Recombinant Xenopus laevis Brachyury protein (t)

What is Xenopus laevis Brachyury protein and what is its functional significance?

Brachyury protein, encoded by the TBXT gene in humans and other organisms, functions as a transcription factor within the T-box family of genes. It serves as the founding member of this family, which in mammals consists of 18 T-box genes . In Xenopus and other organisms, Brachyury plays a critical role in mesoderm formation during embryonic development. The protein is expressed at the onset of gastrulation throughout nascent mesoderm, with transcripts persisting in notochord and posterior mesoderm .

Functionally, Brachyury appears to have a conserved role in defining the midline of bilaterian organisms and establishing the anterior-posterior axis - a function observed in both chordates and molluscs . Loss of Brachyury function in mouse, zebrafish, and Xenopus embryos results in loss of posterior mesoderm and impaired notochord differentiation, highlighting its essential developmental role . At the molecular level, Brachyury functions as a sequence-specific DNA-binding protein that acts as a transcription activator .

What is the structural characterization of Brachyury protein?

The Brachyury protein features a modified immunoglobulin-like β-sandwich fold with additional helical elements between the first and second strands and at the C-terminus . Nearly half of the brachyury protein is intrinsically disordered, which presents challenges for structural studies . The DNA binding domain has been successfully crystallized in complex with DNA, revealing how the protein interacts with its target sequences.

The crystal structure of human brachyury protein was solved in 2017 by Opher Gileadi and colleagues at the Structural Genomics Consortium in Oxford . More recent structural studies have achieved higher resolution (2.25 Å for wild-type human brachyury DNA complex) . The structures of wild-type and variant (G177D) brachyury show high similarity to previous T-box family DNA complexes, including the highly related Xenopus brachyury structure (92% sequence identity and 0.9 Å RMSD) .

How does Brachyury protein recognize and bind to DNA?

Brachyury binds to a specific DNA element - a near palindromic sequence TCACACCT - through a region in its N-terminus called the T-box . Crystallographic studies show that two copies of brachyury bind to the DNA in a 2-fold symmetrical arrangement with a small interface between subunits located towards the N-terminal end of the first β-sheet . DNA contacts are made via loops between strands A and B, c and c', and other regions .

Binding affinity studies using electrophoretic mobility shift assays (EMSA) show that the protein binds to palindromic repeats with high affinity (apparent Kd of around 1 nM), while binding to a single site probe or a natural target promoter sequence (such as from Fibroblast growth factor FGF8) shows lower affinity (Kd of 30-40 nM) . Surface Plasmon Resonance (SPR) experiments confirm this high-affinity binding, with measured dissociation constants of 14.8 ± 2.4 nM for wild-type brachyury .

What expression patterns does Brachyury exhibit during Xenopus development?

In Xenopus embryos, Brachyury (Xbra) is expressed in a ring around the blastopore at the early gastrula stage. This expression pattern is controlled by the Brachyury promoter, which directs expression to specific regions of the developing embryo . Experiments with reporter constructs containing the Xbra2 promoter show that 2.1 kb of 5' flanking sequence is sufficient to direct expression to many sites where the endogenous gene is expressed, though with some differences - notably a gap in expression on the dorsal side of the ring around the blastopore .

By the tailbud stage, Xbra mRNA is found in the posterior mesoderm but is no longer expressed in the notochord . This dynamic expression pattern reflects the changing roles of Brachyury during development and the complex regulation of its expression.

How is the Xenopus Brachyury promoter regulated by signaling molecules?

The Xenopus Brachyury promoter (Xbra2) exhibits a sophisticated response to developmental signaling molecules, particularly activin and FGF. Experimental evidence shows that the promoter is activated by FGF and low concentrations of activin but is suppressed by high concentrations of activin . This concentration-dependent response to activin is particularly significant as it suggests that activin functions as a morphogen during mesoderm induction.

Just 381 bp of Xbra2 5' flanking sequence contains elements that mediate this dose-dependent response to activin . Studies using reporter constructs in animal cap explants show that low levels of activin mRNA (1 pg) strongly activate the Xbra2 promoter, intermediate levels (5 pg) give much lower activation, and high concentrations (50 pg) reduce activity to below baseline levels . This mirrors the expression pattern of the endogenous Xbra2 gene, indicating that the promoter fragment contains all essential regulatory elements for this response.

What methods are optimal for producing and purifying recombinant Xenopus Brachyury protein?

For producing recombinant Brachyury protein, researchers typically focus on the DNA binding domain due to the challenges presented by the intrinsically disordered regions. Based on structural studies, constructs spanning residues 41-224 (for human brachyury) have been successfully crystallized in complex with DNA . For Xenopus Brachyury, a similar approach focusing on the conserved DNA binding domain would be appropriate.

A common methodology involves:

• Gene cloning into a bacterial expression vector with an affinity tag (His-tag or GST-tag)

• Expression in E. coli under optimized conditions (temperature, IPTG concentration)

• Affinity purification using the appropriate resin

• Size-exclusion chromatography to remove aggregates and obtain pure, homogeneous protein

• Verification of proper folding using circular dichroism or thermal shift assays

When studying DNA binding, it's important to ensure that the recombinant protein retains its ability to recognize target sequences. Electrophoretic mobility shift assays (EMSA) and Surface Plasmon Resonance (SPR) have been successfully used to characterize the DNA binding properties of brachyury proteins .

How can researchers investigate the cooperative DNA binding behavior of Brachyury?

Brachyury exhibits cooperative binding to palindromic DNA sequences, with two protein molecules binding to a single DNA molecule. This cooperativity is evident in EMSA experiments where both singly and doubly bound species can be observed, with both appearing at approximately the same point in a protein titration series .

To investigate this phenomenon, researchers can employ several approaches:

• EMSA with varying DNA constructs: Test DNA sequences with modified spacing between half-sites to determine optimal binding geometry.

• Surface Plasmon Resonance (SPR): Use kinetic models to characterize binding. A bivalent binding model has been successfully employed to fit data for brachyury binding to palindromic DNA .

• Isothermal Titration Calorimetry (ITC): To measure thermodynamic parameters of binding and determine cooperativity factors.

• Mutagenesis of interface residues: Target the small interface formed between brachyury subunits when bound to DNA to assess its contribution to cooperative binding.

The cooperative binding behavior may result from "through DNA" effects, where the widening of the minor groove for binding at one site lowers the energy barrier for binding at a nearby site . This model is supported by the observation that the two T-box sites are arranged in an inverted orientation and in the closest possible proximity for binding both sites without steric clashes .

What are the functional differences between wild-type Brachyury and disease-associated variants?

Thermal stability studies reveal only a small difference, with the wild-type protein showing slightly higher thermostability (ΔTm of ~0.7 degrees) . Previous studies using isogenic cellular systems expressing either wild-type or G177D brachyury found no significant differences in downstream target genes identified by ChIP-sequencing .

These findings suggest that the association of G177D with chordoma may involve mechanisms beyond simple changes in DNA binding affinity or protein stability. Researchers investigating the functional differences should consider:

• Protein-protein interactions with cofactors

• Post-translational modifications

• Cellular localization patterns

• Context-dependent effects in specific cell types

What techniques are most effective for identifying Brachyury target genes in Xenopus?

Several complementary approaches can be used to identify brachyury target genes in Xenopus:

• ChIP-seq: Chromatin immunoprecipitation followed by sequencing can identify genome-wide binding sites. This requires either antibodies against the endogenous protein or expression of tagged versions of brachyury.

• RNA-seq after brachyury manipulation: Compare transcriptomes after overexpression or knockdown of brachyury to identify regulated genes.

• Reporter assays: Test candidate promoters using constructs similar to those used for the Xbra2 promoter studies . This approach can validate direct regulation and identify specific regulatory elements.

• ATAC-seq: Assay for Transposase-Accessible Chromatin can identify regions of open chromatin that may contain brachyury binding sites.

• Promoter analysis: Search for the known brachyury binding motif (TCACACCT or similar sequences) in potential target genes .

A combined approach using both ChIP-seq to identify binding sites and RNA-seq to confirm transcriptional effects provides the most robust identification of direct target genes.

How should DNA binding experiments with recombinant Brachyury be designed and analyzed?

When designing DNA binding experiments with recombinant Brachyury, several factors should be considered:

• DNA sequence selection: Include both palindromic arrangements of T-box binding elements (which show higher affinity binding) and single-site sequences. The palindromic sequence TCACACCT is recognized by brachyury . For crystallography, oligonucleotides containing palindromic arrangements of varying length (22-30 nucleotides) have been successfully used .

• Protein construct design: Focus on the DNA binding domain to avoid issues with intrinsically disordered regions. For human brachyury, residues 41-224 have been used successfully .

• Binding assay selection: EMSA and SPR provide complementary information. EMSA can distinguish between singly and doubly bound species, while SPR provides quantitative binding kinetics .

• Data analysis: For SPR data with palindromic DNA, a bivalent binding model is appropriate to account for the cooperative binding behavior . For EMSA, analyze the appearance of both shifted bands to assess cooperativity.

A typical experimental workflow might include:

• Initial EMSA screening with various DNA sequences

• Quantitative binding measurements using SPR

• Crystallography of protein-DNA complexes for structural insights

• Validation in cellular contexts using reporter assays

What are key considerations for comparing Brachyury function across different species?

• Sequence divergence outside the DNA binding domain: While the T-box is highly conserved, other regions may differ substantially between species.

• Expression pattern differences: The spatial and temporal expression patterns may vary between species, reflecting differences in developmental timing and anatomy.

• Regulatory network context: The upstream regulators and downstream targets of brachyury may differ between species, potentially altering its functional role.

• Experimental systems: Different model organisms offer distinct advantages and limitations for studying brachyury function. Xenopus offers advantages for embryological manipulations, while mammalian systems may be more relevant for understanding human disease associations.

When designing comparative studies, researchers should:

• Use sequence alignments to identify conserved and divergent regions

• Compare DNA binding properties using identical experimental conditions

• Examine expression patterns in equivalent developmental stages

• Test functional conservation through cross-species rescue experiments

What are promising approaches for developing small molecule modulators of Brachyury function?

Recent structural studies of human brachyury have identified small molecule binders that could be developed into chemical probes or protein degradation warheads . These approaches offer new possibilities for manipulating brachyury function in experimental and potentially therapeutic contexts.

Key strategies include:

• Structure-based drug design targeting specific pockets in the brachyury protein

• Development of proteolysis targeting chimeras (PROTACs) for selective brachyury degradation

• Screening for molecules that disrupt brachyury-DNA interactions

• Identifying compounds that interfere with brachyury dimerization or protein-protein interactions

These approaches could provide valuable tools for dissecting brachyury function in development and disease, particularly in contexts like chordoma where brachyury has been implicated as a potential therapeutic target.

How might new genomic technologies enhance our understanding of Brachyury's role in development?

Emerging genomic technologies offer new opportunities to understand brachyury's role in development:

• Single-cell RNA-seq: Provides resolution to examine brachyury expression and its targets at the individual cell level during development.

• CUT&RUN or CUT&Tag: Offers improved signal-to-noise ratio compared to ChIP-seq for identifying brachyury binding sites.

• Long-read sequencing: Allows better resolution of complex genomic regions and isoform identification.

• CRISPR screens: Enable systematic functional analysis of brachyury target genes and regulatory elements.

• Spatial transcriptomics: Preserves spatial information about gene expression patterns, critical for understanding brachyury's role in establishing the body axis.

These technologies, combined with traditional embryological approaches in model systems like Xenopus, promise to provide a more comprehensive understanding of how brachyury coordinates mesoderm formation and axis establishment during vertebrate development.