C7ORF49 Human

What is C7ORF49/CYREN and what is its primary function?

C7ORF49, now officially named CYREN, functions as a cell cycle regulator of non-homologous end joining (NHEJ) in humans . This protein plays a critical role in DNA repair pathway choice during the cell cycle. Methodologically, its function was initially characterized through cell cycle-specific DNA repair assays and protein interaction studies.

The protein's full name is "Cell cycle regulator of non-homologous end joining," encoded by the CYREN gene (previously C7ORF49) . As a modulator of retrovirus infection encoded in the human genome, it represents an important component of cellular defense mechanisms .

What are the known post-translational modifications of CYREN?

CYREN undergoes several post-translational modifications that likely regulate its function and interactions. Based on curated databases, these modifications include:

Researchers investigating this protein should consider these modifications when designing experiments, particularly when using antibody-based detection methods or when studying protein-protein interactions that might be affected by these modifications .

How evolutionarily conserved is the CYREN protein?

While the search results don't provide specific information about CYREN's evolutionary conservation, methodological approaches to study this would include comparative genomic analyses across species and humanized yeast models. Researchers interested in evolutionary aspects could employ techniques described for studying human gene function in yeast models.

The humanization approach involves cloning orthologous human cDNA into yeast expression vectors and transforming them into yeast strains with conditional knockout alleles of corresponding yeast genes . Alternatively, CRISPR/Cas9 can be used for precise insertion of human coding sequences at corresponding genomic loci in yeast, replacing yeast genes while maintaining native regulation . These methods provide a growth-based readout of functional conservation.

What experimental approaches are most effective for studying CYREN's role in DNA repair pathway choice?

For researchers investigating CYREN's function in DNA repair pathway regulation, multiple complementary approaches are recommended:

• Conditional knockout systems: Generate cell lines with inducible CYREN depletion to observe acute effects on DNA repair pathway choice at different cell cycle stages.

• Live-cell imaging: Utilize fluorescently tagged CYREN and other DNA repair factors to monitor their recruitment to DNA damage sites in real-time.

• Humanized yeast models: As described in the literature, yeast models offer rapid assessment of CYREN function through complementation assays . The approach involves:

  • Cloning the human CYREN cDNA into yeast expression vectors

  • Transforming these constructs into yeast strains with conditional knockout alleles of related DNA repair genes

  • Analyzing functional complementation through growth-based readouts

• Protein-protein interaction studies: Identify CYREN's binding partners across the cell cycle using techniques such as BioID, immunoprecipitation coupled with mass spectrometry, or yeast two-hybrid screens.

These methodological approaches, particularly when used in combination, provide comprehensive insights into CYREN's functional roles in coordinating DNA repair.

How do CYREN variants affect cancer susceptibility and progression?

The iPTMnet database indicates that variants affecting the S123 phosphorylation site of CYREN have been identified in uterine and colorectal cancers :

For researchers investigating these associations, the following methodological approaches are recommended:

• Case-control genetic studies: Compare the frequency of these variants in cancer patients versus healthy controls.

• Functional characterization: Generate cell lines expressing these variants using CRISPR/Cas9 gene editing and assess:

  • DNA repair efficiency

  • Cell cycle progression

  • Genomic stability

  • Response to DNA-damaging therapies

• Phospho-specific antibody studies: Develop antibodies that specifically recognize phosphorylated S123 to determine how this modification affects CYREN function and whether the cancer-associated variants disrupt normal phosphorylation patterns.

• Clinical correlation studies: Evaluate whether these variants correlate with treatment response or patient outcomes in uterine or colorectal cancer cohorts.

What is known about the interplay between CYREN's post-translational modifications?

The iPTMnet data reveals that CYREN undergoes multiple types of post-translational modifications, including acetylation, O-glycosylation, sumoylation, and phosphorylation at various sites . To methodically investigate potential crosstalk between these modifications, researchers could:

• Develop modification-specific antibodies: Generate antibodies that recognize specific modified forms of CYREN to track their abundance under different cellular conditions.

• Perform mass spectrometry-based proteomic analysis: Use techniques such as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) to quantitatively analyze the combinatorial patterns of modifications.

• Generate modification site mutants: Create point mutations at modification sites (e.g., S123A to prevent phosphorylation) and observe the effects on other modifications.

• Analyze modification dynamics during the cell cycle: Synchronize cells at different cell cycle stages and assess how the pattern of modifications changes, correlating these changes with CYREN's activity in regulating NHEJ.

These approaches would provide insights into how different modifications might regulate CYREN's function in a coordinated manner.

How can humanized yeast models be utilized to study CYREN function?

Humanized yeast models offer a powerful system for studying human gene function, including CYREN. The methodological approach involves:

• Construct preparation: Clone the human CYREN cDNA into yeast expression vectors compatible with high-throughput cloning .

• Yeast transformation: Transform these constructs into yeast strains containing conditional knockout alleles of genes involved in related DNA repair processes .

• Functional complementation assessment: If the yeast gene is essential, viable yeast cells expressing the human gene alone would suggest functional replaceability (Figure 1A in the referenced paper) .

• CRISPR/Cas9 gene replacement: For more precise analysis, use CRISPR/Cas9 for markerless insertion of the human CYREN coding sequence at the corresponding genomic locus, replacing the yeast gene while maintaining native regulation .

• Growth-based readout: Analyze yeast growth patterns to assess functional complementation under different conditions, such as in the presence of DNA-damaging agents.

This approach leverages yeast's rapid growth, easy conversion between haploid and diploid forms, and availability of genome-wide mutant libraries to efficiently test CYREN function .

What techniques are most effective for studying CYREN's cell cycle-dependent activity?

To methodically investigate CYREN's cell cycle-dependent regulation of non-homologous end joining, researchers should consider:

• Cell synchronization methods:

  • Double thymidine block for G1/S synchronization

  • Nocodazole treatment for G2/M synchronization

  • Serum starvation for G0/G1 enrichment

• Live-cell imaging with cell cycle markers:

  • Co-express fluorescently tagged CYREN with established cell cycle phase markers

  • Use time-lapse microscopy to track localization and activity through the cell cycle

• Cell cycle-specific DNA damage induction:

  • Use micro-irradiation at specific cell cycle phases

  • Apply selective DNA-damaging agents at defined cell cycle points

• Quantitative analysis of NHEJ activity:

  • Deploy reporter assays specific for NHEJ repair at different cell cycle stages

  • Compare repair outcomes in the presence and absence of CYREN

• Proteomics approaches:

  • Perform immunoprecipitation of CYREN followed by mass spectrometry at different cell cycle stages

  • Identify cell cycle-specific interaction partners

These methodological approaches would provide a comprehensive understanding of how CYREN's activity is regulated throughout the cell cycle.

How do mutations in CYREN contribute to cancer development and progression?

The iPTMnet database indicates variants at the S123 phosphorylation site of CYREN associated with uterine and colorectal cancers . For researchers investigating these connections, methodological approaches include:

• Cancer genome analysis: Mine large-scale cancer genome databases (TCGA, ICGC) for additional CYREN mutations and assess their frequency across cancer types.

• Functional characterization of cancer-associated variants:

  • Generate isogenic cell lines expressing wild-type or mutant CYREN

  • Assess DNA repair efficiency, genomic stability, and cell proliferation

  • Evaluate resistance to DNA-damaging therapeutic agents

• Mouse models: Develop knockin mouse models with cancer-associated CYREN variants to study their effects on tumor initiation and progression in vivo.

• Patient-derived xenografts (PDX): Establish PDX models from tumors with CYREN mutations to test therapeutic vulnerabilities.

• Clinical correlation studies: Analyze associations between CYREN mutations and:

  • Patient survival

  • Treatment response

  • Metastatic potential

  • Tumor mutational burden

These approaches would help establish whether CYREN mutations are passenger events or driver mutations in cancer development.

What is the potential of targeting CYREN in cancer therapy?

For researchers exploring CYREN as a therapeutic target, methodological considerations include:

• Vulnerability assessment:

  • Conduct synthetic lethality screens in cancer cells with CYREN mutations

  • Identify genetic or pharmacological interventions that selectively kill cells with altered CYREN function

• Small molecule screening:

  • Develop high-throughput assays to identify compounds that modulate CYREN activity

  • Focus on compounds that affect CYREN's interaction with the DNA repair machinery

• Structure-based drug design:

  • Determine the three-dimensional structure of CYREN alone and in complex with interaction partners

  • Design compounds that specifically disrupt cancer-promoting interactions

• Combination therapy evaluation:

  • Test CYREN modulators in combination with established DNA-damaging agents

  • Assess potential for synergistic effects that could increase therapeutic efficacy while reducing side effects

• Biomarker development:

  • Identify patient populations most likely to benefit from CYREN-targeted therapies based on genetic or molecular profiles

  • Develop companion diagnostics to guide treatment decisions

These methodological approaches would establish whether CYREN represents a viable therapeutic target in specific cancer contexts.

What are the most promising future research directions for CYREN/C7ORF49?

Based on the current knowledge about CYREN/C7ORF49, researchers might consider these methodological approaches for future investigations:

• Comprehensive interactome mapping: Employ proximity labeling methods like BioID or APEX to identify the complete network of CYREN interactors across different cellular conditions and cell cycle stages.

• Structural biology approaches: Determine the three-dimensional structure of CYREN using X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy to gain insights into its molecular function.

• Single-cell analysis of CYREN activity: Develop reporters to monitor CYREN function at the single-cell level to understand cell-to-cell variability in its regulation of DNA repair.

• Evolutionary analysis across species: Perform comparative genomic and functional studies to understand how CYREN evolved and whether its function in regulating NHEJ is conserved across species.

• Integration with machine learning approaches: Apply computational methods to predict how various genetic backgrounds might influence CYREN function and to identify potential synthetic lethal interactions that could be therapeutically relevant.