Recombinant Mouse Zinc finger protein ZIC 5 (Zic5)

What is the structural organization of mouse ZIC5 protein and how does it compare to other ZIC family members?

Mouse ZIC5 is a transcription factor characterized by five conserved C2H2-type zinc finger domains that enable DNA binding and transcriptional regulation. Like other ZIC family proteins, these zinc fingers are highly conserved and constitute the primary functional domain. ZIC5 is located on chromosome 13 in a divergently transcribed gene pair with the closely related ZIC2, suggesting they share regulatory elements and have similar expression patterns .

For experimental characterization of ZIC5 structure:

• Use bioinformatic tools to compare amino acid sequences of ZIC family members

• Employ circular dichroism spectroscopy to assess secondary structure elements

• Utilize X-ray crystallography or NMR spectroscopy for detailed structural analysis of the zinc finger domains

• Apply chromatin immunoprecipitation sequencing (ChIP-seq) to identify DNA binding motifs

What are the primary developmental roles of ZIC5 in mouse models?

Mouse ZIC5 plays crucial roles in early development, particularly in neural crest formation and neural tube development. Loss-of-function studies in mice demonstrate that Zic5 knockout results in multiple developmental abnormalities including:

• Neural crest defects affecting craniofacial development

• Neural tube closure defects leading to malformations

• Hydrocephaly development

• Skeletal abnormalities

These phenotypes indicate ZIC5's importance in early embryonic patterning. For studying these roles:

• Generate or obtain conditional knockout models to examine tissue-specific functions

• Use lineage tracing with Zic5-Cre drivers to map developmental contributions

• Implement CRISPR-Cas9 genome editing for precise mutations that mimic human variants

• Apply single-cell RNA sequencing to identify cell populations affected by ZIC5 deficiency

How can I validate the specificity of anti-ZIC5 antibodies for mouse studies?

Antibody validation is critical for reliable ZIC5 research. Recommended validation methods include:

• Western blot analysis using recombinant mouse ZIC5 protein as a positive control

• Comparison of signal between wild-type and Zic5 knockout tissue samples

• Immunoprecipitation followed by mass spectrometry to confirm target identification

• Peptide competition assays to verify binding specificity

• Cross-reactivity testing against other ZIC family proteins, particularly ZIC2 due to sequence similarity

• Immunohistochemistry comparing known expression patterns with literature findings

When selecting commercial antibodies, request validation data specific to mouse ZIC5 and always include appropriate controls in your experiments.

What are the optimal conditions for expressing and purifying recombinant mouse ZIC5 protein?

Recombinant mouse ZIC5 expression requires careful optimization due to its zinc finger domains. Recommended approach:

Expression system selection:

• E. coli systems (BL21-DE3): Best for producing protein fragments like individual zinc finger domains

• Mammalian expression systems (HEK293T): Superior for full-length protein with proper folding and post-translational modifications

• Baculovirus-insect cell systems: Excellent for larger-scale production with proper folding

Purification protocol:

• Express with appropriate affinity tag (His, GST, or FLAG)

• Include zinc supplementation (typically 100μM ZnCl₂) in all buffers to maintain zinc finger structure

• Use mild detergents and reducing agents to maintain protein stability

• Implement a two-step purification strategy:

  • Initial affinity chromatography

  • Secondary size exclusion or ion exchange chromatography

Quality control measures:

• SDS-PAGE and Western blot to confirm size and identity

• Circular dichroism to verify proper folding

• DNA binding assays to confirm functionality

• Mass spectrometry to verify sequence and modifications

What methods are effective for studying ZIC5 transcriptional activity in neural development?

To study mouse ZIC5 transcriptional activity in neural development:

• Reporter assays: Design luciferase constructs containing putative ZIC5 binding sites. The 15bp sequence (5′-ACAGCCAGCCAATCA) located between -200 and -186bp upstream of Xenopus Zic5 has been identified as a functional Notch response element and can serve as a model for studying ZIC5 regulation .

• ChIP-seq analysis: To identify genome-wide ZIC5 binding sites in neural progenitors or neural crest cells.

• EMSA (Electrophoretic Mobility Shift Assay): Useful for confirming direct DNA binding, as demonstrated in studies of the 15bp Notch response element in the Zic5 promoter .

• RNA-seq following ZIC5 manipulation: Compare transcriptomes after ZIC5 overexpression or knockdown to identify downstream targets.

• Single-cell approaches: Apply scRNA-seq with trajectory analysis to identify ZIC5-dependent developmental transitions in neural progenitor populations.

• In utero electroporation: For spatiotemporal manipulation of ZIC5 expression in developing mouse embryos.

How can I design experiments to study the interactions between ZIC5 and Wnt/β-catenin signaling?

To investigate ZIC5's interactions with the Wnt/β-catenin pathway:

• Co-immunoprecipitation assays: To detect physical interactions between ZIC5 and β-catenin or TCF4. Evidence suggests ZIC5 can strengthen β-catenin-TCF4 associations to stimulate Wnt/β-catenin signaling .

• TOPflash/FOPflash reporter assays: These Wnt-responsive luciferase reporters can measure how ZIC5 overexpression or knockdown affects canonical Wnt signaling output.

• Proximity ligation assays: To visualize and quantify interactions between ZIC5 and Wnt pathway components in situ.

• Domain mapping experiments: Generate truncation or point mutants of ZIC5 to identify regions required for Wnt pathway interaction.

• Functional rescue experiments: Test if ZIC5 overexpression can rescue phenotypes caused by Wnt pathway inhibition and vice versa.

• Context-dependent analysis: Compare ZIC5-Wnt interactions across different cell types relevant to development and disease.

• Chromatin conformation capture (3C/4C/Hi-C): Investigate if ZIC5 influences chromatin architecture at Wnt target gene loci.

How does post-translational modification, particularly SUMOylation, regulate mouse ZIC5 function?

ZIC5 is regulated by SUMOylation, which alters its DNA and protein binding properties to promote neural crest specification . To investigate this regulation:

• Identify SUMOylation sites: Use bioinformatic prediction tools (SUMOplot, GPS-SUMO) followed by site-directed mutagenesis of potential lysine residues.

• In vitro SUMOylation assays: Reconstitute SUMOylation of recombinant ZIC5 using purified E1, E2, and E3 SUMO ligases.

• SUMOylation-deficient mutants: Generate lysine-to-arginine mutations at SUMOylation sites and compare:

  • DNA binding affinity (EMSA, ChIP-seq)

  • Protein interaction patterns (IP-MS)

  • Transcriptional activity (reporter assays)

  • Neural crest induction capacity

• Identification of SUMO-dependent interactome: Compare protein interactions of wild-type versus SUMOylation-deficient ZIC5 using proximity-dependent biotin identification (BioID) or immunoprecipitation coupled with mass spectrometry.

• Temporal dynamics: Analyze SUMOylation patterns during neural development using:

  • Immunoprecipitation with SUMO-specific antibodies

  • Proximity ligation assays to visualize SUMOylated ZIC5 in situ

Research indicates that several other neural crest regulators (Pax6, Sox9, Sox10) are also SUMOylated, suggesting a wider regulatory network where coordinated SUMOylation drives neural crest formation .

What are the mechanisms through which Notch signaling regulates ZIC5 expression in neural development?

The Notch signaling pathway directly regulates ZIC5 expression during neural development. Research has identified:

• Novel Notch Response Element (NRE): A 15bp sequence (5′-ACAGCCAGCCAATCA) located between -200 and -186bp upstream of the Xenopus Zic5 gene functions as an NRE .

• Experimental verification methods:

  • Promoter deletion analysis identified this element as essential for Notch-mediated activation

  • Luciferase reporter assays showed mutation of this element abolished Notch-induced transcriptional activation

  • EMSA confirmed increased protein binding to this element upon NICD (Notch intracellular domain) expression

• To investigate this mechanism in mouse models:

  • Perform comparative genomics to identify conserved NREs in mouse Zic5 promoter

  • Generate reporter constructs with wild-type and mutated mouse NREs

  • Use CRISPR-Cas9 to introduce targeted mutations in the endogenous NRE

  • Apply pharmacological Notch inhibitors (γ-secretase inhibitors) to assess effects on ZIC5 expression

• Ketamine disruption model: Research demonstrates that ketamine disrupts neural crest induction by targeting the Notch-Zic5 signaling pathway . This provides an experimental model to study Notch-ZIC5 interactions in developmental contexts.

How does ZIC5 contribute to cancer progression, and what experimental models are appropriate for studying this role?

ZIC5 is implicated in multiple cancer types with distinct mechanisms:

Experimental models:

Key experimental approaches:

• Modulate ZIC5 expression using shRNA, siRNA, or CRISPR-Cas9 in cancer cell lines

• Perform RNA-seq to identify downstream effectors (like COL1A1)

• Use ChIP-seq to map direct regulatory targets

• Assess phenotypic outcomes: proliferation, migration, invasion, and drug resistance

• Evaluate combinatorial targeting of ZIC5 with standard therapeutics (e.g., enzalutamide for prostate cancer)

How can researchers resolve contradictory findings about ZIC5 function across different model systems?

When confronting contradictory findings about ZIC5 across different experimental systems:

• Evaluate model-specific contexts:

  • Developmental stage differences: ZIC5 may have stage-specific functions

  • Species-specific functions: Compare mouse, Xenopus, and human ZIC5 sequences and expression patterns

  • Cell type specificity: ZIC5 may act differently in neural versus non-neural contexts

• Technical reconciliation approaches:

  • Use multiple knockdown/knockout methods (siRNA, shRNA, CRISPR) to confirm phenotypes

  • Validate with rescue experiments using orthologous genes

  • Implement equivalent experimental conditions across models

• Molecular mechanism resolution:

  • Map interaction partners in each model system using IP-MS

  • Identify context-dependent post-translational modifications

  • Compare chromatin landscapes and accessibility at target genes

• Computational integration:

  • Perform meta-analysis of transcriptomic datasets

  • Use systems biology approaches to model ZIC5 regulatory networks

  • Apply machine learning to identify context-dependent features

What are the best practices for distinguishing direct versus indirect targets of ZIC5 transcriptional regulation?

To rigorously identify direct versus indirect ZIC5 targets:

• Integrated genomic approaches:

  • Combine ChIP-seq (ZIC5 binding) with RNA-seq (expression changes)

  • Focus on genes with both binding evidence and expression changes

  • Use CUT&RUN or CUT&Tag for higher resolution binding data

• Temporal analysis:

  • Implement time-course experiments after ZIC5 induction

  • Early responders (0-4 hours) are more likely direct targets

  • Use transcription inhibitors to block secondary responses

• Motif analysis and validation:

  • Identify enriched DNA motifs in ChIP-seq peaks

  • Validate with reporter assays containing wild-type and mutated motifs

  • Perform EMSA to confirm direct binding to motifs

• Functional genomics:

  • Use CRISPR interference at ZIC5 binding sites to test regulatory function

  • Implement enhancer deletion strategies to validate regulatory elements

  • Perform massively parallel reporter assays to test multiple binding sites

• Single-cell approaches:

  • Apply scRNA-seq with RNA velocity to map regulatory trajectories

  • Use single-cell ATAC-seq to correlate chromatin accessibility with ZIC5 binding

How can discrepancies between in vitro and in vivo findings about ZIC5 function be addressed methodologically?

When in vitro and in vivo ZIC5 studies yield conflicting results:

• Contextual factors to consider:

  • Absence of tissue architecture and cell-cell interactions in vitro

  • Different concentrations of growth factors and signaling molecules

  • Altered chromatin structure in cultured cells

  • Lack of developmental timing cues in vitro

• Bridging experimental approaches:

  • Use 3D organoid cultures as intermediate models

  • Implement ex vivo tissue explants that maintain architectural integrity

  • Develop co-culture systems to recapitulate cellular interactions

• Conditional and inducible systems:

  • Use doxycycline-inducible expression systems in vivo

  • Implement tamoxifen-inducible Cre-loxP systems for temporal control

  • Apply optogenetic or chemogenetic tools for precise manipulation

• Technical validation strategies:

  • Confirm antibody specificity in both contexts

  • Use multiple independent methods to measure the same outcome

  • Apply equivalent analytical pipelines to both datasets

• Molecular context assessment:

  • Compare post-translational modification status between conditions

  • Analyze protein interaction networks in both contexts

  • Evaluate chromatin accessibility and histone modifications

What are the implications of ZIC5's role in the Notch signaling pathway for therapeutic development?

The relationship between ZIC5 and Notch signaling has significant therapeutic implications:

• Neural development disorders:

  • The identification of ZIC5 as a Notch target during neural crest formation provides a mechanistic understanding of neurodevelopmental disorders

  • Ketamine's disruption of the Notch-Zic5 pathway suggests a mechanism for anesthetic neurotoxicity in developing brains

  • Potential therapeutic strategies could include Notch pathway modulation to rescue ZIC5-dependent developmental defects

• Cancer applications:

  • Notch signaling is dysregulated in multiple cancers where ZIC5 is overexpressed

  • Combined targeting of Notch and ZIC5 might provide synergistic therapeutic effects

  • Screening for small molecules that disrupt the Notch-ZIC5 regulatory axis

Experimental approaches:

• Develop high-throughput screening assays using the identified 15bp NRE to identify compounds that modulate this interaction

• Test existing Notch inhibitors (γ-secretase inhibitors) for effects on ZIC5 expression and function

• Investigate combination therapies targeting both Notch and ZIC5-dependent pathways in cancer models

How can single-cell technologies advance our understanding of ZIC5 function in developmental and disease contexts?

Single-cell technologies offer powerful approaches to dissect ZIC5 functions:

• Single-cell RNA sequencing applications:

  • Identify cell populations expressing ZIC5 with unprecedented resolution

  • Map developmental trajectories dependent on ZIC5 expression

  • Discover rare cell populations affected by ZIC5 perturbation

• Multimodal single-cell analysis:

  • scATAC-seq to correlate chromatin accessibility with ZIC5 expression

  • CITE-seq to connect surface marker profiles with ZIC5-dependent states

  • Spatial transcriptomics to map ZIC5 expression in tissue context

• Lineage tracing with single-cell resolution:

  • Implement CRISPR lineage recording systems in ZIC5-expressing cells

  • Use inducible genetic labeling to trace the fate of ZIC5+ progenitors

  • Apply split-pool barcoding to connect clonal relationships with transcriptional states

• Disease applications:

  • Analyze patient samples to identify ZIC5-high cell populations in tumors

  • Map resistance mechanisms in cancer cells following therapy

  • Discover new cellular targets for ZIC5-directed therapeutics

What strategies can be employed to target ZIC5 therapeutically in cancer contexts?

Based on ZIC5's role in cancer progression, several therapeutic strategies emerge:

• Direct targeting approaches:

  • Develop small molecule inhibitors that disrupt ZIC5 DNA binding

  • Design decoy oligonucleotides to compete for ZIC5 binding

  • Implement RNA interference or antisense oligonucleotides to reduce expression

• Pathway-based strategies:

  • In prostate cancer: Target the ZIC5-AR feed-forward loop; research shows ZIC5 inhibition reduces AR and AR-V7 protein expression and enhances sensitivity to enzalutamide treatment

  • In hepatocellular carcinoma: Target ZIC5-COL1A1 axis, as ZIC5 promotes HCC proliferation through COL1A1 upregulation

  • Target Wnt/β-catenin pathway downstream of ZIC5

• Combination approaches:

  • Pair ZIC5 inhibition with standard-of-care therapeutics

  • Combine with immune checkpoint inhibitors to enhance immunogenicity

  • Dual targeting of ZIC5 and epigenetic modifiers

• Biomarker-based patient selection:

  • Implement ZIC5 expression analysis for patient stratification

  • Develop companion diagnostics to identify potential responders

  • Monitor ZIC5-dependent pathways as response biomarkers

Experimental models for therapeutic testing:

• Patient-derived xenografts from ZIC5-high tumors

• Genetically engineered mouse models with tissue-specific ZIC5 overexpression

• 3D tumor organoids for high-throughput drug screening