Recombinant Mouse Transcription cofactor vestigial-like protein 2 (Vgll2)

What is the functional domain structure of mouse Vgll2?

Vgll2 is a mammalian homolog of the Drosophila vestigial protein that contains specific domains that interact with TEA domain (TEAD) transcription factors. The protein functions in a tissue-specific manner, primarily in skeletal muscle tissues. As a transcription cofactor, Vgll2 contains regions that facilitate protein-protein interactions with transcriptional machinery. For optimal experimental characterization, expressing and purifying different domains separately can help elucidate their specific functions in various assay systems .

How does recombinant Vgll2 differ structurally from endogenous Vgll2?

Recombinant mouse Vgll2 typically includes tag sequences (such as poly-histidine tags) that facilitate purification and detection but may affect protein folding in certain contexts. When designing experiments, researchers should consider whether to use the full-length protein or specific functional domains based on their experimental goals. If studying interaction with TEAD transcription factors, ensure the relevant binding domains remain accessible and properly folded in the recombinant version .

What are the recommended storage conditions for maintaining recombinant Vgll2 stability?

Similar to other recombinant proteins, Vgll2 stability can be maintained through proper storage protocols. Based on standard practices for similar proteins, recombinant Vgll2 should be stored in a manual defrost freezer, avoiding repeated freeze-thaw cycles that can lead to protein degradation and loss of activity. For long-term storage, aliquoting the protein in single-use volumes is recommended to prevent degradation from repeated thawing and refreezing .

What expression systems are most effective for producing functional recombinant Vgll2?

For mammalian transcription factors like Vgll2, bacterial expression systems may yield high quantities but often struggle with proper folding of complex mammalian proteins. For functional studies, mammalian or insect cell expression systems typically produce properly folded and post-translationally modified Vgll2. When conducting experiments requiring high protein purity, researchers should implement multi-step purification protocols including affinity chromatography followed by size exclusion to remove aggregates and ensure homogeneity .

How can I optimize solubility of recombinant Vgll2 during expression and purification?

Vgll2 solubility challenges can be addressed through several strategies: (1) Express the protein with solubility enhancing tags like MBP or SUMO; (2) Optimize buffer conditions with stabilizing agents like glycerol (10-15%) and reducing agents to maintain disulfide bonds in their reduced state; (3) Consider expressing functional domains separately if the full-length protein proves difficult to solubilize; (4) Adjust induction conditions including temperature, inducer concentration, and duration to enhance proper folding during expression .

What is the most reliable method to verify the functional activity of purified recombinant Vgll2?

To verify Vgll2 functionality, researchers should assess its ability to interact with TEAD transcription factors using co-immunoprecipitation or pull-down assays. Additionally, electrophoretic mobility shift assays (EMSAs) can demonstrate whether the Vgll2-TEAD complex properly binds target DNA sequences. For more comprehensive analysis, cell-based reporter assays measuring the activation of Vgll2-responsive promoters provide functional validation in a cellular context that more closely reflects physiological conditions .

How does Vgll2 interact with TEAD transcription factors to regulate gene expression?

Vgll2 serves as a cofactor for TEAD transcription factors, forming a complex that regulates the expression of target genes involved in muscle fiber differentiation and metabolism. The interaction occurs through specific binding domains and influences the recruitment of additional transcriptional machinery. To experimentally characterize these interactions, researchers can employ proximity ligation assays, FRET-based techniques, or chromatin immunoprecipitation (ChIP) to identify genomic binding sites. The Vgll2-TEAD complex specifically regulates genes involved in slow-twitch muscle fiber formation by counteracting repressors like Sox6, Sp3, and Purβ .

What are the key downstream genes regulated by Vgll2 in skeletal muscle?

RNA-Seq analysis of wild-type versus Vgll2 knockout mice has revealed numerous genes regulated by this transcription cofactor. Under sedentary conditions, 196 genes (78 upregulated, 118 downregulated) show differential expression in Vgll2 KO mice compared to wild-type. Following mechanical overload, this expands dramatically to 1,889 differentially expressed genes (1,336 upregulated, 553 downregulated). Key targets include genes involved in:

• Muscle fiber type determination

• Oxidative metabolism

• Mitochondrial function

• Contractile protein isoform expression

These findings suggest Vgll2's critical role extends beyond muscle fiber type regulation to broader metabolic adaptations in skeletal muscle .

How can I conduct ChIP-seq experiments to identify genome-wide binding sites of Vgll2?

For successful ChIP-seq experiments with Vgll2:

• Use antibodies with verified specificity for Vgll2 or epitope tags on recombinant proteins

• Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

• Ensure sufficient chromatin fragmentation (200-500bp fragments)

• Include appropriate controls (IgG, input chromatin)

• Perform sequencing with sufficient depth (≥20 million reads)

• Analyze data with established pipelines to identify binding motifs and genomic locations

Since Vgll2 acts as a cofactor rather than direct DNA-binding protein, consider performing sequential ChIP (re-ChIP) experiments to identify regions co-bound by Vgll2 and TEAD factors .

How can recombinant Vgll2 be used to study muscle fiber type transitions in response to exercise?

Recombinant Vgll2 can serve as a valuable tool for studying muscle adaptation by:

• Developing in vitro models where exogenous Vgll2 is added to myotubes to monitor changes in gene expression profiles

• Creating reporter systems to track Vgll2-dependent transcriptional activation during muscle development and adaptation

• Performing rescue experiments in Vgll2-deficient systems to confirm specific functions

RNA-Seq data from wild-type and Vgll2 KO mice demonstrates that Vgll2 is essential for proper muscle fiber type transitions in response to mechanical overload, with KO mice showing altered expression patterns of fiber type-specific genes. This suggests recombinant Vgll2 could potentially modulate similar gene expression patterns in experimental models .

What is the molecular mechanism through which Vgll2 regulates the fast-to-slow fiber type transition?

Vgll2 regulates fiber type transition through multiple mechanisms:

• Repression of slow-fiber repressors (Sox6, Sp3, Purβ) during development

• Activation of genes involved in oxidative metabolism

• Modulation of contractile protein isoform expression

• Regulation of mitochondrial density and function

To determine if your recombinant Vgll2 preparation can recapitulate these effects, researchers should:

• Compare gene expression profiles in Vgll2-null myotubes with and without recombinant protein supplementation

• Analyze mitochondrial content and function following Vgll2 administration

• Assess contractile protein isoform switching using immunoblotting or immunofluorescence techniques

How does the gene expression profile differ between wild-type and Vgll2 knockout mice under mechanical overload conditions?

RNA-Seq analysis reveals dramatic differences in gene expression profiles:

These findings indicate that Vgll2 deficiency significantly amplifies the transcriptional response to mechanical overload, suggesting Vgll2 plays a complex role in muscle adaptation beyond simply promoting a fast-to-slow fiber shift. The dramatic increase in differentially expressed genes under mechanical overload conditions (from 196 to 1,889) suggests Vgll2 may function as both activator and repressor depending on cellular context and loading conditions .

How can I design experiments to study the role of Vgll2 phosphorylation in regulating its activity?

To investigate Vgll2 phosphorylation:

• Perform in silico analysis to identify potential phosphorylation sites

• Generate phospho-mimetic (Ser/Thr to Asp/Glu) and phospho-null (Ser/Thr to Ala) mutants

• Compare the activity of wild-type and mutant proteins in:

  • Binding assays with TEAD transcription factors

  • Reporter gene assays measuring transcriptional activity

  • Cell-based assays examining muscle differentiation or fiber type specification

• Employ mass spectrometry to identify actual phosphorylation sites in vivo and in vitro

• Use phospho-specific antibodies to track phosphorylation status under different conditions (rest vs. exercise)

What approaches can be used to investigate potential therapeutic applications of Vgll2 in muscle wasting conditions?

Investigating Vgll2 as a therapeutic target requires:

• Developing cell-permeable Vgll2-derived peptides that can recapitulate specific functions

• Testing recombinant Vgll2 delivery systems (viral vectors, nanoparticles) in muscle atrophy models

• Screening small molecules that can enhance endogenous Vgll2 activity or expression

• Comparing gene expression profiles between Vgll2-treated and untreated muscle in atrophy models

• Assessing functional outcomes including muscle mass, strength, and fiber type composition

RNA-Seq data from Vgll2 studies provides crucial baseline information about which genes and pathways might be targeted in therapeutic approaches. The 1,889 differentially expressed genes identified in Vgll2 KO mice under mechanical overload provide a molecular signature that could be used to evaluate potential therapeutic interventions .

How can multiomics approaches be integrated to better understand Vgll2 function in muscle adaptation?

A comprehensive multiomics strategy should include:

• Transcriptomics: RNA-Seq to identify Vgll2-dependent gene expression changes

• Proteomics: Mass spectrometry to identify Vgll2 interaction partners and assess protein-level changes

• Metabolomics: Analysis of metabolic profiles to understand how Vgll2 affects muscle metabolism

• Epigenomics: ATAC-Seq and ChIP-Seq to examine chromatin accessibility and histone modifications at Vgll2-regulated loci

• Single-cell approaches: scRNA-Seq to identify cell-type specific responses to Vgll2 manipulation

Integration of these datasets requires sophisticated bioinformatics approaches including pathway analysis, network modeling, and machine learning to identify key nodes in the Vgll2 regulatory network. This approach has been particularly informative in understanding how Vgll2 regulates the complex transcriptional networks involved in muscle fiber type specification and adaptation to mechanical stress .

What are common pitfalls when working with recombinant Vgll2 and how can they be avoided?

Common challenges and solutions include:

• Protein insolubility: Use carrier proteins like BSA or optimize buffer conditions with stabilizing agents

• Loss of activity during storage: Store in small aliquots at -80°C with cryoprotectants

• Inconsistent results in functional assays: Verify protein quality by SDS-PAGE and activity assays before each experiment

• Non-specific interactions: Include appropriate blocking agents and controls in binding studies

• Poor antibody recognition: Validate antibodies with positive and negative controls including Vgll2 knockout samples

For recombinant protein preparations, consider using carrier-free formulations for applications where additional proteins might interfere with experimental outcomes .

How can contradictory results between in vitro and in vivo Vgll2 studies be reconciled?

To address discrepancies between in vitro and in vivo findings:

• Consider the cellular context: Muscle cells in culture lack the three-dimensional architecture and mechanical inputs present in vivo

• Examine expression levels: Ensure recombinant Vgll2 is used at physiologically relevant concentrations

• Account for post-translational modifications: In vitro systems may not recapitulate all modifications present in vivo

• Consider temporal dynamics: Acute vs. chronic effects of Vgll2 manipulation may differ

• Evaluate compensatory mechanisms: In vivo systems may activate alternative pathways to compensate for Vgll2 manipulation

RNA-Seq data from knockout mice studies show that Vgll2 deficiency has broader effects under mechanical overload than in sedentary conditions, highlighting the importance of considering physiological context when interpreting results .

What controls are essential when performing RNA-Seq analysis to identify Vgll2-regulated genes?

Essential controls for RNA-Seq experiments include:

• Biological replicates: Minimum of 3-4 per condition to account for biological variability

• RNA quality controls: RIN scores >8 to ensure RNA integrity

• Library preparation controls: Inclusion of spike-in controls to monitor technical variation

• Sequencing depth controls: Sufficient depth (>20M reads per sample) for detecting moderately expressed genes

• Validation controls: qRT-PCR confirmation of key differentially expressed genes

• Phenotypic controls: Correlation of gene expression changes with observable phenotypes

When analyzing Vgll2-dependent gene expression, appropriate statistical thresholds (typically adjusted p-value <0.05 and fold change >1.5) should be applied to identify truly significant changes. These approaches were successfully employed in studies comparing wild-type and Vgll2 knockout mice under both sedentary and mechanical overload conditions .

How might single-cell transcriptomics advance our understanding of Vgll2 function in heterogeneous muscle tissues?

Single-cell RNA-Seq could reveal:

• Cell type-specific responses to Vgll2 manipulation

• Temporal dynamics of gene expression during muscle adaptation

• Identification of rare cell populations particularly sensitive to Vgll2 function

• Cellular communication networks influenced by Vgll2 activity

• Distinctions between myonuclear and satellite cell responses

This approach would be particularly valuable in understanding the heterogeneous response to mechanical overload, where Vgll2 has been shown to regulate the expression of 1,889 genes, potentially affecting different muscle cell subpopulations in distinct ways .

What potential roles might Vgll2 play in muscle regeneration and stem cell differentiation?

Investigating Vgll2 in muscle regeneration requires:

• Tracking Vgll2 expression during different phases of muscle injury and repair

• Examining the effect of Vgll2 manipulation on satellite cell activation and differentiation

• Assessing whether Vgll2 influences the determination of fiber type during regeneration

• Exploring potential interactions between Vgll2 and inflammation mediators during repair

• Comparing regenerative capacity in wild-type versus Vgll2-deficient muscles

Given Vgll2's established role in muscle differentiation and fiber type specification, it likely plays important but currently uncharacterized roles in the regenerative process and myogenic stem cell fate determination .

How do the functional impacts of Vgll2 vary across different muscle types and under different loading conditions?

To comprehensively characterize muscle-specific and loading-dependent effects:

• Compare Vgll2 expression and function across:

  • Fast-twitch dominant muscles (EDL, gastrocnemius)

  • Slow-twitch dominant muscles (soleus)

  • Mixed fiber type muscles (plantaris)

• Examine responses to various loading protocols:

  • Mechanical overload

  • Endurance exercise

  • Resistance training

  • Disuse/unloading

• Analyze fiber type transitions using immunohistochemistry for myosin heavy chain isoforms

• Correlate transcriptional profiles with functional outcomes