Recombinant Mouse T-cell leukemia homeobox protein 2 (Tlx2)

What is Recombinant Mouse T-cell leukemia homeobox protein 2 (TLX2)?

Recombinant Mouse TLX2 is a DNA-binding nuclear transcription factor belonging to the homeobox family of proteins. It is produced through heterologous expression systems for research applications. The recombinant protein typically contains amino acids 1-284 of the native mouse sequence and may include purification tags such as Strep-Tag for isolation and detection purposes. TLX2 is crucial for the development of the peripheral nervous system and plays significant roles in neuronal differentiation processes. As a Hox protein, it contributes to patterns of embryonic development by regulating downstream target genes .

What is the protein sequence of Mouse TLX2?

The full amino acid sequence of Mouse TLX2 (AA 1-284) is: MEPAVLAAHH LPHHEPISFG IDQILSGPEP PGGGLGPGQS GQSHGESAAF SSGFHGASGY APAGSLASLP RGSGVGPGGV IRVPAHRPLP VPPPSGAAPA VPGPSGLGGA GGLAGLTFPW MDSGRRFAKD RLTAALSPFS GTRRIGHPYQ NRTPPKRKKP RTSFSRSQVL ELERRFLRQK YLASAERAAL AKALRMTDAQ VKTWFQNRRT KWRRQTAEER EAERHRAGRL LLHLQQDALP RPLRPPLPPD PLCLHNSSLF ALQNLQPWAE DNKVASVSGL ASVV .

How is TLX2 genetically regulated?

TLX2 is subject to transcriptional control by other homeodomain proteins, particularly PHOX2B. Research has confirmed a direct functional link between PHOX2B and TLX2 genes. PHOX2B binds to cell-specific elements in the 5′ regulatory region of the TLX2 gene, leading to its transactivation. This interaction has been confirmed both in vitro through transient transfections and electrophoretic-mobility-shift assays, and in vivo through chromatin immunoprecipitation assays. Quantitative real-time PCR has demonstrated up-regulation of endogenous TLX2 mRNA levels following PHOX2B over-expression. Notably, PHOX2B proteins carrying mutations responsible for congenital central hypoventilation syndrome (CCHS) show severe impairment in activating TLX2 expression .

What is the role of TLX2 in normal development?

TLX2, also known as HOX11L1 or Neural crest homeobox protein, is crucial for the development of the peripheral nervous system. It appears to be particularly important in the differentiation of autonomic nervous system (ANS) specific neuronal lineages. TLX2 functions downstream of bone morphogenetic protein (BMP) signaling pathways. In the context of intestinal development, TLX2 is a downstream target of regulation by both PHOX2A and PHOX2B proteins during neuronal differentiation. Loss-of-function of TLX2 may contribute to abnormal development and possibly play a role in tumorigenesis, particularly in gastrointestinal stromal tumors .

What orthologs of TLX2 exist across species?

TLX2 shows significant sequence homology across various mammalian species. The human TLX2 has approximately 79% sequence identity with both mouse and rat orthologs. This high degree of conservation suggests evolutionarily preserved functions in vertebrate development. The mouse ortholog is also referred to as Hox11L1, Ncx, or Enx in some literature. These orthologs share similar expression patterns and developmental roles, particularly in neural-crest-derived cells that give rise to autonomic nervous system neurons .

What expression systems are optimal for producing Recombinant Mouse TLX2?

For producing Recombinant Mouse TLX2, cell-free protein synthesis (CFPS) systems have proven effective, particularly those derived from Nicotiana tabacum (tobacco). The ALiCE® expression system, based on lysates obtained from tobacco, contains all the necessary protein expression machinery to produce even difficult-to-express proteins like transcription factors. This approach offers advantages over traditional cell-based systems, particularly for proteins that might be toxic to host cells. For researchers planning expression experiments, it's advisable to include appropriate purification tags such as Strep-Tag for one-step affinity chromatography purification. Alternative systems including bacterial (E. coli), insect, or mammalian expression platforms may be considered based on the specific experimental requirements and downstream applications .

What purification strategies work best for Recombinant Mouse TLX2?

The most effective purification strategy for Recombinant Mouse TLX2 involves one-step affinity chromatography utilizing tags engineered into the recombinant construct. Strep-Tagged TLX2 can be efficiently purified using Strep-Tactin columns, which provide high specificity and yield. For researchers dealing with inclusion bodies, denaturation with agents like urea followed by on-column refolding might be necessary. Consideration should be given to buffer composition, particularly pH and salt concentration, to maintain protein solubility and activity. Quality control steps should include SDS-PAGE analysis to verify purity and Western blotting to confirm identity. For applications requiring higher purity, secondary purification steps such as ion exchange chromatography or size exclusion chromatography may be employed .

How can researchers validate the activity of purified Recombinant Mouse TLX2?

Validating the activity of purified Recombinant Mouse TLX2 requires multiple complementary approaches:

• DNA-binding assays: Electrophoretic mobility shift assays (EMSAs) can confirm the ability of TLX2 to bind its target DNA sequences.

• Reporter gene assays: Constructs containing TLX2-responsive elements driving reporter gene expression can be used in cell-based systems.

• Chromatin immunoprecipitation (ChIP): This technique confirms in vivo binding of TLX2 to target promoters.

• Transcriptional activation assays: Measuring increased expression of known target genes following TLX2 addition.

• Functional complementation: Restoring TLX2 function in TLX2-deficient cell lines or animal models.
  Each validation approach provides different insights into TLX2 functionality, and researchers should select methods based on their specific research questions .

What applications is Recombinant Mouse TLX2 commonly used for?

Recombinant Mouse TLX2 is utilized in diverse research applications including:

• ELISA: For detecting antibodies against TLX2 or measuring TLX2 levels in biological samples.

• Western Blotting: As a positive control or for antibody validation studies.

• SDS-PAGE: For protein characterization and quality control.

• Chromatin immunoprecipitation: To study DNA-protein interactions.

• Transcription factor activity assays: To investigate gene regulation mechanisms.

• Blocking experiments: The protein fragment can be used at 100x molar excess to validate antibody specificity in immunohistochemistry, immunocytochemistry, and Western blot applications.

• Cancer research: To investigate the role of TLX2 in tumorigenesis.

• Developmental biology: To study neural crest and autonomic nervous system development .

How stable is Recombinant Mouse TLX2 under laboratory conditions?

Recombinant Mouse TLX2 exhibits moderate stability under standard laboratory conditions, though as a transcription factor, it requires careful handling. The protein should be stored at -80°C for long-term preservation, with the addition of glycerol (10-50%) to prevent freeze-thaw damage. Once thawed, the protein remains relatively stable for 48-72 hours at 4°C. Multiple freeze-thaw cycles should be avoided as they significantly reduce activity. The addition of reducing agents (such as DTT or β-mercaptoethanol) at low concentrations can help maintain disulfide bonds in their native state. Protease inhibitor cocktails are recommended when working with cell lysates or during extended procedures. For applications requiring longer activity periods, researchers should consider the addition of stabilizing agents such as trehalose or bovine serum albumin. Rigorous quality control testing (enzymatic activity assays, binding assays) should be performed before critical experiments .

What is the role of TLX2 in cancer development and progression?

TLX2 demonstrates significant involvement in multiple cancer types with complex and sometimes contradictory roles:

How does TLX2 expression correlate with clinical outcomes in different cancer types?

• High TLX2 expression correlates with poor OS in colorectal adenocarcinoma (HR 1.62; 95% CI 1.09–2.41; P = 0.0172)

• High TLX2 expression correlates with poor OS in kidney renal clear cell carcinoma (HR 1.45; 95% CI 1.07–1.95; P = 0.016)

• High TLX2 expression correlates with poor OS in ovarian cancer (HR 1.34; 95% CI 1.03–1.73; P = 0.029)

• High TLX2 expression correlates with poor OS in uterine carcinosarcoma (HR 2.40; 95% CI 1.21–4.76; P = 0.012)

• Low TLX2 expression correlates with better OS in skin cutaneous melanoma (HR 0.71; 95% CI 0.54–0.94; P = 0.015)
  Progression-Free Survival (PFS) Correlations:

• High TLX2 expression correlates with poor PFS in adrenocortical carcinoma (HR 3.10; 95% CI 1.67–5.77; P = 0.0004)

• High TLX2 expression correlates with poor PFS in colorectal adenocarcinoma (HR 1.47; 95% CI 1.03–2.11; P = 0.035)

• High TLX2 expression correlates with poor PFS in rectal adenocarcinoma (HR 2.09; 95% CI 1.07–4.08; P = 0.03)

• High TLX2 expression correlates with poor PFS in uterine carcinosarcoma (HR 3.01; 95% CI 1.54–5.92; P = 0.001)

• Low TLX2 expression correlates with better PFS in sarcoma (HR 0.67; 95% CI 0.48–0.93; P = 0.018)
  These correlations suggest that TLX2 may serve as a valuable prognostic biomarker in multiple cancer types, with potential implications for treatment planning and patient stratification .

What mechanisms underlie TLX2's role in tumor development?

The mechanisms through which TLX2 influences tumor development appear to be multifaceted and context-dependent:

• Genetic Alterations: The most frequent TLX2 alteration in pan-cancer is amplification, suggesting gene dosage effects may contribute to oncogenesis.

• Co-occurrence with Other Genetic Alterations: TLX2 alterations frequently co-occur with alterations in NXF2B, MSLNL, PCGF1, INO80B-WBP1, LBX2-AS1, MRPL53, LBX2, TTC31, WDR54, and WBP1, indicating potential functional interactions in cancer development.

• Epigenetic Regulation: High methylation levels of TLX2 have been observed in 17 different tumor types, suggesting epigenetic silencing may play a role in some contexts.

• Immune System Modulation: TLX2 expression is associated with immune cell infiltration and immune checkpoint genes, potentially affecting tumor immune surveillance.

• Pathway Involvement: Research suggests TLX2 may influence various oncogenic pathways and contribute to chemoresistance mechanisms.

• Competing Endogenous RNA (ceRNA) Networks: A potential ceRNA network involving LINC01010/miR-146a-5p/TLX2 has been proposed in ovarian cancer, suggesting complex regulatory interactions at the RNA level.
  These diverse mechanisms highlight TLX2's complex role in cancer biology and suggest it may represent a valuable therapeutic target for various human cancers .

How do mutations in TLX2 affect its function in neural development?

Mutations in TLX2 can significantly alter its developmental functions, particularly in neural crest-derived tissues:

• Loss-of-function mutations in TLX2 may contribute to abnormal development of the peripheral nervous system, particularly affecting autonomic neurons. These mutations potentially disrupt the transcriptional programs necessary for proper neuronal differentiation.

• Regulatory region mutations affecting the binding of upstream regulators like PHOX2B can impair TLX2 expression, as demonstrated by the reduced ability of mutant PHOX2B proteins (associated with congenital central hypoventilation syndrome) to activate TLX2 expression.

• Homeodomain mutations that affect DNA binding capacity would compromise TLX2's ability to regulate downstream target genes critical for neuronal development.

• Protein-protein interaction domain mutations may disrupt TLX2's ability to form functional complexes with co-factors necessary for transcriptional activation or repression.
  The developmental consequences of TLX2 dysfunction may include abnormalities in enteric nervous system formation, potentially contributing to gastrointestinal disorders and predisposing to gastrointestinal stromal tumors through disruption of normal developmental pathways .

What is known about the interaction between TLX2 and PHOX2B?

The interaction between TLX2 and PHOX2B represents a critical regulatory relationship in autonomic nervous system development:

• Transcriptional Regulation: PHOX2B directly regulates TLX2 expression by binding to cell-specific elements in the 5′ regulatory region of the TLX2 gene, as confirmed through transient transfections and electrophoretic-mobility-shift assays.

• In Vivo Confirmation: Chromatin immunoprecipitation assays have verified this protein-DNA interaction occurs in living cells under physiological conditions.

• Functional Consequences: Quantitative real-time PCR experiments demonstrate that PHOX2B overexpression leads to upregulation of endogenous TLX2 mRNA levels.

• Developmental Context: Both genes are expressed in neural-crest-derived cells and are involved in downstream steps of BMP (bone morphogenetic protein) signaling, suggesting a coordinated role in neuronal differentiation programs.

• Pathological Implications: PHOX2B proteins carrying mutations responsible for congenital central hypoventilation syndrome (CCHS) show severe impairment in activating TLX2 expression, suggesting this regulatory relationship has clinical relevance.
  This PHOX2B-TLX2 relationship represents a key component of the transcription-factor cascade underlying the differentiation of neuronal lineages of the autonomic nervous system during embryogenesis .

What analytical methods are appropriate for studying TLX2 expression in cancer?

Researchers investigating TLX2 expression in cancer contexts should employ a multi-faceted analytical approach:

How can researchers integrate TLX2 expression data with other molecular features?

Integration of TLX2 expression data with other molecular features requires sophisticated multi-omics approaches:

• Correlation with Genomic Instability Markers:

  • Analyze relationships between TLX2 expression and tumor mutational burden (TMB)

  • Assess association with microsatellite instability (MSI) status

  • Apply Wilcoxon test to determine statistical significance of these associations

• Methylation Analysis:

  • Correlate TLX2 expression with TLX2 gene promoter methylation levels

  • Perform regression analysis to quantify methylation-expression relationships

  • Consider TCGA methylation data (450K or EPIC arrays) for pan-cancer analysis

• Immune Microenvironment Integration:

  • Correlate TLX2 expression with immune cell infiltration scores

  • Analyze relationships with immune checkpoint gene expression

  • Apply GSEA (Gene Set Enrichment Analysis) to identify immune-related pathways associated with TLX2 expression

• Pathway Analysis:

  • Identify molecular pathways associated with TLX2 expression

  • Assess potential involvement in chemoresistance mechanisms

  • Apply pathway enrichment analysis tools (KEGG, Reactome, Gene Ontology)

• Regulatory Network Analysis:

  • Construct competing endogenous RNA (ceRNA) networks involving TLX2

  • Example: LINC01010/miR-146a-5p/TLX2 network in ovarian cancer

  • Validate regulatory relationships experimentally

What experimental approaches can validate computational findings about TLX2?

Validating computational findings about TLX2 requires rigorous experimental approaches:

• Cell Line Expression Analysis:

  • Measure TLX2 expression in cancer cell lines compared to normal cell counterparts

  • Use qRT-PCR for mRNA quantification

  • Perform Western blotting for protein-level confirmation

  • Example: TLX2 expression was significantly upregulated in ovarian cancer cell lines compared to ovarian epithelial cell lines

• Functional Studies:

  • Conduct knockdown experiments using siRNA or shRNA against TLX2

  • Perform overexpression studies using expression vectors

  • Assess phenotypic effects on:

    • Cell proliferation (MTT/CCK-8 assays)

    • Apoptosis (flow cytometry with Annexin V/PI)

    • Migration and invasion (Transwell assays)

    • Colony formation ability

• Chromatin Immunoprecipitation (ChIP):

  • Confirm direct binding of TLX2 to predicted target genes

  • Validate transcription factor binding sites

  • Quantify enrichment using qPCR or sequencing (ChIP-seq)

• Reporter Gene Assays:

  • Construct reporter plasmids containing promoters of potential TLX2 target genes

  • Measure transcriptional activation/repression in response to TLX2 expression

  • Assess the impact of mutations in key binding sites

• In Vivo Validation:

  • Develop xenograft models with TLX2 manipulation

  • Analyze tumor growth, metastasis, and immune infiltration

  • Correlate with patient data to confirm clinical relevance

How should researchers handle contradictory results in TLX2 studies?

Resolving contradictory results in TLX2 research requires systematic approaches:

• Contextual Analysis:

  • Recognize that TLX2 may have tissue-specific and context-dependent functions

  • For example, high TLX2 expression correlates with poor outcomes in multiple cancers (COAD, KIRC, OC, UCS) but low expression is beneficial in others (SKCM)

  • Analyze the biological context (tissue type, developmental stage, disease state) that might explain these differences

• Methodological Evaluation:

  • Assess differences in experimental methodologies:

    • Sample preparation techniques

    • Detection methods (antibodies, primers, probes)

    • Normalization strategies

    • Statistical approaches

  • Standardize protocols when possible to improve comparability

• Sample Heterogeneity Consideration:

  • Evaluate tumor heterogeneity within and between samples

  • Consider single-cell approaches to resolve cell-type specific effects

  • Stratify analyses by molecular subtypes or clinical parameters

• Integrated Data Analysis:

  • Combine multiple data types (genomic, transcriptomic, proteomic)

  • Employ meta-analysis techniques to synthesize findings across studies

  • Apply machine learning approaches to identify patterns in complex datasets

• Collaborative Validation:

  • Engage in multi-laboratory validation studies

  • Establish consensus protocols and reporting standards

  • Create centralized repositories for TLX2-related data to facilitate comparison

What tools and resources are most valuable for TLX2 research?

Researchers investigating TLX2 can leverage various tools and resources: