Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YFR056C (YFR056C)

What is YFR056C and where is it located in the S. cerevisiae genome?

YFR056C is a putative uncharacterized protein in Saccharomyces cerevisiae (baker's yeast). It is located on chromosome VI near the right telomeric region, approximately 5-7.5 kb from the telomere . The gene is part of a subtelomeric region that can be subject to silencing mechanisms, which has implications for its expression patterns and regulation. As with other yeast genes, the systematic name (YFR056C) indicates its chromosomal location, with "Y" designating yeast, "FR" indicating chromosome VI right arm, "056" denoting the relative position on the chromosome, and "C" signifying that it is transcribed from the complementary DNA strand.

How is YFR056C expression regulated in wild-type yeast cells?

YFR056C expression appears to be influenced by its subtelomeric location and chromatin structure. Studies have shown that YFR056C can be down-regulated over 2.5-fold in certain conditions . The gene's expression is partially regulated by Sir proteins, particularly Sir3, which are involved in heterochromatin formation and gene silencing. Chromatin immunoprecipitation (ChIP) analyses of RNA polymerase II (RNAPII) occupancy at YFR056C have indicated that repression of its transcription is only partially SIR3-dependent . This suggests multiple layers of regulation, including both Sir-dependent heterochromatin formation and potentially other transcriptional regulatory mechanisms.

What experimental approaches are recommended for initial characterization of YFR056C?

For initial characterization of an uncharacterized protein like YFR056C, a multi-faceted approach is recommended:

• Gene expression analysis: qRT-PCR to quantify expression under various growth conditions and genetic backgrounds.

• Protein localization: Construction of GFP-fusion proteins to determine subcellular localization, similar to approaches used for other yeast proteins like Yll056cp .

• Phenotypic analysis of deletion mutants: Creating yFR056C deletion strains and analyzing their growth under various stress conditions.

• Protein purification and biochemical characterization: Expression of recombinant YFR056C with affinity tags for purification and subsequent biochemical assays.

• Protein-protein interaction studies: Yeast two-hybrid screening or affinity purification coupled with mass spectrometry.

When designing these experiments, it's crucial to consider the influence of the gene's subtelomeric location on its expression when interpreting results, as telomere-proximal genes can exhibit position effects.

How can I effectively express recombinant YFR056C for functional studies?

For optimal expression of recombinant YFR056C, consider the following methodological approach:

• Expression system selection: While E. coli is commonly used, expressing in S. cerevisiae itself can be advantageous for proper folding and post-translational modifications. Use a strain with the endogenous YFR056C deleted to avoid interference.

• Vector design considerations:

  • Include affinity tags (His6, GST, or FLAG) for purification

  • Consider inducible promoters like GAL1 to control expression timing

  • Include appropriate secretion signals if extracellular studies are needed

• Optimization protocol:

  Parameter	Recommended Approach	Considerations
  Temperature	Test 25°C, 30°C, and 37°C	Lower temperatures may increase proper folding
  Induction time	4-24 hours depending on stability	Monitor by western blot
  Media composition	YPD vs. minimal media with supplements	Nutrient availability affects expression levels
  Cell lysis	Glass bead disruption or enzymatic methods	Preserve protein structure and activity

• Purification strategy: Implement a two-step purification process using affinity chromatography followed by size-exclusion chromatography to obtain high purity protein.

• Activity verification: Develop functional assays based on predicted protein functions from bioinformatic analyses and homology modeling.

Similar approaches have been successful for characterizing other previously uncharacterized yeast proteins such as the atypical short-chain dehydrogenase/reductase YLL056C .

What strategies can I use to investigate the function of YFR056C in relation to telomeric silencing?

To investigate YFR056C's potential role in telomeric silencing and chromosome boundary formation:

• Genetic interaction analysis:

  • Create double mutants with known silencing factors (sir2Δ, sir3Δ, sir4Δ, rpd3Δ, sas2Δ)

  • Analyze synthetic genetic interactions similar to those observed between SAS-I complex and RPD3

  • Monitor for genetic interactions that show synthetic lethality or rescue phenotypes

• Chromatin state analysis:

  • Perform ChIP-seq for histone modifications (H3K9ac, H4K16ac) around the YFR056C locus

  • Map heterochromatin boundary elements using Sir protein spreading assays

  • Implement reporter gene silencing assays with constructs inserted at varied distances from telomeres

• Telomeric boundary element testing:

  • Use boundary element assays similar to those designed for testing Rpd3 function

  • Insert URA3 reporter genes at varying distances from telomeres in wild-type and YFR056C mutant strains

  • Assess repression using 5-FOA resistance and growth on uracil-deficient media

• Transcriptome analysis:

  • Compare RNA-seq data from wild-type and YFR056C mutant strains

  • Focus analysis on subtelomeric genes to identify pattern changes

  • Quantify expression changes in genes like YFR055W that are neighboring YFR056C

This approach will help determine whether YFR056C plays a structural role in chromatin organization or has a more direct function in telomeric silencing mechanisms.

How can I differentiate between direct and indirect effects when studying YFR056C's influence on neighboring genes?

Differentiating between direct and indirect effects of YFR056C on neighboring gene expression requires controlled experimental approaches:

• Temporal analysis of gene expression changes:

  • Implement an inducible/repressible YFR056C system using promoters like tetO or GAL1

  • Collect time-course data after YFR056C induction/repression

  • Primary (direct) effects typically occur rapidly, while secondary effects emerge later

• Protein-DNA interaction mapping:

  • Perform ChIP-seq with tagged YFR056C to identify direct binding sites

  • Compare binding profiles with expression changes in neighboring genes

  • Implement DNA footprinting assays to confirm specific DNA interactions

• Structure-function analysis:

  • Create point mutations in predicted functional domains of YFR056C

  • Assess the impact on both direct binding targets and downstream genes

  • Design domain swap experiments with similar proteins to identify functional regions

• Reconstitution experiments:

  • For suspected enzymatic activities, perform in vitro reconstitution with purified components

  • Similar to approaches used for characterizing the aldehyde reductase activity of Yll056cp

  • Test substrate specificity and kinetic parameters

• Controlled reference experiment design:

  Experiment Type	Control Design	Data Interpretation
  Gene deletion	Include non-affected gene deletions	Compare specificity of effects
  ChIP experiments	Include non-specific antibody controls	Establish binding specificity
  Expression analysis	Analyze non-neighboring genes	Distinguish positional from functional effects
  Genetic interactions	Test interactions with unrelated pathways	Identify pathway-specific effects

This systematic approach helps establish causality in complex genetic networks and distinguishes YFR056C's direct functions from secondary effects due to its genomic location.

How should I interpret contradictory results between genetic and biochemical analyses of YFR056C?

When faced with contradictory results between genetic and biochemical approaches studying YFR056C:

Remember that seemingly contradictory results often lead to new biological insights about complex regulatory mechanisms.

What bioinformatic approaches can help predict the function of YFR056C?

To predict the function of the uncharacterized protein YFR056C using bioinformatics:

• Sequence-based analysis:

  • Perform sensitive homology searches using PSI-BLAST, HHpred, and HMMER against diverse databases

  • Identify conserved domains using InterPro, Pfam, and CDD

  • Analyze protein disorder regions with DISOPRED and MobiDB

• Structural prediction and analysis:

  • Generate 3D structure predictions using AlphaFold2 or RoseTTAFold

  • Identify potential binding pockets and catalytic sites

  • Perform molecular docking studies with predicted ligands

  • Compare predicted structures with known proteins to identify functional homologs

• Genomic context analysis:

  Analysis Type	Tools/Resources	Interpretation Focus
  Synteny analysis	SyntTax, Genomicus	Conservation of gene neighborhood
  Co-expression networks	SPELL, YeastNet	Functional associations
  Genetic interaction maps	TheCellMap.org	Pathway membership
  Phylogenetic profiling	PhyloPro	Co-evolution patterns

• Integration with experimental data:

  • Correlate expression patterns with published datasets using tools like Expression Atlas

  • Match subcellular localization predictions with the Yeast GFP Fusion Localization Database

  • Compare with data from systematic functional genomics projects like the Saccharomyces Genome Deletion Project

• Machine learning approaches:

  • Apply function prediction algorithms like FFPred, SIFTER, and DeepGOPlus

  • Use protein language models like ESM and ProtT5 to identify functional features

  • Validate predictions with available experimental data

When interpreting predictions for YFR056C, consider its subtelomeric location, as proteins in these regions often have roles in stress response, nutrient metabolism, or adaptation to environmental conditions .

How can I design a CRISPR-Cas9 system to study YFR056C function in S. cerevisiae?

For effective CRISPR-Cas9 manipulation of YFR056C in S. cerevisiae:

• System design considerations:

  • Select appropriate Cas9 expression vectors (constitutive vs. inducible)

  • Choose RNA polymerase III promoters (SNR52, RPR1) for guide RNA expression

  • Design specific sgRNAs with minimal off-target effects

• sgRNA design for YFR056C:

  Application	sgRNA Target Region	Considerations
  Gene knockout	Early coding sequence	PAM sites with highest specificity
  N-terminal tagging	Near start codon	Preserve protein function
  C-terminal tagging	Near stop codon	Avoid disrupting regulatory elements
  Point mutations	Specific codons	Design appropriate repair templates
  Promoter modulation	Upstream regulatory region	Map regulatory elements first

• Repair template design:

  • Include 40-60 bp homology arms flanking the cut site

  • For tagging, ensure in-frame fusion with appropriate linkers

  • Consider using selectable markers (URA3, KanMX) with loxP sites for marker recycling

  • Implement silent mutations in the PAM site or sgRNA target region to prevent re-cutting

• Validation strategies:

  • PCR screening and sequencing to confirm edits

  • RT-qPCR to assess expression changes

  • Western blotting for tagged protein variants

  • Phenotypic assays based on predicted function

• Technical optimization for telomeric regions:

  • Telomeric regions can be challenging for CRISPR editing due to heterochromatin

  • Consider temporarily disrupting silencing factors (e.g., sir2Δ backgrounds)

  • Test multiple sgRNAs targeting different sites within YFR056C

  • Optimize transformation methods specific for subtelomeric targets

When implementing CRISPR-Cas9 for YFR056C, special attention should be paid to its genomic context since its location near telomeric regions may affect editing efficiency .

What are the most effective approaches for studying protein-protein interactions involving YFR056C?

To comprehensively characterize protein-protein interactions involving YFR056C:

• Complementary methodological framework:

  Method	Advantages	Limitations	Best Application
  Yeast two-hybrid	High-throughput screening	False positives/negatives	Initial interaction discovery
  Affinity purification-MS	Captures complexes in native context	May miss transient interactions	Core complex identification
  BioID/TurboID	Detects proximity in living cells	Identifying nearby proteins	Spatial interactome mapping
  FRET/BRET	Real-time interaction dynamics	Requires fluorescent tags	Interaction kinetics studies
  Co-immunoprecipitation	Validates specific interactions	Antibody limitations	Confirmation of direct interactions
  Split-protein complementation	In vivo validation	May stabilize weak interactions	Visualizing interactions in cells

• Specialized approaches for telomeric proteins:

  • ChIP-reChIP to detect co-occupancy at chromatin regions

  • Sequential IPs to isolate specific subcomplexes

  • Proximity-dependent labeling specifically at telomeres

  • Consider Sir-protein interactions due to YFR056C's telomeric location

• Protein domain mapping:

  • Create truncated variants to identify interaction domains

  • Use peptide arrays to pinpoint specific binding motifs

  • Perform mutagenesis of predicted interaction surfaces

  • Test effects of post-translational modifications on interactions

• Quantitative interaction analysis:

  • Implement surface plasmon resonance or isothermal titration calorimetry

  • Determine binding kinetics and affinities

  • Assess competition between different interaction partners

  • Evaluate environmental influences (pH, salt, temperature) on binding

• Functional validation:

  • Confirm biological relevance of interactions through genetic approaches

  • Test if deleting interaction partners affects YFR056C function

  • Create separation-of-function mutations that specifically disrupt individual interactions

  • Analyze epistatic relationships between YFR056C and interactors

Remember to integrate interaction data with existing information about YFR056C's subtelomeric location and potential involvement in telomeric silencing .

How can YFR056C research contribute to our understanding of telomeric regulation in eukaryotes?

YFR056C research can significantly advance our understanding of telomeric regulation in eukaryotes through:

• Boundary element mechanisms:

  • YFR056C's location places it at potential chromatin boundary regions

  • Studies could reveal novel mechanisms for separating heterochromatin from euchromatin

  • Comparison with known boundary elements like tRNA genes and Rpd3-dependent boundaries may reveal evolutionary conservation

• Comparative genomics approach:

  Organism	Telomeric Structure	Research Value	Connection to YFR056C
  S. cerevisiae	Relatively simple telomeres	Model for basic mechanisms	Direct homology
  S. pombe	Complex heterochromatin	Alternative silencing mechanisms	Functional analogs
  Mammalian cells	Long telomeres with shelterin	Medical relevance	Conserved principles
  Other fungi	Diverse telomeric arrangements	Evolutionary insights	Degree of conservation

• Integration with telomere maintenance pathways:

  • Investigate potential roles in telomerase regulation

  • Study interactions with the Sir protein complex

  • Examine relationships with histone modifying enzymes like Rpd3 and Sas2

  • Assess impact on telomere position effect and gene silencing

• Heterochromatin spreading mechanisms:

  • YFR056C could function in regulating heterochromatin spreading

  • Research could reveal dynamic aspects of boundary formation

  • May provide insights into Sir3 protein spreading observed in various chromatin contexts

  • Could connect to broader epigenetic regulation mechanisms

• Translational research potential:

  • Understanding telomeric regulation has implications for aging and cancer

  • YFR056C research may reveal conserved mechanisms applicable to human disease

  • Could identify new therapeutic targets for telomere-related disorders

  • May connect to previously unrecognized aspects of genome stability

This research direction has particular value given the observation that YFR056C expression is affected by Sir-dependent mechanisms and may be involved in chromatin boundary dynamics at telomeres .

What are emerging technologies that could accelerate functional characterization of YFR056C?

Emerging technologies that could revolutionize YFR056C characterization include:

• Advanced genome editing approaches:

  • Prime editing for precise modifications without double-strand breaks

  • Base editing for specific nucleotide changes

  • CRISPR activation/interference (CRISPRa/CRISPRi) for modulating expression without DNA modification

  • Multiplexed editing to simultaneously modify YFR056C and potential interactors

• Single-cell technologies:

  • Single-cell RNA-seq to capture heterogeneity in YFR056C expression

  • Single-cell proteomics to detect cell-to-cell variation in protein levels

  • Single-cell epigenomics to map chromatin states at the YFR056C locus

  • Live-cell imaging with advanced microscopy techniques

• Spatial biology methods:

  Technology	Application	Advantage for YFR056C Research
  Super-resolution microscopy	Precise localization	Visualize telomeric positioning
  Spatial transcriptomics	Gene expression in context	Map expression relative to nuclear landmarks
  Proximity labeling	In situ interaction mapping	Identify neighbors in telomeric regions
  4D nucleome mapping	Chromatin organization over time	Track dynamics of telomeric regions

• Protein structure and function technologies:

  • AlphaFold2 and RoseTTAFold for accurate structure prediction

  • Cryo-EM for complex structural determination

  • High-throughput protein engineering with deep mutational scanning

  • Cell-free protein synthesis for rapid functional testing

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Network analysis to position YFR056C in cellular pathways

  • Mathematical modeling of telomeric silencing incorporating YFR056C

  • Machine learning for predicting phenotypic outcomes of YFR056C perturbations

These technologies can help overcome challenges associated with studying proteins in telomeric regions, where traditional approaches may be limited due to heterochromatin formation and position effects .