Recombinant Xenopus laevis Transducin-like enhancer protein 4 (tle4), partial

What is the molecular structure and function of Xenopus laevis TLE4?

Xenopus laevis TLE4 functions as a transcriptional co-repressor belonging to the Groucho/TLE family. Like other members of this family, TLE4 likely contains several conserved domains including the Q domain (for oligomerization), GP domain (glycine/proline-rich), CcN domain (containing nuclear localization signals), SP domain (serine/proline-rich), and WD40 repeat domain (for protein-protein interactions) . TLE4 does not bind DNA directly but interacts with DNA-binding transcription factors through specific motifs, particularly the eh1 motif found in proteins like FoxG1, to form repressive complexes that modify chromatin structure and inhibit gene expression . As a co-repressor, TLE4 plays essential roles in developmental processes, particularly in neural development and specification.

How is TLE4 expressed during Xenopus development?

TLE4 shows temporally and spatially regulated expression during Xenopus development. While the specific expression pattern in Xenopus laevis has not been fully characterized in the provided search results, related studies in Xenopus tropicalis suggest that TLE family members, including TLE4, exhibit specific expression domains during embryogenesis . For detection of TLE4 mRNA in Xenopus tropicalis, researchers have used EST clone TGas107e13 . The expression patterns of TLE family members are particularly important in brain development, where they show region-specific expression that contributes to proper neural patterning and specification.

What distinguishes TLE4 from other TLE family members in Xenopus?

Xenopus contains several TLE family members, including TLE1, TLE2, TLE4, and the short form AES . These proteins share conserved domains but likely have distinct expression patterns and interaction partners that confer specific functions. Sequence analysis through BLAST searches can identify the specific homologues in the Xenopus genome . Based on studies in other systems, TLE4 likely has unique expression domains and interaction partners that distinguish it functionally from other family members. The table below summarizes the known TLE family members in Xenopus:

What are the most effective approaches for cloning and expressing recombinant Xenopus TLE4?

For cloning and expressing recombinant Xenopus TLE4, researchers can follow similar approaches as used for other TLE family members. Based on methods described for TLE family proteins, effective strategies include:

• Identification of full-length sequence through EST database searches

• PCR amplification of the coding sequence from cDNA libraries or embryonic cDNA

• Cloning into appropriate expression vectors (e.g., pCS2 with suitable restriction sites like EcoRI/XbaI)

• Addition of epitope tags (such as Flag or HA) for detection and purification purposes

• Expression in prokaryotic systems (E. coli) for biochemical studies or eukaryotic systems (insect cells, mammalian cells) for functional studies requiring proper folding and post-translational modifications

For partial recombinant TLE4, specific domains can be amplified and expressed separately, depending on the experimental requirements. When designing expression constructs, care should be taken to maintain critical functional domains intact.

What methods are optimal for detecting TLE4 expression in Xenopus tissues?

For comprehensive analysis of TLE4 expression in Xenopus tissues, researchers should employ multiple complementary approaches:

• mRNA detection: In situ hybridization using antisense RNA probes generated from EST clones (like TGas107e13 for X. tropicalis TLE4) . This technique allows visualization of spatial expression patterns in tissue sections or whole embryos.

• Protein detection: Immunostaining with specific antibodies against TLE4. If Xenopus-specific antibodies are unavailable, antibodies against conserved regions of mammalian TLE4 may cross-react.

• Quantitative analysis: Quantitative PCR (qPCR) for relative expression levels across tissues or developmental stages .

• Western blotting: For analyzing protein size and expression levels in tissue extracts.

• RNA immunoprecipitation (RIP): Can be used to study associations of TLE4 mRNA with regulatory complexes .

These approaches can be combined to build a comprehensive picture of TLE4 expression patterns during development.

What functional assays are most informative for studying TLE4 activity?

To investigate the functional activity of recombinant TLE4, researchers should consider these methodological approaches:

• Co-immunoprecipitation assays: To identify protein interaction partners of TLE4, similar to approaches used for other TLE family members . These assays can confirm interactions with specific transcription factors containing eh1 or other TLE-interaction motifs.

• Transcriptional repression assays: Reporter gene assays using promoters of putative target genes to assess TLE4's repressive activity.

• Loss-of-function studies: Morpholino (MO) knockdown approaches similar to those used for other TLE family members in Xenopus . MOs can be designed to specifically target TLE4 mRNA.

• Gain-of-function studies: mRNA injection for overexpression of wild-type or mutant TLE4 to assess developmental effects .

• Domain mapping experiments: Creation of deletion or point mutation constructs to identify critical functional domains, similar to approaches used for FoxG1-TLE interactions .

• Chromatin immunoprecipitation (ChIP): To identify genomic regions bound by TLE4-containing complexes.

How does TLE4 contribute to neural development in Xenopus?

While specific details of TLE4's role in Xenopus neural development are not fully detailed in the provided search results, evidence from related studies suggests important functions in this process. In mammalian systems, TLE4 shows specific expression in deep cortical layers (layer VI) and contributes to neuronal specification . In Xenopus, TLE family members interact with neural transcription factors like FoxG1 to regulate telencephalon development . By analogy, TLE4 likely plays roles in:

• Regional specification within the developing brain

• Neuronal subtype specification

• Repression of inappropriate gene expression during neural differentiation

• Maintenance of neural progenitor populations

Loss-of-function studies (e.g., with morpholinos) would be necessary to precisely define TLE4's roles in Xenopus neural development, similar to studies performed for TLE2 .

What protein-protein interactions mediate TLE4 function during development?

TLE4, like other TLE family members, likely interacts with multiple transcription factors to mediate its repressive functions. While specific TLE4 interactions in Xenopus are not fully characterized in the provided search results, we can infer potential interactions based on studies of related TLE proteins:

• FoxG1 interaction: TLE family members interact with FoxG1 through its N-terminal eh1 motif . This interaction is crucial for ventral telencephalon development in Xenopus tropicalis.

• Transcription factors with eh1 motifs: Many homeodomain proteins contain eh1 motifs that mediate TLE interaction.

• Repression complexes: TLE4 likely forms part of larger repressive complexes that include histone deacetylases and other chromatin modifiers.

• Translational regulation: Interestingly, TLE4 mRNA has been found associated with 4E-T in RNA immunoprecipitation studies, suggesting potential roles in translational regulation complexes .

The functional significance of these interactions can be studied through co-immunoprecipitation, domain mapping, and functional assays in developing embryos.

What phenotypes result from TLE4 perturbation in Xenopus embryos?

• Neural development defects: Based on studies of TLE2, knockdown of TLE4 might affect specific brain regions, particularly in the forebrain where TLE family members are known to function with FoxG1 .

• Cell fate specification defects: Given TLE4's role as a transcriptional co-repressor, its loss might lead to inappropriate gene expression and altered cell fate decisions.

• Telencephalon development: Knockdown of TLE2 in Xenopus tropicalis reduces ventral telencephalon development . TLE4 might similarly affect specific brain regions.

• Potential redundancy: Functional redundancy among TLE family members might mask some phenotypes, necessitating combinatorial knockdown approaches.

Careful phenotypic analysis would require examination of region-specific markers, particularly those of brain regions where TLE4 is normally expressed.

How can recombinant TLE4 be used to study epigenetic mechanisms during development?

Recombinant TLE4 can serve as a valuable tool for investigating epigenetic mechanisms during Xenopus development through several advanced approaches:

• Chromatin modification analysis: TLE co-repressors typically function by recruiting histone deacetylases and other chromatin modifiers. Recombinant TLE4 can be used in biochemical assays to identify specific chromatin modifiers recruited to target sites.

• ChIP-sequencing approaches: Using tagged recombinant TLE4 in ChIP-seq experiments can identify genome-wide binding sites and, when combined with histone modification ChIP, reveal associated chromatin states.

• Domain-specific functions: Partial recombinant TLE4 constructs containing specific domains can help dissect which regions are responsible for recruiting different epigenetic modifiers.

• Competitor studies: Recombinant TLE4 fragments can be used as competitors to disrupt specific interactions in developing embryos, allowing temporal control over TLE4 function.

• Proteomics approaches: Recombinant TLE4 can serve as bait in pull-down experiments coupled with mass spectrometry to identify novel components of TLE4-containing repressive complexes.

What are the emerging techniques for studying TLE4 dynamics in live Xenopus embryos?

Advanced imaging and functional genomics approaches are opening new possibilities for studying TLE4 dynamics in developing Xenopus embryos:

• Fluorescently tagged TLE4: Generation of fluorescent fusion proteins (e.g., TLE4-GFP) allows visualization of protein localization and dynamics in live embryos.

• CRISPR-Cas9 genome editing: While not specifically mentioned in the search results for TLE4, CRISPR approaches are increasingly used in Xenopus. TLE4 could be tagged endogenously or knocked out using this technology.

• Optogenetic approaches: Light-controlled recruitment or inhibition of TLE4 function could allow precise temporal and spatial control of its activity.

• Single-cell transcriptomics: Combining TLE4 perturbation with single-cell RNA-seq can reveal cell-type specific responses to TLE4 activity.

• Proximity labeling: Techniques like BioID or APEX2 fused to TLE4 can identify proteins in close proximity in living cells, revealing the composition of TLE4 complexes in different developmental contexts.

These emerging techniques offer opportunities for more dynamic and precise analysis of TLE4 function than traditional fixed-tissue approaches.

How can comparative studies between mammalian and Xenopus TLE4 advance our understanding of conserved developmental mechanisms?

Comparative studies of TLE4 between Xenopus and mammals provide valuable insights into evolutionarily conserved mechanisms of development:

• Structural conservation analysis: Comparison of domain structures, interaction interfaces, and post-translational modification sites between Xenopus and mammalian TLE4 can reveal conserved functional elements.

• Expression pattern comparison: Analysis of TLE4 expression in homologous structures between amphibian and mammalian brains can identify conserved developmental roles.

• Functional rescue experiments: Testing whether mammalian TLE4 can rescue Xenopus TLE4 knockdown phenotypes (and vice versa) can define functionally conserved properties.

• Interaction partner conservation: Identifying whether TLE4 interacts with the same transcription factors across species can reveal conserved regulatory networks.

• Cross-species chromatin studies: Comparing TLE4 binding sites and associated epigenetic marks between species can identify conserved target genes and regulatory mechanisms.

Such comparative approaches leverage Xenopus as a tractable developmental system while providing insights relevant to mammalian development and potential disease mechanisms.

What are the main challenges in producing soluble, functional recombinant TLE4?

Researchers working with recombinant Xenopus TLE4 commonly encounter several technical challenges:

• Protein solubility: TLE proteins contain multiple domains with different biochemical properties, potentially leading to aggregation during expression and purification. Using solubility tags (like MBP or SUMO) or expressing individual domains separately may improve solubility.

• Proper folding: Expressing full-length TLE4 in prokaryotic systems might result in improper folding. Eukaryotic expression systems (insect cells, mammalian cells) often yield better results for functional studies.

• Co-factor requirements: TLE4 functions as part of larger complexes, and isolated recombinant protein may lack necessary co-factors for full activity. Co-expression with binding partners might be necessary for certain functional studies.

• Post-translational modifications: Functional TLE4 likely requires specific post-translational modifications that may be absent in recombinant systems. Mass spectrometry can identify which modifications are present or absent in recombinant preparations.

• Protein stability: TLE4 may be subject to regulated degradation. Including protease inhibitors throughout purification and storage is essential for maintaining intact protein.

How can researchers overcome specificity issues when studying TLE4 among highly similar family members?

Distinguishing TLE4 from other TLE family members presents several challenges that can be addressed through these strategies:

• Isoform-specific antibodies: Develop antibodies against unique regions of TLE4 that differ from other family members. Careful validation is essential to ensure specificity.

• Custom morpholinos or CRISPR guides: Design knockdown or knockout reagents targeting unique sequences in TLE4 mRNA or gene. Validate specificity using rescue experiments with morpholino-resistant constructs.

• Domain-swapping experiments: Create chimeric proteins with domains from different TLE family members to identify which domains confer specific functions.

• Unique binding partner identification: Perform immunoprecipitation followed by mass spectrometry to identify proteins that specifically interact with TLE4 but not other family members.

• Isoform-specific qPCR primers: Design primers spanning unique regions or exon junctions to specifically quantify TLE4 expression levels.

• Cross-species conservation analysis: Identify TLE4-specific sequence features that are conserved across species but distinct from other TLE family members.

What controls are essential when performing functional studies with recombinant TLE4?

Rigorous controls are critical for ensuring reliable results in functional studies of recombinant TLE4: