SPAC869.09 Antibody

What is SPAC869.09 and why is it significant in fission yeast research?

SPAC869.09 is a gene in Schizosaccharomyces pombe that becomes activated during cellular quiescence. According to epigenome mapping studies, SPAC869.09 is among the genes that are moderately induced during quiescence and requires the histone methyltransferase Set1 for this induction . The protein is particularly interesting because it undergoes significant chromatin state changes between vegetative and quiescent cells, showing increased H3K4me3 levels and RNA polymerase II occupancy in its transcription start site (TSS) during quiescence .

SPAC869.09 is located in a chromosomal region that contains several other genes activated during quiescence, including mel1, mel2, and other genes involved in cellular adaptation to nutrient limitation . Understanding the regulation and function of SPAC869.09 can provide insights into cellular adaptation mechanisms during stress conditions and nutrient deprivation.

How can I validate a SPAC869.09 antibody for fission yeast research?

Validating a SPAC869.09 antibody requires a systematic approach:

• Expression verification: Generate a tagged version of SPAC869.09 (e.g., with GFP or FLAG tag) as a positive control to confirm antibody reactivity.

• Knockout controls: The gold standard for antibody validation is testing in knockout strains. YCharOS studies showed that knockout cell lines are superior to other types of controls for both Western blots and immunofluorescence imaging .

• Epitope mapping: Determine the specific epitope recognized by the antibody, which is particularly important for understanding potential cross-reactivity.

• Multiple technique validation: Test the antibody in multiple applications:

  • Western blot: To verify correct molecular weight

  • Immunoprecipitation: To confirm native protein recognition

  • Immunofluorescence: To verify subcellular localization

  • ChIP: If studying chromatin association

• Specificity testing: Check for cross-reactivity with closely related proteins, especially those in the same chromosomal region (e.g., SPAC869.07c, SPAC869.08) .

A recent study showed that approximately 12 publications per protein target included data from antibodies that failed to recognize their intended targets , emphasizing the critical importance of proper validation.

What techniques are most effective for detecting SPAC869.09 expression in fission yeast?

Given the characteristics of SPAC869.09 as revealed in epigenome mapping studies, several complementary techniques are recommended:

• ChIP-seq: Particularly effective since SPAC869.09 shows significant changes in H3K4me3 levels and RNA polymerase II occupancy. Combine antibodies against SPAC869.09 with those targeting histone modifications (especially H3K4me3) to correlate protein presence with chromatin state .

• RNA-seq with spike-in normalization: This allows accurate quantification of expression changes between vegetative and quiescent cells .

• Western blotting: Use this as a basic technique to detect protein levels, but be aware that whole-cell extracts may not reflect the chromatin-bound fraction.

• Integrated Genomics Viewer (IGV) visualization: This approach was used effectively in research to visualize changes in histone marks (H3K4me3, H3K9ac) and RNA Pol II occupancy at SPAC869.09 between vegetative and quiescent cells .

• Immunofluorescence microscopy: Especially useful when combined with cell cycle markers to distinguish between vegetative and quiescent states.

Each technique has specific requirements for antibody characteristics, so validation should be performed for each intended application.

What controls are essential when using a SPAC869.09 antibody in quiescence studies?

When studying SPAC869.09 in the context of quiescence, several essential controls are needed:

• Temporal controls:

  • Vegetative cells (V cells) as baseline control

  • Early quiescence (e.g., 24h after nitrogen starvation)

  • Established quiescence (e.g., 1-2 weeks in quiescence)

• Genetic controls:

  • Wild-type strain

  • set1Δ mutant (since SPAC869.09 induction requires Set1)

  • Strains with mutations in other quiescence-related pathways (e.g., cAMP-PKA pathway)

• Specificity controls:

  • SPAC869.09 deletion strain

  • Antibody preincubation with recombinant SPAC869.09 protein

• Positive controls:

  • Other quiescence-induced genes (e.g., mel1, SPAC869.07c)

  • Core quiescence genes with established expression patterns

• Epigenetic controls:

  • ChIP for H3K4me3 levels at the SPAC869.09 locus

  • RNA polymerase II occupancy measurements

These controls allow for comprehensive validation of antibody specificity and experimental relevance, particularly in distinguishing true quiescence-related signals from artifacts.

How does SPAC869.09 expression relate to chromatin modifications during quiescence?

SPAC869.09 exhibits a striking relationship with chromatin modifications during quiescence:

• H3K4me3 enrichment: SPAC869.09 shows significantly increased H3K4me3 levels at its transcription start site (TSS) in quiescent cells compared to vegetative cells. This modification is strongly associated with active transcription .

• RNA polymerase II occupancy: Increased RNA Pol II (both S2 and S5 phosphorylated forms) occupancy is observed at the SPAC869.09 locus during quiescence, indicating active transcription .

• Set1 dependency: The induction of SPAC869.09 in quiescent cells requires the histone methyltransferase Set1, which is responsible for H3K4 methylation .

• Chromatin state changes: SPAC869.09 transitions to an "Active" chromatin state during quiescence, as defined by the combination of increased H3K4me3 and RNA Pol II occupancy .

• Relationship to other histone marks: Unlike H3K4me3, other histone modifications such as H3K9me2, H3K9me3, and H2A.Z show relatively minor changes at the SPAC869.09 locus during quiescence .

This pattern suggests that SPAC869.09 is part of a specific gene expression program activated during quiescence, with H3K4me3 playing a crucial regulatory role.

How can computational approaches aid in designing a specific antibody against SPAC869.09?

Developing a specific antibody against SPAC869.09 can benefit from several computational approaches:

• Epitope prediction and selection:

  • Use algorithms to identify antigenic regions unique to SPAC869.09

  • Select epitopes that are accessible in the protein's native conformation

  • Avoid regions with high sequence similarity to other fission yeast proteins

• Structure-based design using RosettaAntibodyDesign (RAbD):

  • RAbD samples diverse antibody sequences and structures to optimize binding specificity

  • The framework allows customization of CDRs (Complementarity-Determining Regions) to enhance target recognition

  • Incorporates canonical cluster analysis for CDR structure prediction

• IsAb antibody design protocol:

  • Implements a step-by-step computational workflow including RosettaAntibody for structure prediction, RosettaRelax for energy minimization, and two-step docking

  • Utilizes alanine scanning to identify hotspots for binding optimization

  • Applies computational affinity maturation to enhance binding properties

• Deep learning approaches:

  • Train models using existing antibody-antigen complex data to predict binding properties

  • Utilize databases like PLAbDab (Patent and Literature Antibody Database) that contain ~150,000 antibody sequences with functional annotations

  • Implement structure-based deep learning to distinguish antibody specificity for different targets

• Epitope binning and cross-reactivity analysis:

  • Use structural information to predict potential cross-reactivity with similar proteins

  • Incorporate both heavy and light chain information to improve prediction accuracy (as light chain contribution significantly improves target specificity)

When designing antibodies computationally, it's crucial to validate predictions experimentally, as a recent study showed that different search methods yield varying results for different antibodies .

What methodologies can help characterize the role of SPAC869.09 in the fission yeast quiescence program?

To comprehensively characterize SPAC869.09's role in quiescence:

• Time-course expression analysis:

  • Monitor SPAC869.09 expression at multiple timepoints during entry into and exit from quiescence

  • Compare its expression pattern with known early (24h) and core (1-2 weeks) quiescence genes

  • Use spike-in normalized RNA-seq for accurate quantification across conditions

• Genetic perturbation studies:

  • Generate SPAC869.09 deletion and overexpression strains

  • Assess impact on:

    • Quiescence entry and maintenance

    • Cell viability during prolonged quiescence

    • Revival capacity upon nutrient addition

  • Compare phenotypes with other quiescence-related genes in the same chromosomal region (e.g., mel1, mel2)

• Epigenetic regulation studies:

  • Perform ChIP-seq for multiple histone modifications in wild-type and set1Δ backgrounds

  • Map the regulatory network controlling SPAC869.09 expression

  • Use IGV visualization to compare epigenetic landscapes across conditions

• Functional genomics approaches:

  • Conduct synthetic genetic array analysis with SPAC869.09 deletion

  • Perform proteomics to identify interaction partners

  • Use CRISPR interference to modulate expression in specific phases

• Metabolic impact assessment:

  • Given that many quiescence-induced genes encode metabolic enzymes , investigate potential metabolic functions

  • Perform metabolomic profiling in wild-type vs. SPAC869.09 deletion strains during quiescence

• Cytoplasmic properties analysis:

  • Investigate relationship between SPAC869.09 and cytoplasmic fluidization during dormancy breaking

  • Use tracer particles to measure changes in cytoplasmic properties in relation to SPAC869.09 expression

This comprehensive approach will provide insights into both the regulation and function of SPAC869.09 in the quiescence program.

How can epigenetic techniques be combined with antibody-based approaches to study SPAC869.09 regulation?

Integrating epigenetic techniques with antibody-based approaches provides powerful insights into SPAC869.09 regulation:

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with H3K4me3 antibody, then with SPAC869.09 antibody

  • Determines if SPAC869.09 protein associates specifically with H3K4me3-marked regions

  • Helps establish causal relationships between histone modification and protein binding

• CUT&RUN or CUT&Tag with bioinformatic integration:

  • More sensitive alternatives to traditional ChIP that require less material

  • Combine data from SPAC869.09 protein localization with histone modification maps

  • Analyze using differential peak calling between vegetative and quiescent cells

• Proximity ligation assay (PLA):

  • Detects physical proximity between SPAC869.09 and specific chromatin marks

  • Useful for confirming interactions suggested by ChIP studies

  • Provides spatial resolution within individual cells

• ChIP-seq with spike-in controls:

  • Use exogenous DNA spike-in to normalize between vegetative and quiescent cells

  • Critical for accurate quantitative comparisons across conditions with different chromatin compaction states

• Mass spectrometry after chromatin immunoprecipitation:

  • Identify proteins that co-precipitate with SPAC869.09

  • Characterize post-translational modifications on SPAC869.09 itself

  • Map the protein interaction network at SPAC869.09-bound chromatin regions

• Chromatin accessibility assays (ATAC-seq) combined with ChIP:

  • Correlate SPAC869.09 binding with changes in chromatin accessibility

  • Determine if SPAC869.09 acts upstream or downstream of chromatin opening

  • Compare with RNA Pol II occupancy data to establish transcriptional consequences

These integrated approaches can reveal how SPAC869.09 contributes to the dramatic epigenome changes observed during quiescence in fission yeast.

What are the challenges in developing antibodies against fission yeast proteins like SPAC869.09?

Developing antibodies against fission yeast proteins presents several unique challenges:

• Limited immunogenicity and epitope accessibility:

  • Yeast proteins may share high homology with host proteins used for immunization

  • The native conformation may mask important epitopes

  • Post-translational modifications in yeast may differ from those in expression systems

• Validation challenges:

  • Approximately 50% of commercial antibodies fail to meet basic characterization standards

  • Proper validation requires knockout controls, which can be labor-intensive to generate in fission yeast

  • Cross-reactivity with related proteins must be carefully assessed

• Technical limitations:

  • Many antibody development platforms are optimized for mammalian proteins

  • Yeast cell walls present barriers for in situ applications

  • Fixation methods for yeast can affect epitope preservation differently than in mammalian cells

• Expression and purification for immunization:

  • Obtaining sufficient quantities of correctly folded SPAC869.09 for immunization

  • Potential toxicity when expressing yeast proteins in bacterial systems

  • Choosing between full-length proteins vs. peptide epitopes for immunization

• Application-specific optimization:

  • Different experimental techniques require distinct antibody properties

  • ChIP-grade antibodies need different characteristics than those for Western blotting

  • Quantitative techniques require antibodies with consistent binding properties

• Reproducibility considerations:

  • Batch-to-batch variation, especially in polyclonal antibodies

  • Recombinant antibodies offer better reproducibility but may be more difficult to develop initially

  • Documentation of validation is often inadequate, leading to wasted research efforts

These challenges highlight the importance of thorough validation and characterization of any antibody against SPAC869.09 or other fission yeast proteins.

How does the H3K4me3 modification influence SPAC869.09 expression in quiescent cells?

The relationship between H3K4me3 modification and SPAC869.09 expression reveals important regulatory mechanisms:

• Correlation between H3K4me3 and activation:

  • SPAC869.09 shows significantly increased H3K4me3 levels at its TSS during quiescence

  • This correlates with increased RNA polymerase II occupancy and gene expression

  • Based on volcano plot analysis, SPAC869.09 is among the genes with super-significant increases in H3K4me3 levels in quiescent cells

• Set1 dependency mechanism:

  • The induction of SPAC869.09 in quiescence requires Set1, the catalytic subunit of Set1C/COMPASS complex responsible for H3K4 methylation

  • Set1 deletion likely prevents the establishment of H3K4me3 marks necessary for SPAC869.09 activation

• Temporal relationships during quiescence entry:

  • H3K4me3 deposition appears to precede full transcriptional activation

  • This suggests a model where epigenetic marking creates a permissive environment for transcription factor binding and RNA Pol II recruitment

• Specificity of H3K4me3 influence:

  • While many histone modifications show minimal changes during quiescence, H3K4me3 exhibits dramatic redistribution

  • This suggests that H3K4me3 plays a specialized role in quiescence-specific gene regulation

  • At SPAC869.09, H3K4me3 changes are much more significant than H3K9ac, H3K9me2/3, or H2A.Z occupancy changes

• Quantitative relationship with gene expression:

  • Bubble plots and pie diagrams show that 81.3% of core quiescence genes have increased H3K4me3 at TSS regions

  • This establishes H3K4me3 as a key predictor of sustained quiescence-specific expression

These findings position H3K4me3 as a critical regulatory mark for SPAC869.09 activation during quiescence, highlighting the importance of studying this particular modification when investigating SPAC869.09 regulation.

What bioinformatic approaches can improve epitope selection for SPAC869.09 antibody development?

Sophisticated bioinformatic strategies can significantly enhance epitope selection for SPAC869.09 antibody development:

• Computational epitope prediction workflow:

  • Combine multiple algorithm outputs (Bepipred, Emini Surface Accessibility, Kolaskar-Tongaonkar Antigenicity) for consensus prediction

  • Weight predictions based on algorithm performance for yeast proteins

  • Prioritize epitopes unique to SPAC869.09 through proteome-wide BLAST analysis

• Structural prediction and epitope accessibility:

  • Use AlphaFold2 or RosettaFold to predict SPAC869.09 structure

  • Calculate surface exposure scores for potential epitopes

  • Model antibody-epitope interactions using RosettaAntibodyDesign

• Comparative genomics approach:

  • Analyze sequence conservation across different yeast species

  • Identify regions unique to S. pombe to minimize cross-reactivity

  • Consider epitopes in regions with species-specific functions

• Integration with experimental data:

  • Utilize protein expression data from proteomic studies

  • Consider post-translational modifications identified in mass spectrometry datasets

  • Factor in known protein-protein interaction sites that might affect epitope accessibility

• Machine learning for epitope ranking:

  • Train models on successfully developed yeast protein antibodies

  • Incorporate features like hydrophilicity, flexibility, and secondary structure

  • Use deep learning approaches similar to those demonstrated for SARS-CoV-2 antibodies

• Database mining for similar targets:

  • Leverage PLAbDab and other antibody databases to identify successfully targeted proteins with similar properties

  • Examine epitopes used in successful antibodies against other quiescence-induced proteins

  • Analyze patterns in effective antibody-antigen interactions for yeast proteins

This systematic bioinformatic approach increases the likelihood of selecting epitopes that will yield specific, high-affinity antibodies against SPAC869.09.

How can antibodies against SPAC869.09 help unravel the transcriptional networks activated during quiescence?

Antibodies against SPAC869.09 can serve as powerful tools for dissecting quiescence-specific transcriptional networks:

• Genome-wide binding profile analysis:

  • ChIP-seq with SPAC869.09 antibodies to identify direct binding sites

  • Compare binding profiles between vegetative and quiescent cells

  • Integrate with RNA-seq data to determine functional consequences of binding

• Protein complex identification:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Identify SPAC869.09 interaction partners in different cellular states

  • Map dynamic changes in protein complexes during quiescence entry and exit

• Transcription factor network mapping:

  • Co-ChIP experiments to identify co-binding relationships with other transcription factors

  • Determine if SPAC869.09 functions in coordination with other quiescence-activated factors

  • Establish hierarchical relationships in transcriptional cascades

• Chromatin state correlation:

  • Multi-omics integration of SPAC869.09 binding with:

    • Histone modification profiles (especially H3K4me3)

    • Chromatin accessibility data

    • RNA polymerase II occupancy

  • Determine whether SPAC869.09 precedes or follows chromatin state changes

• Functional genomics integration:

  • Compare binding profiles in wild-type versus mutant backgrounds (e.g., set1Δ)

  • Assess changes in SPAC869.09 binding upon perturbation of quiescence-related pathways

  • Identify genetic dependencies for SPAC869.09 function

• Single-cell applications:

  • Use antibodies for single-cell protein quantification

  • Correlate with single-cell transcriptomics to identify cell-to-cell variability

  • Map heterogeneity in quiescence program activation across a population

This comprehensive approach using SPAC869.09 antibodies can reveal not only the direct targets and functions of SPAC869.09 but also its position within the broader transcriptional networks governing quiescence in fission yeast.