Recombinant Danio rerio Protein Jade-3 (phf16), partial

What is JADE3 (PHF16) and what are its key structural features in Danio rerio?

JADE3, also called PHF16 (Plant Homeodomain Finger protein 16), is an 823 amino acid protein containing two mid-molecule plant homeodomain (PHD) zinc finger domains. These zinc finger domains have the capacity for binding and recognition of histone modifications . JADE3 is a member of the Jade protein family, which consists of three highly similar proteins (JADE1, JADE2, and JADE3) that function in the HBO1 histone acetyltransferase complex to direct histone acetylation . The PHD domains are critical for JADE3's function, as deletion of either PHD domain affects the protein's activity .

How does JADE3 differ functionally from other members of the Jade protein family?

Despite structural similarities among Jade family proteins, JADE3 exhibits unique functional properties:

• JADE3, but not JADE1 or JADE2, significantly inhibits Influenza A virus (IAV) and activates NF-κB signaling .

• RNA-seq analysis reveals that JADE3 overexpression causes significant upregulation of 1155 genes and downregulation of 1124 genes compared to controls, demonstrating a distinct gene expression profile from JADE2 .

• While JADE2-expressing cells show only modest increases in phosphorylated p65 (a component of NF-κB), JADE3-expressing cells exhibit substantial increases, and JADE1-expressing cells show no increase .

• Although all three JADE proteins participate in the HBO1 complex, speculation about their redundant functions has been challenged by evidence of JADE3's unique antiviral phenotype .

What are the primary functions of JADE3 in Danio rerio?

JADE3 in Danio rerio appears to have several key functions:

• Antiviral activity: JADE3 has demonstrated significant antiviral activity towards influenza A virus, SARS-CoV-2, and murine norovirus, suggesting its antiviral function is not cell-type or virus specific .

• NF-κB pathway activation: JADE3 expression activates the NF-κB signaling pathway, consistent with its antiviral function .

• Histone acetylation: As part of the HBO1 complex, JADE3 directs histone acetylation, particularly of H4, which is important for gene regulation .

• Gene expression regulation: JADE3 significantly alters the expression of thousands of genes, suggesting a broad role in transcriptional regulation .

What molecular biology techniques are commonly used to study JADE3 in zebrafish?

Several molecular techniques are particularly valuable for studying JADE3 in Danio rerio:

• CRISPR/Cas9 genome editing: This technique has been successfully used to generate zebrafish mutants, allowing for functional studies of genes in vivo . Similar approaches could be applied to generate JADE3 mutants.

• PCR amplification: Specific primers can be designed based on zebrafish cDNA sequences, as was done for other genes in Danio rerio .

• Subcellular fractionation: This technique can determine the localization of proteins, as demonstrated with PIH proteins in zebrafish, which were found to be cytoplasmic rather than present in flagella .

• In vitro transcription: Generation of mRNAs for target genes can be performed using designed primers containing RNA polymerase promoter sequences .

• Immunoblot analysis: This can be used to confirm protein expression or the null status of mutants .

How can researchers effectively express and purify recombinant Danio rerio JADE3 protein?

For effective expression and purification of recombinant Danio rerio JADE3:

• Clone the JADE3 gene into an appropriate expression vector, similar to how other zebrafish genes have been subcloned into pCRII-TOPO plasmid for analysis .

• Consider using bacterial expression systems for initial attempts, but be prepared to shift to eukaryotic systems if proper folding of PHD domains becomes an issue.

• Include affinity tags (His, GST, etc.) to facilitate purification while ensuring they don't interfere with the zinc-coordinating PHD domains.

• Optimize expression conditions carefully, as PHD domains require proper zinc coordination for structural integrity.

• Include zinc in buffers throughout purification to maintain PHD domain structure.

• Consider using molecular chaperones to enhance proper folding of complex proteins.

• Verify protein activity through functional assays, such as histone peptide binding or NF-κB activation assays.

What experimental approaches should be used to investigate JADE3's role in antiviral immunity?

To thoroughly investigate JADE3's antiviral function:

• Viral challenge assays: Compare viral replication in JADE3-expressing versus control cells for multiple virus types. The significant antiviral activity of JADE3 has been demonstrated using mNeon reporter IAV at 24 hours post-infection .

• Genetic complementation: In JADE3-knockout cells, restore JADE3 expression to confirm specificity of antiviral effects. Complementation with JADE3 cDNA has been shown to restore IAV titers to levels comparable with parental cell lines .

• Domain mutation analysis: Create JADE3 mutants lacking one or both PHD domains to determine their necessity for antiviral function. Evidence suggests both PHD domains are required for JADE3's antiviral activity .

• Transcriptome analysis: Perform RNA-seq to identify genes regulated by JADE3 that may contribute to antiviral responses. JADE3 overexpression significantly alters gene expression profiles .

• NF-κB pathway investigation: Measure phosphorylation of p65 and expression of NF-κB target genes in the presence or absence of JADE3 .

• Comparative analysis with other JADE proteins: Compare antiviral activities of JADE1, JADE2, and JADE3 to highlight JADE3's unique properties .

How can researchers determine the specific histone modifications regulated by JADE3 in the context of the HBO1 complex?

To investigate JADE3's role in histone modification:

• Chromatin immunoprecipitation (ChIP): Identify genomic regions where JADE3 associates with modified histones.

• Mass spectrometry analysis: Characterize specific histone modifications influenced by JADE3.

• In vitro histone acetyltransferase assays: Reconstitute the HBO1 complex with JADE3 and measure acetyltransferase activity on nucleosome substrates.

• Comparative analysis: The HBO1 complex with JADE proteins directs H4 acetylation, while the complex with BRPF proteins directs H3K14 acetylation . Compare these activities in detail.

• JADE3 mutant analysis: Create JADE3 mutants lacking specific domains and assess their impact on histone modifications.

• Correlative studies: Correlate changes in histone modifications with gene expression changes using RNA-seq data from cells with modified JADE3 expression.

What are the optimal approaches for using CRISPR/Cas9 to study JADE3 function in zebrafish models?

For effective CRISPR/Cas9-based studies of JADE3 in zebrafish:

• Guide RNA design: Design specific gRNAs targeting the JADE3 gene, following successful approaches used for other zebrafish genes. CRISPR/Cas9 has been effectively used to generate zebrafish mutants of genes like ktu and twister .

• Mutation verification: Confirm mutations through sequencing and immunoblot analysis to ensure null status, as was done for PIH protein mutants .

• Phenotypic characterization: Analyze developmental phenotypes, antiviral responses, and gene expression patterns in JADE3 mutant zebrafish.

• Domain-specific mutations: Generate mutations specifically targeting the PHD domains to study their function in vivo.

• Rescue experiments: Reintroduce wild-type JADE3 or domain mutants to confirm phenotype specificity and domain functions.

• Tissue-specific analysis: Examine JADE3 function in different tissues, as gene expression patterns can be tissue-specific in zebrafish .

How should researchers design experiments to investigate JADE3's role in the NF-κB signaling pathway?

To effectively study JADE3's involvement in NF-κB signaling:

• Phosphorylation analysis: Monitor phosphorylation of NF-κB components (particularly p65 at Serine 536) in cells with varying JADE3 expression. JADE3-expressing cells show increased phosphorylated p65, even after treatment with recombinant human TNF-α .

• Inhibitor studies: Use specific inhibitors of the NF-κB pathway to determine whether JADE3's antiviral activity depends on this pathway.

• Protein-protein interaction studies: Identify direct interactions between JADE3 and components of the NF-κB pathway using co-immunoprecipitation or proximity ligation assays.

• Reporter gene assays: Use NF-κB-responsive reporter constructs to quantify pathway activation by JADE3.

• Domain requirement analysis: The antiviral activity of JADE3 and its ability to increase phosphorylated p65 requires both PHD domains, suggesting these domains are essential for NF-κB activation .

• Transcriptome analysis: Identify NF-κB target genes regulated by JADE3 using RNA-seq. JADE3 overexpression causes significant changes in gene expression profiles that may include NF-κB targets .

What challenges might researchers face when studying the PHD domains of JADE3, and how can these be addressed?

When studying JADE3's PHD domains, researchers should consider:

• Structural integrity: PHD domains are zinc finger domains that require zinc for proper folding . Ensure buffers contain appropriate zinc concentrations during protein purification and handling.

• Functional redundancy: Although both PHD domains are required for JADE3's antiviral function , they may have distinct roles in other contexts. Design experiments that can distinguish between the functions of each domain.

• Histone binding specificity: PHD domains can recognize specific histone modifications. Use histone peptide arrays or pull-down assays to determine the binding specificity of each JADE3 PHD domain.

• Structural determination: Consider X-ray crystallography or NMR for structural analysis of JADE3 PHD domains, which can reveal binding mechanisms.

• Mutational analysis: Create point mutations in conserved residues within each PHD domain to identify key functional residues.

• Chimeric proteins: Generate chimeric proteins swapping PHD domains between JADE family members to determine whether domain specificity contributes to JADE3's unique functions.

How can tissue-specific expression patterns of JADE3 in zebrafish be effectively analyzed?

To analyze tissue-specific expression of JADE3 in zebrafish:

• In situ hybridization: This technique can visualize gene expression in specific tissues of zebrafish embryos, as used for DNAH genes which showed distinct expression patterns between embryos and testes .

• RT-PCR analysis: Quantify JADE3 expression in different tissues, similar to how zebrafish embryos and testes showed distinct expression patterns of DNAH genes .

• Transgenic reporter lines: Generate zebrafish lines expressing fluorescent reporters under the JADE3 promoter to visualize expression patterns in vivo.

• Single-cell RNA-seq: This can provide high-resolution data on cell-type-specific expression of JADE3 during development.

• Comparative analysis: Compare JADE3 expression patterns with other genes of interest. For example, in zebrafish, some genes show organ-specific expression patterns, with dnah11 specifically detected in Kupffer's vesicle, while dnah8 and dnah3 were specifically detected in testis .

• Developmental time-course: Analyze JADE3 expression throughout zebrafish development to identify stage-specific expression patterns.

What methods should be used to investigate potential interaction partners of JADE3 in zebrafish?

To identify and characterize JADE3 interaction partners:

• Co-immunoprecipitation: Pull down JADE3 from zebrafish cells or tissues and identify associated proteins by mass spectrometry.

• Yeast two-hybrid screening: Use JADE3 or its domains as bait to screen for interacting proteins.

• Proximity labeling: Use BioID or APEX2 fused to JADE3 to identify proteins in close proximity in vivo.

• Pull-down assays: Use recombinant JADE3 to pull down potential interactors from zebrafish lysates.

• Domain mapping: Determine which domains of JADE3 are required for specific protein interactions.

• Comparative analysis: Compare JADE3 interactome with those of JADE1 and JADE2 to identify unique interaction partners that might explain JADE3's distinct functions .

• Functional validation: Confirm the biological significance of identified interactions through genetic or biochemical approaches.

How can researchers effectively compare JADE3 function between in vitro and in vivo experimental settings?

To compare JADE3 function between in vitro and in vivo settings:

• Parallel experimental design: Develop complementary in vitro and in vivo experiments addressing the same questions about JADE3 function.

• Cell-type considerations: In vitro, use cell types relevant to the in vivo tissues of interest.

• Genetic approaches: Use CRISPR/Cas9 to create comparable JADE3 mutations in cell lines and zebrafish .

• Functional readouts: Develop assays that can be performed both in vitro and in vivo, such as gene expression analysis or protein interaction studies.

• Developmental context: Consider that JADE3 function may be developmentally regulated in vivo, requiring stage-specific analysis.

• Tissue-specific effects: Examine JADE3 function in multiple tissues, as gene expression patterns can be organ-specific in zebrafish .

• Systemic interactions: In vivo studies can reveal systemic effects not observable in cell culture, particularly for proteins involved in immune responses like JADE3 .