Recombinant Pan paniscus Zinc fingers and homeoboxes protein 1 (ZHX1), partial

What is Zinc fingers and homeoboxes protein 1 (ZHX1)?

ZHX1 is a nuclear homodimeric transcriptional repressor that belongs to the zinc-fingers and homeoboxes (ZHX) family. This protein contains multiple zinc finger domains and homeobox domains that facilitate DNA binding and transcriptional regulation. ZHX1 was first identified through immunoscreening with a monoclonal B92 antibody and has since been characterized as a critical regulator in various biological processes, particularly in cancer development and progression. The protein functions primarily as a transcriptional repressor, modulating the expression of downstream target genes involved in proliferation, apoptosis, and cellular differentiation .

How does ZHX1 from Pan paniscus compare to human ZHX1?

Recombinant Pan paniscus (bonobo) ZHX1 shares high sequence homology with human ZHX1, reflecting their close evolutionary relationship. While the core functional domains are highly conserved between these species, subtle amino acid differences may exist that could affect protein-protein interactions or DNA binding affinity. Researchers typically use recombinant versions with greater than 85% purity as determined by SDS-PAGE analysis . These minor structural variations may provide valuable insights into the evolutionary conservation of ZHX1 function across primates and potentially reveal species-specific regulatory mechanisms.

What expression systems are commonly used for producing recombinant ZHX1?

Recombinant ZHX1 from Pan paniscus and other species can be expressed in multiple host systems including:

Each expression system provides different advantages depending on the research application, with the most physiologically relevant version typically produced in mammalian cells .

How does ZHX1 function as a transcriptional regulator in different cellular contexts?

ZHX1 exhibits context-dependent regulatory functions across different tissue and cell types. As a transcriptional repressor, ZHX1 binds to specific DNA sequences through its zinc finger domains and recruits co-repressor complexes to inhibit gene expression. Research indicates that ZHX1 can form homodimers with itself or heterodimers with other ZHX family members (ZHX2 and ZHX3) through its homeobox domains, potentially expanding its regulatory repertoire. The protein has been shown to interact with the A subunit of nuclear factor-Y (NF-YA) and may regulate cell cycle-related genes. In certain contexts, ZHX1 has been demonstrated to modulate pathways related to stemness, inflammation, epithelial-mesenchymal transition (EMT), and apoptosis, suggesting diverse regulatory mechanisms depending on the cellular environment .

What experimental approaches best elucidate ZHX1 binding partners and transcriptional targets?

Several complementary approaches are recommended to comprehensively identify ZHX1 binding partners and downstream targets:

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq): This technique identifies genome-wide DNA binding sites of ZHX1, revealing direct transcriptional targets. The analysis typically requires a high-quality antibody against ZHX1 or an epitope-tagged version of the protein.

• RNA-sequencing after ZHX1 modulation: Comparing transcriptomes after ZHX1 knockdown or overexpression identifies genes whose expression is influenced by ZHX1, though this approach doesn't distinguish direct from indirect targets.

• Co-Immunoprecipitation coupled with Mass Spectrometry: This method identifies proteins that physically interact with ZHX1, revealing potential co-factors and regulatory partners.

• Proximity Labeling approaches: BioID or APEX2 fused to ZHX1 can identify proximal proteins in living cells, providing insights into the composition of ZHX1-containing complexes.

These approaches together can create a comprehensive map of ZHX1's regulatory network, particularly when conducted across multiple cell types to capture context-dependent interactions .

How do post-translational modifications affect ZHX1 function?

Post-translational modifications (PTMs) of ZHX1 represent an important but understudied aspect of its regulation. Current research suggests that ZHX1 may be regulated by:

Understanding these modifications is crucial as they likely serve as molecular switches that dictate ZHX1's function in different physiological and pathological contexts. Researchers should employ recombinant ZHX1 expressed in mammalian systems when studying PTMs to ensure proper modification patterns .

What are the optimal conditions for maintaining recombinant ZHX1 stability?

Recombinant ZHX1 stability is critical for experimental success. Recommended storage and handling conditions include:

• Storage at -80°C for long-term preservation with minimal freeze-thaw cycles

• Working aliquots can be maintained at -20°C with protease inhibitors

• For experiments, maintain protein in buffers containing:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 150 mM NaCl

  • 10% glycerol as a stabilizer

  • 1 mM DTT or 5 mM β-mercaptoethanol to maintain reduced state

  • 0.1% Nonidet P-40 or Triton X-100 to prevent aggregation

The addition of 0.5-1 mg/ml BSA as a carrier protein can further enhance stability for dilute solutions. Researchers should validate protein integrity by SDS-PAGE before crucial experiments to ensure the protein retains its expected molecular weight and hasn't degraded .

What experimental controls are critical when studying ZHX1 function in vitro?

Robust experimental design for ZHX1 functional studies requires several controls:

• Protein quality controls: Inclusion of denatured protein samples and commercially validated ZHX1 standards to confirm specificity.

• Functional validation controls:

  • Heat-inactivated ZHX1 to confirm activity-dependent effects

  • Mutant versions with disrupted zinc finger or homeobox domains to validate domain-specific functions

  • Competitive binding assays with known ZHX1 interacting partners

• Specificity controls:

  • Related family members (ZHX2, ZHX3) to determine family-specific versus member-specific effects

  • Non-specific DNA binding proteins to distinguish specific transcriptional effects

• Cell-based functional controls:

  • Rescue experiments after knockdown to confirm specificity

  • Dose-response studies to establish concentration-dependent effects

These controls collectively ensure that observed effects are specifically attributable to ZHX1 function rather than experimental artifacts or non-specific activities .

What are the recommended approaches for ZHX1 knockdown and overexpression studies?

Several complementary approaches can be employed for modulating ZHX1 expression in experimental systems:

When using Pan paniscus ZHX1 for cross-species studies, researchers should consider species-specific controls and validation of cross-reactivity with relevant pathway components. For each approach, dose-dependent effects should be carefully documented to establish physiologically relevant experimental conditions .

How does ZHX1 expression vary across different cancer types?

ZHX1 exhibits complex and sometimes contradictory expression patterns across different malignancies:

These divergent patterns suggest context-dependent functions that may be influenced by tissue-specific cofactors, genomic alterations, or microenvironmental cues. Researchers studying ZHX1 should carefully consider these tissue-specific variations when designing experiments and interpreting results .

What is the relationship between ZHX1 and immune cell infiltration in cancer?

ZHX1 expression has been significantly correlated with immune cell infiltration in various cancer types, particularly in lung adenocarcinoma (LUAD):

• Positive correlations with immune infiltrates in LUAD:

  • CD8+ T cells (r=0.247, P=3.31E−08)

  • CD4+ T cells (r=0.099, P=2.97E−02)

  • Macrophages (r=0.204, P=6.05E−06)

  • Neutrophils (r=0.214, P=2.14E−06)

  • Dendritic cells (r=0.2, P=8.08E−06)

• Functional implications:

  • ZHX1 may influence the tumor immune microenvironment through regulation of chemokines and cytokines

  • Correlation with inflammation signatures in acute myeloid leukemia (ρ=0.547, P=0.031)

  • Potential role in modulating immune checkpoint expression

These findings suggest ZHX1 may represent a link between tumor-intrinsic signaling and immune surveillance mechanisms. Researchers investigating ZHX1 in cancer models should consider analyzing immune parameters alongside tumor cell-intrinsic effects to fully understand its functional impact .

How can conflicting reports about ZHX1's role as tumor suppressor versus oncogene be reconciled?

The apparently contradictory roles of ZHX1 across different cancers can be analyzed through several hypothetical frameworks:

• Context-dependent cofactor availability: ZHX1's function may depend on tissue-specific binding partners that dictate whether it activates or represses specific gene sets.

• Dose-dependent effects: Moderate ZHX1 expression might suppress tumor growth while complete loss or overexpression could promote oncogenesis through different mechanisms.

• Cancer evolution stage-specific roles: ZHX1 might function as a tumor suppressor in early carcinogenesis but acquire oncogenic functions during progression or metastasis.

• Pathway-specific regulation: In cholangiocarcinoma, ZHX1 has been shown to potentially act through EGR1, suggesting that downstream effectors may differ between cancer types.

• Technical considerations: Different antibodies, detection methods, or reference controls might contribute to apparently contradictory results.

Researchers investigating these paradoxical functions should design experiments that:

• Compare multiple cancer types within the same experimental system

• Analyze ZHX1 function across cancer progression stages

• Employ unbiased -omics approaches to identify context-specific targets

• Consider heterogeneity within tumor samples that might mask cell population-specific effects

What is the potential of ZHX1 as a therapeutic target?

The therapeutic targeting of ZHX1 represents a complex but promising research direction:

• Targeting approaches:

  • Small molecule inhibitors of ZHX1-DNA interaction

  • Peptide-based disruptors of protein-protein interactions

  • PROTAC approaches for targeted degradation

  • siRNA/antisense oligonucleotides for expression modulation

• Therapeutic considerations:

  • Cancer-type specific strategies required (inhibition for cholangiocarcinoma vs. activation for lung adenocarcinoma)

  • Potential for combination with immunotherapies given immune infiltration correlations

  • Biomarker development for patient stratification

• Challenges:

  • Transcription factors traditionally considered "undruggable"

  • Context-dependent functions requiring precise therapeutic window

  • Potential off-target effects on related zinc finger proteins

Researchers exploring ZHX1 as a therapeutic target should employ comprehensive preclinical models that recapitulate the appropriate tissue microenvironment and immune context to accurately predict therapeutic responses .

How does Pan paniscus ZHX1 compare to other primate ZHX1 proteins in functional studies?

Comparative functional analysis of ZHX1 across primates can provide evolutionary insights into conserved and divergent mechanisms:

Cross-species functional analyses should include detailed comparisons of:

• DNA binding specificity using ChIP-seq or SELEX approaches

• Protein-protein interaction networks through comparative proteomics

• Transcriptional output using RNA-seq after expression in conserved cell systems

These studies may reveal evolutionary adaptations in transcriptional regulation and potentially identify highly conserved regions as critical functional domains that could serve as therapeutic targets .