Recombinant Dog T-box transcription factor TBX2 (TBX2), partial

What is the basic structure and function of TBX2 in mammalian systems?

TBX2 belongs to a phylogenetically conserved family of transcription factors characterized by a common DNA-binding domain called the T-box. This protein functions primarily as a transcriptional repressor that regulates critical developmental processes including cell fate decisions, migration, and tissue morphogenesis .

The protein contains:

• A highly conserved T-box DNA-binding domain

• N-terminal and C-terminal domains with repression functions

• Specific domains for protein-protein interactions with co-repressors

In developmental contexts, TBX2 is strongly expressed in mesenchymal progenitors and plays crucial roles in organogenesis, particularly in lung, heart, and limb development . Mutations leading to reduced TBX2 levels result in developmental abnormalities including congenital heart and skeletal defects .

How do recombinant TBX2 proteins differ between species, and what implications does this have for research using dog TBX2?

Recombinant TBX2 proteins show high conservation in the T-box domain across mammalian species but exhibit variations in N- and C-terminal regions. These differences can affect:

• DNA binding specificity and affinity

• Protein-protein interaction capabilities

• Post-translational modification sites

When working with dog TBX2, researchers should note that while the T-box domain shares approximately 98% homology with human TBX2, terminal regions may contain species-specific sequences that affect function. This is particularly important when:

• Designing binding assays for target gene identification

• Testing interactions with co-factors

• Evaluating antibody cross-reactivity between species

• Extrapolating findings to human disease models

Appropriate controls using species-matched systems are recommended for comparative studies to account for these variations .

What expression systems are most effective for producing functional recombinant dog TBX2 protein?

Production of functional recombinant dog TBX2 requires careful consideration of expression systems to ensure proper folding and post-translational modifications. Based on current methodologies:

Recommended Expression Systems:

For functional transcription factor assays, mammalian systems are preferred as they provide the native cellular environment for proper protein folding and modification. If using bacterial systems, fusion partners like MBP or SUMO can improve solubility. Co-expression with chaperone proteins may enhance proper folding for complex domains.

For optimal results when expressing the T-box domain alone (for DNA binding studies), prokaryotic systems may be sufficient, while full-length TBX2 studies typically benefit from eukaryotic expression systems .

What experimental approaches can resolve contradictory data on TBX2 target genes in different tissue contexts?

Resolving contradictory findings regarding TBX2 target genes requires multi-faceted approaches that account for context-specific regulation. The following methodological framework is recommended:

• Integrated genomic analysis:

  • Perform parallel ChIP-seq and RNA-seq in the specific tissue/cell type of interest

  • Compare binding profiles across multiple contexts to identify context-specific binding

  • Validate with CUT&RUN for higher resolution of binding sites

• Determinants of context-specificity:

  • Analyze chromatin accessibility (ATAC-seq) to determine if differential targeting results from varied chromatin states

  • Profile co-factors using co-immunoprecipitation followed by mass spectrometry

  • Investigate post-translational modifications that may alter TBX2 function in different contexts

• Validation experiments:

  • Apply CRISPR-mediated mutagenesis of putative binding sites

  • Use reporter assays with mutated binding elements

  • Perform proteomic analysis of TBX2 complexes in different cellular contexts

This approach has successfully resolved apparent contradictions in TBX2 targeting between lung mesenchymal progenitors and cancer cells, revealing that TBX2 represses E-cadherin in cancer contexts but not in developmental contexts .

How can researchers distinguish direct versus indirect effects of TBX2 overexpression in gain-of-function studies?

Distinguishing direct from indirect effects in TBX2 gain-of-function studies requires careful experimental design:

Recommended methodology:

• Temporal analysis:

  • Implement inducible expression systems (e.g., Tet-On/Off)

  • Perform time-course analysis of transcriptional changes following TBX2 induction

  • Early response genes (0-6h) are more likely to be direct targets

• DNA binding dependence:

  • Generate DNA-binding deficient TBX2 mutants by introducing point mutations in the T-box domain

  • Compare phenotypes between wild-type and binding-deficient TBX2

  • Effects requiring intact DNA binding are likely direct transcriptional effects

• Direct binding verification:

  • Perform ChIP-seq to identify genome-wide binding sites

  • Correlate binding with gene expression changes

  • Verify binding site functionality through reporter assays

• Acute manipulation:

  • Use degron-tagged TBX2 for rapid protein depletion

  • Monitor immediate transcriptional consequences

These approaches have been successfully applied to delineate TBX2's direct repression of E-cadherin during epithelial-mesenchymal transition, confirming TBX2 directly binds to the E-cadherin promoter and represses its transcription .

What are the optimal experimental conditions for studying TBX2-mediated repression of target genes?

TBX2 primarily functions as a transcriptional repressor, and optimizing experimental conditions is crucial for accurate mechanistic studies:

Key parameters to control:

• Cellular context:

  • Select cell types with appropriate cofactor expression

  • Consider the chromatin state of potential target genes

  • Account for endogenous TBX2 levels that may mask effects

• Expression level considerations:

  • Use titratable expression systems to avoid non-physiological effects

  • Compare effects at different TBX2 concentrations

  • Include physiologically relevant controls

• Repression assay optimization:

  • For reporter assays, use minimal promoters containing verified TBX2 binding sites

  • Include positive controls with known repressed targets (e.g., E-cadherin promoter fragments)

  • Test multiple time points (24h, 48h, 72h) for maximal repression detection

• Co-repressor considerations:

  • Verify expression of known TBX2 co-repressors in your cellular system

  • Consider co-expressing essential co-factors if lacking

Experimental designs should incorporate these parameters to avoid false negatives. Studies have shown that TBX2-mediated repression of E-cadherin occurs through binding to sites in the proximal promoter region (-131/+61), and this can serve as a positive control for repression assays .

How can researchers effectively model the developmental functions of TBX2 using recombinant proteins?

Modeling developmental functions of TBX2 requires approaches that recapitulate its context-specific activities:

• Organoid systems:

  • Generate tissue-specific organoids (lung, cochlear, cardiac)

  • Introduce recombinant TBX2 at defined developmental stages

  • Monitor alterations in differentiation trajectories through single-cell transcriptomics

• Ex vivo explant cultures:

  • Harvest embryonic tissue at relevant developmental stages

  • Apply recombinant TBX2 protein using controlled delivery methods

  • Analyze cell fate decisions using lineage tracing techniques

• Chimeric systems:

  • Generate mosaic tissues with cells expressing different levels of TBX2

  • Analyze cell-autonomous and non-cell-autonomous effects

  • Use fluorescent reporters for real-time monitoring

Particularly informative are developmental systems where TBX2 marks multipotent progenitors, such as in lung mesenchyme. Research shows that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung and maintains their undifferentiated state . These systems can be used to test how recombinant TBX2 proteins affect lineage specification and differentiation.

What experimental approaches best characterize the role of TBX2 in epithelial-mesenchymal transition (EMT) in cancer models?

Given TBX2's established role in promoting EMT in cancer, these methodological approaches are recommended:

• Graduated expression systems:

  • Establish cell lines with doxycycline-inducible TBX2 expression

  • Create a dose-response curve to identify the threshold for EMT induction

  • Monitor changes in epithelial and mesenchymal markers at both protein and mRNA levels

• Functional EMT assessment:

  • Migration assays (wound healing, transwell)

  • Invasion assays (Matrigel invasion)

  • 3D culture morphology analysis

  • Quantification of cell-cell adhesion strength

• Mechanism delineation:

  • ChIP-seq to identify direct EMT-related targets

  • Rescue experiments with E-cadherin re-expression

  • Co-expression with EMT-blocking factors

• In vivo validation:

  • Orthotopic xenograft models with inducible TBX2

  • Circulating tumor cell analysis

  • Metastasis quantification

Research has demonstrated that TBX2 directly binds to the E-cadherin promoter and represses its transcription, providing a molecular mechanism for TBX2-induced EMT. This binding occurs at specific sites in the proximal promoter region and can be verified through ChIP analysis followed by luciferase reporter assays .

How should researchers approach studying interactions between TBX2 and other signaling pathways in developmental contexts?

TBX2 operates within complex regulatory networks, requiring systematic approaches to decipher its crosstalk with other pathways:

• Pathway-specific reporting systems:

  • Utilize pathway-specific reporter constructs (e.g., TOPFlash for Wnt signaling)

  • Monitor pathway activity following TBX2 modulation

  • Use small molecule inhibitors or activators to manipulate pathway status

• Protein-protein interaction mapping:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Use proximity labeling techniques (BioID, APEX) for in vivo interactions

  • Validate interactions through fluorescence resonance energy transfer (FRET)

• Genetic interaction analysis:

  • Combined knockdown/overexpression experiments

  • Epistasis analysis to determine hierarchical relationships

  • CRISPR screens to identify synthetic interactions

• Developmental timing considerations:

  • Stage-specific analysis of pathway interactions

  • Temporal manipulation of TBX2 expression

  • Detailed phenotypic analysis at multiple developmental timepoints

Research has shown TBX2 maintains progenitor populations in part through maintaining pro-proliferative WNT signaling via repression of WNT antagonist genes like Frzb and Shisa3 . Additionally, the Wnt/β-catenin and PI3K/AKT pathways have been implicated in regulating TBX2 expression in cancer contexts, suggesting bidirectional regulation .

What are the most reliable methods for detecting recombinant dog TBX2 protein expression and activity?

Accurate detection and activity assessment of recombinant dog TBX2 requires appropriate technical approaches:

Detection methods comparison:

Activity assessment methods:

• DNA binding assays:

  • Electrophoretic mobility shift assay (EMSA)

  • Microscale thermophoresis (MST)

  • Surface plasmon resonance (SPR)

  • DNA pulldown followed by Western blot

• Transcriptional repression assays:

  • Luciferase reporter assays using known TBX2 target promoters

  • RT-qPCR measurement of endogenous target genes

• Chromatin association:

  • Chromatin immunoprecipitation (ChIP)

  • CUT&RUN for higher resolution

  • DNA adenine methyltransferase identification (DamID)

The combination of binding and functional assays provides the most complete assessment of recombinant TBX2 activity. Research has validated luciferase reporter assays using the E-cadherin promoter (-108 to +125) as a reliable system for measuring TBX2 repressive activity .

What are the critical considerations when designing experiments to assess TBX2-dependent phenotypes?

Experimental design for TBX2-dependent phenotype assessment requires careful controls and considerations:

• Appropriate controls:

  • DNA-binding deficient mutants

  • Transcriptionally inactive mutants

  • Dose-matched irrelevant transcription factor controls

  • Empty vector controls with matched selection

• Expression level considerations:

  • Verify physiologically relevant expression levels

  • Use inducible systems to control expression timing and level

  • Implement rescue experiments with varying TBX2 concentrations

• Phenotype assessments:

  • Include both cellular and molecular readouts

  • Quantitative measurements (proliferation, migration rates)

  • Multi-parameter analysis of phenotypic changes

  • Time-course analysis to distinguish primary and secondary effects

• Validation strategies:

  • Conduct loss-of-function experiments to complement gain-of-function

  • Utilize multiple cell lines or model systems

  • Test in relevant physiological contexts

A critical aspect is distinguishing direct TBX2-dependent effects from secondary consequences. Studies of TBX2 in hair cells of the cochlea provide a good example of this approach, where Tbx2 overexpression was tested at different developmental stages, and molecular rescues were performed to validate the specificity of observed phenotypes .

What are the best approaches for purifying active recombinant TBX2 protein for biochemical and structural studies?

Purification of functional recombinant TBX2 presents technical challenges that require specific strategies:

Recommended purification workflow:

• Expression optimization:

  • Test multiple tags (His, GST, MBP, SUMO)

  • Evaluate different positions for tags (N-terminal vs. C-terminal)

  • Optimize expression temperature (16-30°C)

  • Consider co-expression with chaperones

• Solubility enhancement:

  • Include stabilizing additives in lysis buffer (10% glycerol, low concentrations of non-ionic detergents)

  • Test various salt concentrations (150-500 mM NaCl)

  • Add reducing agents to prevent disulfide bond formation

  • Consider mild solubilization agents for inclusion bodies

• Purification strategy:

  • Multi-step purification scheme (affinity → ion exchange → size exclusion)

  • Avoid harsh elution conditions

  • Include protease inhibitors throughout

  • Minimize purification time to prevent degradation

• Activity verification:

  • DNA binding assays (EMSA, fluorescence anisotropy)

  • Circular dichroism to verify proper folding

  • Dynamic light scattering to assess monodispersity

For structural studies, consider domain-based approaches, as the full-length protein may have disordered regions that impede crystallization. The T-box domain alone is typically more amenable to structural analysis while maintaining specific DNA-binding activity .

What strategies are most promising for developing inhibitors of TBX2 function for cancer therapeutics?

Given TBX2's role as an oncogene in multiple cancers, several approaches show promise for therapeutic development:

• Direct TBX2 targeting approaches:

  • Small molecule inhibitors targeting the DNA-binding domain

  • Peptide-based inhibitors disrupting protein-protein interactions

  • Degraders (PROTACs) targeting TBX2 for proteasomal degradation

• Rational screening approaches:

  • Fragment-based drug discovery focused on the T-box domain

  • In silico screening based on T-box crystal structures

  • High-throughput reporter assays measuring TBX2 repressive activity

• Drug repurposing strategies:

  • Screen FDA-approved drugs for TBX2 inhibitory activity

  • Focus on compounds that disrupt transcription factor-DNA interactions

  • Identify drugs that affect TBX2 expression or stability

• Combination therapy approaches:

  • Target both TBX2 and downstream effectors

  • Combine with epigenetic modifiers to overcome TBX2-mediated repression

  • Use pathway inhibitors for signaling networks that regulate TBX2

Recent research has demonstrated the efficacy of a drug repurposing approach to identify compounds that can inhibit TBX2-dependent cancers, which is expected to be more cost-effective with reduced side effects compared to de novo drug development .