively characterize SPBC11C11.05 function?

A multi-omics approach provides a systems-level understanding of SPBC11C11.05 function:

• Integrated Transcriptomics and Proteomics:

  • RNA-seq and proteomics analysis of SPBC11C11.05 deletion or depletion

  • Identification of consistently affected genes and proteins

  • Network analysis to identify affected pathways

  • Comparison with existing datasets, such as cell cycle-regulated gene expression in S. pombe

• Chromatin and Transcription Integration:

  • Combination of ChIP-seq, ATAC-seq, and RNA-seq data

  • Correlation of SPBC11C11.05 binding with chromatin states and gene expression

  • Analysis of effects on RNA Polymerase II and III distribution

  • Focus on regions where Pol II primes chromatin for Pol III transcription

• Genetic Interaction Mapping:

  • Systematic genetic interaction analysis with genes involved in transcription, chromatin regulation, cell cycle control, and stress response

  • Synthetic genetic array (SGA) analysis to identify functional relationships

  • Validation of key interactions through targeted double mutant analysis

  • Integration with physical interaction data from AP-MS or Y2H studies

• Evolutionary and Comparative Genomics:

  • Comparison of SPBC11C11.05 function with related proteins in other species

  • Analysis of conservation patterns for key domains or residues

  • Examination of the evolution of transcriptional regulation mechanisms between S. pombe and other model organisms

  • Leveraging the extensive comparative data available between S. pombe and S. cerevisiae

Integration across these multiple data types will provide complementary perspectives on SPBC11C11.05 function and place it within the broader context of cellular processes.

What approaches can resolve potential contradictions in experimental data regarding SPBC11C11.05?

When facing contradictory experimental results regarding SPBC11C11.05, consider these systematic approaches:

• Condition-Specific Effects Analysis:

  • Test whether contradictions arise from different experimental conditions

  • Systematic variation of growth conditions (media, temperature, growth phase)

  • Specific examination of cell cycle stages and stress conditions

  • Studies have shown that the requirement for Pol II CTD S2 phosphorylation is stronger when cells exit from stationary phase compared to exponential growth

• Strain Background Verification:

  • Confirm genetic backgrounds of strains used in different studies

  • Verify gene deletions or modifications by sequencing

  • Test for suppressor mutations that might arise in SPBC11C11.05 mutants

  • Create fresh mutants to eliminate accumulated genetic changes

• Methodological Triangulation:

  • Apply multiple orthogonal techniques to address the same question

  • For example, combine ChIP-seq, in vitro binding assays, and genetic approaches

  • Consider technical limitations of each method when interpreting results

  • Include appropriate controls for each methodology

• Quantitative Analysis Refinement:

  • Employ rigorous statistical approaches to data analysis

  • Increase biological and technical replicates

  • Use spike-in controls for normalization when appropriate

  • Implement advanced computational methods for data integration

• Targeted Hypothesis Testing:

  • Design experiments specifically to address contradictions

  • Create separation-of-function mutants to test specific aspects

  • Use conditional alleles to distinguish primary from secondary effects

  • Apply time-course analyses to determine order of events

By systematically addressing contradictions through these approaches, researchers can develop a more nuanced understanding of SPBC11C11.05 function that accounts for context-dependent effects and technical limitations.

How can research findings on SPBC11C11.05 be connected to broader questions in S. pombe biology?

Connecting SPBC11C11.05 research to broader biological questions enhances its impact:

• Evolution of Transcriptional Regulation:

  • S. pombe ELL complex (containing Ell1, Eaf1, and Ebp1) represents an evolutionary precursor of the metazoan SEC complex

  • Understanding SPBC11C11.05's potential role in transcription could provide insights into the evolution of transcriptional regulation

  • Comparisons with S. cerevisiae can reveal conserved and divergent mechanisms, as these species diverged 400-600 million years ago

• Coordination of Different RNA Polymerase Systems:

  • Research has revealed that Pol II transcription primes efficient Pol III transcription at tRNA genes

  • SPBC11C11.05 might contribute to this coordination mechanism

  • This connects to fundamental questions about how cells integrate different transcriptional machineries

• Cell Cycle Control Mechanisms:

  • S. pombe is a premier model for cell cycle studies

  • If SPBC11C11.05 affects cell cycle-regulated genes, this would connect to central questions in cell division control

  • The identification of 747 cell cycle-regulated genes provides a framework for understanding potential SPBC11C11.05 targets

• Stress Response and Adaptation:

  • Stress response in S. pombe involves the Sty1 MAP kinase pathway and transcription factors like Atf1

  • SPBC11C11.05 might function in stress adaptation mechanisms

  • This connects to broader questions about cellular resilience and environmental response

• Growth Transition Biology:

  • The transition between stationary phase and active growth involves dramatic transcriptional reprogramming

  • Pol II and its associated activities are required for efficient recruitment of Pol III upon exit from stationary phase

  • SPBC11C11.05 might participate in this growth transition process

By connecting specific findings about SPBC11C11.05 to these broader biological questions, researchers can position their work within the larger context of S. pombe biology and eukaryotic cell function.