Recombinant Danio rerio Polycomb complex protein BMI-1-B (bmi1b)

What is the molecular function of BMI-1-B in Danio rerio?

BMI-1-B functions as a core component of the Polycomb Repressive Complex 1 (PRC1) in zebrafish. Like its mammalian counterpart, it plays critical roles in epigenetic regulation by participating in histone modification, specifically H2A ubiquitination. The protein contains a RING domain that contributes significantly to its binding interactions with other proteins, including transcription factors . BMI-1-B participates in chromatin remodeling processes that regulate gene expression during development and cellular differentiation, particularly in hematopoietic stem cells. The protein's action involves direct interaction with specific DNA elements and recruitment of additional factors that mediate transcriptional repression of target genes .

How does BMI-1-B expression change during zebrafish development?

BMI-1-B expression demonstrates dynamic patterns throughout zebrafish development with tissue-specific regulation. During early embryogenesis, expression is detected at approximately 36 hours post-fertilization (hpf) in regions corresponding to definitive hematopoietic stem cell (HSC) development, including the aorta-gonad-mesonephros (AGM) equivalent region in zebrafish . Expression patterns overlap significantly with hematopoietic markers such as c-Myb and Runx1, reflecting its importance in HSC ontogeny. As development progresses, BMI-1-B expression becomes more restricted to specific tissue compartments where stem cell maintenance is essential. Methodologically, whole-mount in situ hybridization using antisense RNA probes provides the most comprehensive visualization of bmi1b expression patterns across developmental stages.

What phenotypes result from BMI-1-B depletion in zebrafish models?

Morpholino knockdown of BMI-1-B in zebrafish embryos results in significant defects in hematopoietic stem cell development, which phenocopies aspects of Runx1/CBFβ deficiency . Specifically, BMI-1-B knockdown impairs the emergence of definitive HSCs in the AGM region by 36 hpf, as evidenced by reduced c-Myb expression. Unlike mammalian BMI-1 knockout models that demonstrate multiple developmental abnormalities including neurological and skeletal defects , zebrafish BMI-1-B knockdown primarily affects hematopoiesis in early studies. This selective phenotype suggests possible functional redundancy with paralogous genes or maternal contribution of protein during early development. Methodologically, phenotypic assessment should include in situ hybridization for hematopoietic markers, flow cytometric analysis of blood cell populations, and long-term functional assessment of HSC capacity.

What expression systems are optimal for recombinant zebrafish BMI-1-B production?

The following table compares expression systems for recombinant BMI-1-B production:

For optimal expression, the RING domain (amino acids 1-57) should be carefully considered as it contributes significantly to protein-protein interactions but may affect solubility .

What methods are most effective for studying BMI-1-B interactions with other PRC1 components?

To study BMI-1-B interactions with other PRC1 components, multiple complementary approaches should be employed. Pull-down assays using glutathione-S-transferase (GST) fusion proteins have successfully demonstrated direct interactions between mammalian BMI-1 and other factors , suggesting similar approaches would be effective for zebrafish BMI-1-B. Co-immunoprecipitation (co-IP) experiments with tagged versions of BMI-1-B and potential binding partners provide evidence of interactions in cellular contexts.

For more detailed characterization, structural approaches combining X-ray crystallography and NMR spectroscopy are recommended, as they have successfully revealed that the central domain of mammalian BMI-1 adopts an ubiquitin-like (UBL) fold and participates in both protein-protein interactions and homo-oligomerization . Advanced techniques such as proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) can confirm these interactions in vivo.

For genome-wide analysis of BMI-1-B binding sites and co-localization with other PRC1 components, ChIP-seq methodology has proven effective, as demonstrated with Ring1b and Runx1/CBFβ in megakaryocytic cells . When implementing these approaches, researchers should include appropriate controls to account for potential non-specific binding.

How can CRISPR-Cas9 be optimized for generating BMI-1-B mutants in zebrafish?

CRISPR-Cas9 genome editing for generating BMI-1-B mutants in zebrafish requires careful design and validation strategies. For optimal guide RNA (gRNA) design, target the early exons encoding the RING domain (amino acids 1-57) or the central UBL domain involved in protein-protein interactions and homo-oligomerization . Multiple gRNAs targeting different regions should be tested for efficiency.

The following methodological workflow is recommended:

• gRNA design: Use zebrafish-specific design tools that account for genome peculiarities and off-target prediction. Select 3-4 targets with minimal off-target potential.

• Delivery method: Microinjection of Cas9 protein with synthetic gRNAs into one-cell stage embryos provides efficient editing while minimizing toxicity.

• Mutation screening: Employ T7 endonuclease I assay or high-resolution melt analysis for initial screening, followed by Sanger sequencing to characterize specific mutations.

• Founder selection: Choose founders with frameshift mutations that disrupt functional domains rather than in-frame deletions that may retain partial activity.

• Validation: Confirm reduction in BMI-1-B protein levels using Western blot and assess phenotypes related to hematopoietic development, comparing with morpholino knockdown results.

• Off-target analysis: Sequence potential off-target sites predicted by bioinformatic tools to ensure phenotypes are specifically related to BMI-1-B disruption.

When analyzing mutant phenotypes, examine hematopoietic stem cell development at approximately 36 hpf using markers such as c-Myb and Runx1, as these have been shown to be affected by BMI-1-B depletion .

How does BMI-1-B contribute to epigenetic regulation in zebrafish?

BMI-1-B contributes to epigenetic regulation through its role in the PRC1 complex, which mediates histone H2A ubiquitination, a key repressive mark. Research in mammalian systems has demonstrated that BMI-1 regulates expression of the INK4a locus by binding directly to a specific BMI-1-responding element (BRE) . In zebrafish, BMI-1-B likely employs similar mechanisms to regulate target genes during development.

To investigate the epigenetic function of BMI-1-B in zebrafish, researchers should employ:

• ChIP-seq analysis: Identify genome-wide binding sites of BMI-1-B and correlate these with histone modifications, particularly H2A ubiquitination. The methodology should follow established protocols used for Ring1b ChIP-seq, which yielded high-quality data (>56 million filtered reads) in previous studies .

• CUT&RUN or CUT&Tag: These newer methodologies offer higher sensitivity and specificity than traditional ChIP, requiring fewer cells and providing better signal-to-noise ratios.

• RNA-seq following BMI-1-B manipulation: Compare transcriptomes before and after BMI-1-B knockdown or knockout to identify regulated genes. This approach revealed significant overlap between genes regulated by Ring1b and Runx1/CBFβ in megakaryocytic cells .

• ATAC-seq: Assess changes in chromatin accessibility following BMI-1-B depletion to understand how it contributes to chromatin compaction.

Evidence suggests that BMI-1-B likely functions through interactions with both DNA elements and transcription factors like Runx1/CBFβ, as demonstrated by the significant overlap in chromatin occupancy between Ring1b and Runx1/CBFβ (70% of Ring1b peaks were bound by CBFβ) . This points to a model where BMI-1-B is recruited to specific genomic loci through multiple mechanisms.

What is the relationship between zebrafish BMI-1-B and cellular stress response?

The relationship between BMI-1-B and cellular stress response in zebrafish remains an emerging area of research, but evidence from mammalian systems provides important insights. Studies in mice have shown that BMI-1 deficiency leads to increased reactive oxygen species (ROS) and diminished oxidative capacity in thymocytes, resulting in enhanced DNA damage response .

To investigate this relationship in zebrafish, researchers should:

• Measure ROS levels: Compare ROS production in control versus BMI-1-B depleted zebrafish embryos or isolated cells using fluorescent probes such as CM-H2DCFDA or CellROX.

• Assess mitochondrial function: Measure oxygen consumption rates and mitochondrial membrane potential in BMI-1-B deficient cells to determine if mitochondrial dysfunction occurs similar to that observed in mammalian BMI-1 knockout models.

• DNA damage assessment: Quantify markers of DNA damage (γH2AX foci, comet assay) in BMI-1-B depleted cells under normal and stress conditions.

• Stress recovery experiments: Expose BMI-1-B deficient and control embryos to various stressors (oxidative, radiation, heat shock) and assess recovery and survival rates.

• Transcriptomic analysis: Perform RNA-seq on BMI-1-B depleted cells under normal and stress conditions to identify dysregulated stress response pathways.

This line of investigation may reveal whether BMI-1-B in zebrafish, like its mammalian counterpart, plays a role in regulating cellular redox status and stress resistance, which would have significant implications for understanding its function in development and disease.

How do BMI-1-B and Ring1b cooperate in zebrafish hematopoietic stem cell development?

The cooperation between BMI-1-B and Ring1b in zebrafish hematopoietic stem cell (HSC) development represents a critical area for understanding the functional mechanisms of PRC1 complex during embryogenesis. Evidence indicates that both proteins are required for proper HSC emergence and maintenance, with morpholino knockdown of either resulting in similar phenotypes characterized by reduced c-Myb expression in the AGM region at 36 hpf .

To elucidate their cooperative functions, researchers should implement:

• Double knockdown/knockout experiments: Compare phenotypes of individual versus combined BMI-1-B and Ring1b depletion to assess potential synergistic or redundant functions. Analyze HSC markers, cell proliferation, apoptosis, and differentiation capacity.

• Rescue experiments: Attempt to rescue BMI-1-B deficiency phenotypes with Ring1b overexpression and vice versa to determine functional dependency.

• Domain mapping: Generate constructs with mutations in specific domains to determine which regions are critical for BMI-1-B and Ring1b cooperation. The RING domain of BMI-1-B (amino acids 1-57) is particularly important for protein interactions .

• Protein complex analysis: Use quantitative proteomics approaches such as BioID or APEX proximity labeling to identify the composition of PRC1 complexes in the presence or absence of BMI-1-B or Ring1b.

• ChIP-seq co-localization analysis: Determine the genome-wide co-occupancy of BMI-1-B and Ring1b, similar to studies showing significant overlap between Ring1b and Runx1/CBFβ binding sites in megakaryocytic cells .

The H2A ubiquitination activity assay is particularly important, as both BMI-1-B interaction with polyhomeotic proteins and BMI-1-B homo-oligomerization via the UBL domain are necessary for this activity . Researchers should assess how disruption of either protein affects this critical epigenetic mark in developing HSCs.

Why might recombinant BMI-1-B show poor solubility or degradation during expression?

Recombinant BMI-1-B solubility and stability challenges often stem from its structural properties and functional domains. The RING domain (amino acids 1-57) and ubiquitin-like (UBL) domain are known to participate in multiple protein-protein interactions and homo-oligomerization, which can affect proper folding in heterologous expression systems . To overcome these challenges, researchers should consider:

• Optimize expression conditions: Test multiple temperatures (16-30°C), induction strengths, and durations to find conditions that favor soluble expression over inclusion body formation.

• Solubility-enhancing fusion partners: Employ tags known to enhance solubility such as MBP (maltose-binding protein), SUMO, or Thioredoxin, rather than simple affinity tags like His6.

• Co-expression with interacting partners: Co-express BMI-1-B with natural binding partners from the PRC1 complex, as proper complex formation may stabilize the protein.

• Domain-based approach: Express individual domains separately, particularly when structural or interaction studies focus on specific regions. The UBL domain has been successfully expressed and crystallized in previous studies .

• Buffer optimization: Screen multiple buffer compositions, particularly testing different pH values (7.0-8.5), salt concentrations (150-500 mM NaCl), and additives such as glycerol (5-10%) or low concentrations of non-ionic detergents.

• Protease inhibition: Include a comprehensive protease inhibitor cocktail during purification, as BMI-1 proteins may be susceptible to specific proteases. Consider adding EDTA for metalloproteases if compatible with downstream applications.

If expression remains challenging, consider native purification from zebrafish tissues with tagged constructs expressed through transgenesis, though this approach significantly reduces yield.

How can inconsistent phenotypes in BMI-1-B functional studies be explained and addressed?

Inconsistent phenotypes in BMI-1-B functional studies may arise from multiple biological and technical factors. To address these challenges, researchers should implement the following strategies:

• Genetic compensation assessment: Recent research has shown that genetic knockout can trigger compensatory upregulation of related genes. Test for upregulation of paralogous genes (e.g., bmi1a) in knockout models that may not occur in acute knockdown approaches.

• Maternal contribution evaluation: BMI-1-B may have maternal contribution that masks zygotic loss-of-function in early development. Use maternal-zygotic mutants or adult conditional knockouts to address this issue.

• Dosage-dependent effects: Implement partial knockdown with titrated morpholino concentrations or heterozygous mutants to detect dose-dependent phenotypes that may be obscured in complete loss-of-function models.

• Genetic background control: Maintain consistent genetic backgrounds across experiments, as modifier genes can significantly influence phenotypic outcomes of BMI-1-B manipulation.

• Temporal control of gene disruption: Use inducible knockout systems (e.g., Cre-ERT2) to disrupt BMI-1-B function at specific developmental stages, avoiding early compensatory mechanisms.

• Functional redundancy testing: Perform combined knockdown/knockout of BMI-1-B with related genes, particularly other PRC1 components, to uncover redundant functions.

• Stress condition testing: Assess phenotypes under both standard and stress conditions, as BMI-1 has been implicated in stress response in mammalian systems , and phenotypes may only manifest under challenging conditions.

How might studies of BMI-1-B in zebrafish inform understanding of human diseases?

Zebrafish BMI-1-B studies offer significant potential for illuminating human disease mechanisms, particularly in hematological disorders, stem cell dysfunction, and cancer. Future research should focus on:

• Modeling BMI-1 dysregulation in human diseases: Generate zebrafish models with BMI-1-B mutations that mimic those found in human disorders, particularly overexpression models relevant to various cancers where BMI-1 upregulation has been observed .

• Drug discovery applications: Utilize zebrafish BMI-1-B models for high-throughput screening of compounds that modulate its function or restore normal phenotypes in mutant backgrounds, providing potential therapeutic leads for human diseases.

• Regenerative medicine insights: Investigate BMI-1-B's role in tissue regeneration processes unique to zebrafish but relevant to human regenerative medicine, particularly focusing on hematopoietic system recovery after injury.

• Aging and senescence mechanisms: Explore BMI-1-B's role in regulating senescence in zebrafish, as mammalian BMI-1 regulates the INK4a locus critical for cell cycle control and senescence , processes highly relevant to human aging and age-related diseases.

• Comparative epigenetic regulation: Perform comparative studies of BMI-1-B genomic targets in zebrafish versus human BMI-1 targets to identify evolutionarily conserved regulatory networks with disease relevance.

The zebrafish model offers unique advantages for these studies, including rapid development, optical transparency for in vivo imaging, and amenability to genetic manipulation and drug screening at scale. The high conservation of PRC1 function between zebrafish and humans suggests that insights gained from BMI-1-B studies will have direct translational relevance.

What emerging technologies will advance understanding of BMI-1-B function?

Emerging technologies are poised to significantly advance our understanding of BMI-1-B function in zebrafish. Researchers should consider implementing:

• Single-cell multi-omics: Integration of single-cell RNA-seq, ATAC-seq, and proteomics will reveal cell-type-specific functions of BMI-1-B during development and in adult tissues, providing unprecedented resolution of its regulatory networks.

• Live imaging of chromatin dynamics: Techniques like CRISPR-based chromatin imaging will allow visualization of BMI-1-B-mediated chromatin remodeling in real-time during zebrafish development, linking molecular changes to cellular behaviors.

• Spatial transcriptomics: These methods will map BMI-1-B-dependent gene expression changes in their spatial context within intact tissues, revealing how BMI-1-B influences tissue organization and cell-cell interactions.

• Optogenetic and chemogenetic control: Developing tools for precise temporal and spatial control of BMI-1-B activity will enable dissection of its immediate versus long-term functions in specific cell populations.

• Cryo-EM analysis of PRC1 complexes: High-resolution structural studies of complete zebrafish PRC1 complexes containing BMI-1-B will reveal mechanistic details of how these protein assemblies function on chromatin.

• Base editing and prime editing: These precise genome editing technologies will allow introduction of specific BMI-1-B variants to model disease-associated mutations without creating double-strand breaks.

• Chromatin conformation capture technologies: Methods such as Hi-C and its derivatives will reveal how BMI-1-B influences 3D genome organization and enhancer-promoter interactions during development.

These advanced technologies, particularly when used in combination, promise to address fundamental questions about BMI-1-B function that cannot be resolved using traditional approaches alone.