Recombinant Danio rerio Protein Jade-1 (phf17), partial

What is Jade-1 protein and what is its significance in zebrafish?

Jade-1 (Gene for Apoptosis and Differentiation in Epithelia; also known as PHF17) is a member of the JADE family of proteins that contain Plant Homeo-domain (PHD) zinc finger domains . In zebrafish (Danio rerio), Jade-1 has been identified as a gene product involved in developmental processes. The zebrafish Jade-1 protein shares significant structural and functional similarities with human Jade-1, making it a valuable model for studying evolutionary conservation of this protein family . The zebrafish ortholog of Jade-1 has been characterized with official gene ID 327437, mRNA reference sequence NM_199805, protein reference sequence NP_956099, and UniProt ID Q803A0 .

What are the structural characteristics of zebrafish Jade-1 protein?

Zebrafish Jade-1, like its human counterpart, contains PHD zinc finger domains that are critical for its function in histone recognition and modification. Based on comparative analysis with human Jade-1, the zebrafish protein likely contains:

• PHD zinc finger domains essential for histone binding and chromatin regulation

• Regions involved in protein-protein interactions with chromatin modifying complexes

• Domains associated with subcellular localization signals

The zebrafish Jade-1 protein shares conserved structural elements with human Jade-1, which is characterized by a PEST domain with multiple serine phosphorylation sites and two PHD zinc fingers that are required for histone acetylation activity .

What expression patterns does Jade-1 show during zebrafish development?

Jade-1 shows specific temporal and spatial expression during zebrafish development. Studies suggest that, similar to its mouse ortholog which is expressed in the embryonic primitive streak, zebrafish Jade-1 is likely expressed during critical developmental stages and in specific tissues including epithelial structures . The expression pattern correlates with its potential roles in cell differentiation, proliferation, and tissue organization during embryogenesis. RT-PCR and in situ hybridization studies have demonstrated tissue-specific expression patterns, suggesting regulated roles in different developmental contexts .

What are the optimal conditions for handling recombinant zebrafish Jade-1 protein?

For optimal handling and storage of recombinant zebrafish Jade-1 protein:

• Short-term storage: Maintain at +4°C in PBS buffer

• Long-term storage: Store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles to prevent protein denaturation

• For experimental use, maintain sterile conditions as the preparation should contain <1.0 EU per μg of protein (as determined by LAL method)

The protein is typically supplied in either liquid form or as a lyophilized powder, with the latter requiring reconstitution in an appropriate buffer before use .

What purification strategies are most effective for isolating recombinant zebrafish Jade-1?

The most effective purification strategies for recombinant zebrafish Jade-1 typically utilize affinity chromatography approaches:

• His-tag affinity purification: Since recombinant zebrafish Jade-1 is commonly produced with a histidine tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ matrices provides high specificity

• Sequential chromatography: For higher purity (>80%), a multi-step purification protocol may be necessary:

  • Initial capture using affinity chromatography

  • Intermediate purification by ion-exchange chromatography

  • Polishing step using size exclusion chromatography

• Quality control: Verification of purified protein using SDS-PAGE, Western blotting, and mass spectrometry ensures identity and integrity

How can researchers verify the functionality of recombinant zebrafish Jade-1 protein?

Functional verification of recombinant zebrafish Jade-1 can be performed through several complementary approaches:

• Histone acetylation assay: Given Jade-1's role in histone modification, in vitro histone acetylation assays using purified nucleosomes or histone peptides can assess enzymatic activity

• Protein-protein interaction studies:

  • Pull-down assays to confirm interaction with known binding partners

  • Co-immunoprecipitation with components of histone acetyltransferase complexes

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) or chromatin immunoprecipitation (ChIP) to verify interaction with chromatin

• Cell-based reporter assays: Transfection of recombinant Jade-1 into cellular models followed by analysis of transcriptional activation/repression of target genes

How does zebrafish Jade-1 compare functionally to mammalian orthologs in chromatin modification studies?

Comparative functional analysis between zebrafish and mammalian Jade-1 reveals both conserved and divergent aspects:

What experimental approaches can determine the role of Jade-1 in zebrafish embryonic development?

To investigate Jade-1's role in zebrafish embryonic development, researchers can employ several complementary approaches:

• Morpholino-mediated knockdown:

  • Design antisense morpholinos targeting jade1 mRNA

  • Microinject into 1-4 cell stage embryos

  • Analyze developmental phenotypes with quantitative scoring systems

• CRISPR/Cas9 gene editing:

  • Create precise jade1 mutants targeting functional domains

  • Establish stable transgenic lines

  • Perform detailed phenotypic analysis across developmental stages

• Rescue experiments:

  • Co-inject mRNA encoding wild-type or mutant Jade-1 with knockdown reagents

  • Quantify rescue efficiency using standardized phenotypic criteria

  • Compare domain-specific mutants to identify critical functional regions

• Transcriptome analysis:

  • Perform RNA-seq on jade1 mutants at key developmental timepoints

  • Identify differentially expressed genes

  • Conduct pathway enrichment analysis to uncover affected developmental processes

These approaches have revealed roles for Jade family proteins in embryonic development in other model organisms, and similar methodologies can be applied to understand zebrafish-specific functions .

What are the analytical considerations when studying the interaction between zebrafish Jade-1 and the HBO1 histone acetyltransferase complex?

When studying Jade-1/HBO1 interactions in zebrafish, researchers should consider:

• Complex composition analysis:

  • Immunoprecipitation followed by mass spectrometry to identify all complex components

  • Comparison with known human HBO1 complex components (HBO1, JADE1/2/3, ING4/5)

  • Quantitative analysis of stoichiometry among complex members

• Functional biochemical assays:

  • In vitro reconstitution of zebrafish Jade-1/HBO1 complex

  • Histone acetyltransferase assays with defined substrates

  • Analysis of substrate specificity (H4K5, H4K8, H4K12) compared to mammalian complexes

• Structural considerations:

  • The PHD zinc finger domains in Jade-1 are critical for histone binding and complex function

  • Mutations in these domains can serve as negative controls in interaction studies

  • Cross-linking mass spectrometry can map precise interaction interfaces

• Chromatin targeting analysis:

  • ChIP-seq to identify genomic binding sites of Jade-1 and HBO1 in zebrafish cells

  • Integration with transcriptomic data to correlate binding with gene expression

  • Comparison with mammalian data to identify conserved and divergent targets

The functional interaction between Jade-1 and HBO1 is likely conserved between zebrafish and mammals, with both participating in histone acetylation that impacts DNA replication, recombination, and repair processes .

What are common technical challenges when working with recombinant zebrafish Jade-1 and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant zebrafish Jade-1:

• Protein solubility issues:

  • Challenge: Jade-1 contains hydrophobic regions that may affect solubility

  • Solution: Optimize buffer conditions with increased salt concentration (150-300 mM NaCl), add mild detergents (0.01-0.05% Tween-20), or use solubility tags (e.g., MBP, SUMO)

• Maintaining protein stability:

  • Challenge: Degradation during purification and storage

  • Solution: Add protease inhibitors during purification, store at recommended temperatures (-20°C to -80°C for long-term), and avoid repeated freeze-thaw cycles

• Achieving high purity:

  • Challenge: Contaminating proteins affecting functional assays

  • Solution: Implement multi-step purification protocols beyond initial affinity purification to achieve >80% purity as verified by SDS-PAGE and Western blotting

• Confirming proper folding:

  • Challenge: Recombinant protein may not adopt native conformation

  • Solution: Perform circular dichroism (CD) spectroscopy and functional assays to verify that the protein has the correct secondary structure and activity

How can researchers distinguish between direct and indirect effects when studying Jade-1 function in zebrafish models?

Distinguishing direct from indirect effects in Jade-1 functional studies requires rigorous experimental design:

• Structure-function relationship analysis:

  • Generate point mutations in specific functional domains (e.g., PHD fingers)

  • Create truncation variants eliminating specific protein regions

  • Compare phenotypic outcomes to identify domain-specific effects

• Temporal control of gene expression:

  • Employ inducible expression systems (e.g., heat shock promoters, Gal4/UAS)

  • Use photoactivatable morpholinos for stage-specific knockdown

  • Analyze immediate versus delayed effects following Jade-1 manipulation

• Direct target identification:

  • Perform ChIP-seq to identify direct binding sites of Jade-1 on chromatin

  • Correlate with transcriptomic changes following Jade-1 perturbation

  • Identify genes showing both binding and expression changes as likely direct targets

• Rescue experiments with increasing specificity:

  • Rescue with full-length protein versus specific domains

  • Use orthologous proteins from other species to test functional conservation

  • Employ disease-associated mutations to connect to pathological mechanisms

These approaches help create a hierarchy of evidence for direct versus indirect effects in complex developmental contexts.

What considerations should be taken into account when comparing results from zebrafish Jade-1 studies with mammalian systems?

When comparing zebrafish and mammalian Jade-1 studies, researchers should consider:

• Evolutionary constraints and divergence:

  • While core functions are likely conserved, regulatory mechanisms may differ

  • Sequence comparison analysis should focus on functional domains rather than whole-protein identity

  • Synteny analysis can provide context for gene evolution and potential functional shifts

• Experimental system differences:

  • Cell type-specific effects may vary between species

  • Developmental timing differences must be considered when comparing embryonic roles

  • Temperature-dependent effects (zebrafish develop at lower temperatures than mammals)

• Technical considerations:

  • Antibody cross-reactivity issues between species

  • Differences in post-translational modifications affecting protein function

  • Species-specific interaction partners that may alter functional outcomes

• Data interpretation framework:

  • Establish clear orthologs through phylogenetic analysis before making functional comparisons

  • Use multiple functional readouts to establish conservation of mechanism

  • Consider tissue-specific roles, as Jade-1 expression patterns may differ between species

How is zebrafish Jade-1 being used to study connections between epigenetic regulation and developmental processes?

Zebrafish Jade-1 research is emerging as a valuable model for understanding epigenetic contributions to development:

• Developmental epigenomics approaches:

  • Genome-wide mapping of histone modifications in jade1 mutant embryos

  • Integration with transcriptomic data to correlate chromatin states with gene expression

  • Identification of critical developmental gene regulatory networks under Jade-1 control

• Cell lineage-specific epigenetic profiling:

  • Single-cell approaches to map Jade-1 activity across different cell populations

  • FACS-based isolation of specific cell types followed by ChIP-seq or ATAC-seq

  • Correlation of epigenetic states with cell fate decisions in normal and jade1-perturbed contexts

• Transgenerational epigenetic studies:

  • Analysis of potential maternal contribution of Jade-1 protein or mRNA

  • Investigation of epigenetic marks established by Jade-1 that persist across cell divisions

  • Examination of potential environmental effects on Jade-1-mediated epigenetic programming

This research direction benefits from zebrafish advantages including external fertilization, transparent embryos, and rapid development that facilitate real-time observation of epigenetic processes.

What are the emerging techniques for studying the dynamics of Jade-1 protein interactions in live zebrafish embryos?

Cutting-edge approaches for studying Jade-1 dynamics in vivo include:

• Fluorescent protein tagging and advanced microscopy:

  • CRISPR knock-in of fluorescent tags at the endogenous jade1 locus

  • Live imaging using light-sheet microscopy for whole-embryo visualization

  • Super-resolution techniques to visualize subnuclear localization and dynamics

• Optogenetic control of Jade-1 function:

  • Light-inducible protein interaction systems to control Jade-1 complex formation

  • Spatiotemporal control of Jade-1 activity in specific cell populations

  • Real-time monitoring of downstream effects following acute manipulation

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify Jade-1 interaction partners in vivo

  • Tissue-specific expression of proximity labeling constructs

  • Developmental stage-specific analysis of the Jade-1 interactome

• Single-molecule tracking:

  • Quantum dot labeling of Jade-1 protein for long-term tracking

  • Analysis of binding kinetics and residence time on chromatin

  • Correlation with chromatin states and transcriptional activity

These technologies offer unprecedented insights into the dynamic nature of Jade-1 function during development.

How can studies of zebrafish Jade-1 inform our understanding of human diseases associated with chromatin dysregulation?

Zebrafish Jade-1 studies provide translational insights into human disease mechanisms:

Zebrafish offer advantages for high-throughput screening of compounds affecting Jade-1 function and downstream pathways. The conservation of Jade-1 interaction with the HBO1 complex suggests that insights from zebrafish studies may be directly applicable to understanding human diseases involving chromatin dysregulation . Comparative studies between zebrafish and human Jade-1 function can highlight both conserved disease mechanisms and species-specific differences that inform therapeutic strategies.