Recombinant Danio rerio Histone-binding protein RBBP7 (rbbp7)

What is the basic structure of zebrafish RBBP7 protein?

RBBP7 is a nuclear protein characterized by a 6-repeat WD40 domain structure. The protein forms a characteristic 7-bladed β-propeller structure with a protruding N-terminal α-helix and a short C-terminal α-helix. The C-terminal region of wild-type RBBP7 typically contains 23 hydrogen bonds that stabilize its structure. This configuration is critical for its function, as the WD40 domains create a platform for protein-protein interactions, particularly with histones and chromatin-associated proteins .

For proper experimental design when working with recombinant RBBP7, researchers should consider that disruption of the C-terminal region (as seen in mutations like p.W401Mfs*5) can significantly alter the protein's structure, reducing hydrogen bonding and eliminating the C-terminal α-helix, thereby affecting its binding capabilities with partner proteins . Experimental approaches targeting specific domains should account for these structural features.

Does zebrafish actually express RBBP7, given conflicting reports in the literature?

This represents an important contradiction in the current literature. Some studies suggest that RBBP7 orthologs are absent in the zebrafish genome. For instance, investigation into zebrafish genes encoding Polycomb Repressive Complex (PRC) subunits indicates that certain genes coding for PRC components, including RBBP7, appear to be absent .

• Incomplete genome annotation in earlier studies

• Different naming conventions across research groups

• Difficulties in identifying true orthologs versus paralogs

When designing experiments with zebrafish RBBP7, researchers should verify the gene's presence using multiple bioinformatic approaches, including synteny analysis and protein sequence alignment with human and mouse RBBP7, rather than relying solely on database annotations .

How does RBBP7 interact with histone H4 in zebrafish, and what methodologies can verify this interaction?

RBBP7 interacts with histone H4 through hydrogen bonds, salt bridges, and van der Waals contacts. The protein's C-terminal hydrophilic/charged α-helix extends the N-terminal α-helix and is particularly important for interaction with the key residue Arg35 of histone H4 .

To experimentally verify this interaction in zebrafish models, researchers can employ:

• Co-immunoprecipitation (Co-IP) assays using antibodies against RBBP7 followed by Western blotting for histone H4

• Chromatin immunoprecipitation (ChIP) to identify genomic regions where RBBP7 and histone H4 co-localize

• Fluorescence resonance energy transfer (FRET) microscopy using tagged versions of both proteins

• In vitro binding assays with recombinant proteins

Critically, researchers should include controls testing the binding of mutant RBBP7 versions (especially those affecting the C-terminal domain), as mutations can significantly impair histone H4 interaction. Studies have shown that the loss of RBBP7's C-terminal hydrophilic region disrupts its normal interaction with histone H4, which has functional consequences for chromatin remodeling and gene expression .

What role does RBBP7 play in Polycomb Repressive Complex activity in zebrafish?

RBBP7 serves as a component of Polycomb Repressive Complex 2 (PRC2) in vertebrates, contributing to epigenetic silencing through histone modifications. In zebrafish development, RBBP7's role in PRC2 activity must be considered in the context of genome duplication and subsequent gene loss events that characterize teleost evolution.

While some studies suggest RBBP7 may be absent in zebrafish , the protein's homologs appear to function similarly to their mammalian counterparts in epigenetic regulation when present. When designing experiments investigating PRC2 activity in zebrafish:

• Consider the potential compensatory role of RBBP4, which shares approximately 90% amino acid identity with RBBP7 and may functionally substitute for RBBP7 in zebrafish

• Examine H3K27me3 marks as an indicator of PRC2 activity, as RBBP proteins are determinants of site-specific H3K27 trimethylation across the genome

• Employ ChIP-seq to map genome-wide distribution of PRC2 components and associated histone modifications

The apparent absence of RBBP7 in some zebrafish genetic analyses but not others raises questions about whether functional redundancy exists or whether annotation errors have occurred. Researchers should approach this contradiction by experimentally verifying the presence and activity of RBBP7 or its functional equivalents when studying PRC2 in zebrafish models .

How do mutations in zebrafish RBBP7 affect its role in histone modification and chromatin structure?

Mutations in RBBP7, particularly those affecting the WD40 domains, can significantly disrupt its epigenetic regulatory functions. Research has shown that mutations like p.W401Mfs*5, which affects the last WD40 domain, lead to:

• Altered protein conformation with reduced hydrogen bonding capability

• Loss of the C-terminal hydrophilic region critical for histone interactions

• Disrupted binding to histone H4 and BRCA1

• Changes in downstream histone modifications

When investigating such mutations experimentally, researchers should:

• Perform molecular modeling to predict structural changes (as demonstrated by the 3D modeling revealing reduction from 23 to 8 hydrogen bonds in mutant proteins)

• Conduct protein-protein interaction assays comparing wild-type and mutant RBBP7

• Analyze histone modification patterns using ChIP-seq or similar techniques

• Assess changes in gene expression profiles resulting from altered chromatin states

Additionally, researchers should note that RBBP7 exhibits methyltransferase activity independent of PRC2. RBBP7 can interact with SUV39H1 to specifically methylate H3K9, leading to heterochromatin silencing. RBBP7 also inhibits DNA methyltransferase 1, affecting DNA methylation patterns. Therefore, mutation analysis should include examination of these non-PRC2 functions as well .

What is the significance of RBBP7's interaction with BRCA1 in DNA damage responses in zebrafish cells?

RBBP7 plays a critical role in genome stability through its interaction with BRCA1, which facilitates DNA repair processes. When designing experiments to study this interaction in zebrafish:

• Consider that molecular docking studies show the energy of interaction between the BRCT domain of BRCA1 and wild-type RBBP7 (-10.9 kcal/mol) is stronger than with mutant RBBP7 (-9.5 kcal/mol)

• Expect that disruption of RBBP7-BRCA1 interaction may lead to decreased BRCA1 levels and increased γH2AX, indicating accumulated DNA damage

• Design cell cycle analysis experiments to detect arrest patterns, as RBBP7 deficiency affects cell cycle progression

To methodologically investigate this interaction:

• Use co-immunoprecipitation to verify RBBP7-BRCA1 binding in zebrafish cells

• Employ immunofluorescence to co-localize these proteins during DNA damage responses

• Conduct functional assays measuring DNA repair efficiency after inducing damage (e.g., with radiation or chemical agents) in cells with normal versus depleted RBBP7 levels

• Monitor γH2AX levels as a marker of DNA damage accumulation

Research indicates that RBBP7 disruption results in heightened DNA damage and increased apoptosis in various cell types following genotoxic stress. Additionally, RBBP7 may influence the expression of DNA damage-responsive genes like GADD45. This suggests RBBP7 plays a multifaceted role in maintaining genomic integrity in zebrafish cells, similar to its function in mammalian systems .

What are the optimal methods for generating recombinant Danio rerio RBBP7 protein for in vitro studies?

When producing recombinant zebrafish RBBP7, researchers should consider several methodological approaches:

Expression Systems:

• E. coli-based expression using pET vectors with a 6xHis tag for easy purification

• Insect cell expression (Sf9 or Hi5 cells) using baculovirus for proper eukaryotic post-translational modifications

• Yeast expression systems for complex protein folding

Purification Strategy:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

• Secondary purification via ion exchange chromatography

• Size exclusion chromatography as a polishing step to remove aggregates

Quality Control Measures:

• SDS-PAGE and Western blotting to confirm identity and purity

• Circular dichroism to verify proper protein folding

• Dynamic light scattering to assess homogeneity

• Functional binding assays with histone H4 peptides to confirm activity

When designing the recombinant construct, researchers should consider the critical nature of the C-terminal domain for RBBP7 function. Studies have shown that mutations affecting this region significantly impair protein-protein interactions. Therefore, C-terminal tags may interfere with normal function and should be avoided in favor of N-terminal tags when possible .

For functional verification, researchers should test recombinant RBBP7's ability to bind histone H4 using in vitro binding assays, as this interaction is fundamental to its biological role. Additionally, testing interaction with other known partners like BRCA1 can provide further confirmation of proper folding and function .

What gene knockdown or knockout strategies are most effective for studying RBBP7 function in zebrafish?

Several approaches can be used to manipulate RBBP7 expression in zebrafish, each with distinct advantages and limitations:

Morpholino (MO) Oligonucleotides:

• Advantages: Rapid assessment of gene function during early development

• Limitations: Efficacy limited to about 3 days post-injection; potential off-target and toxicity effects, including activation of the Tp53 pathway

• Methodological note: Co-injection with tp53-MOs can partially limit non-specific effects

CRISPR/Cas9 Gene Editing:

• Advantages: Creates heritable mutations; allows precise modification of specific genomic locations

• Methodology: Design guide RNAs targeting early exons to ensure complete loss of function; validate editing efficiency using T7 endonuclease assays or sequencing

• Analysis approach: Generate homozygous mutant lines to study complete loss-of-function phenotypes

Conditional Knockout Systems:

• For studying RBBP7 in specific tissues or developmental stages

• Options include the Gal4/UAS system or Cre/loxP approaches adapted for zebrafish

siRNA Approaches:

• In cell culture models, siRNA-mediated knockdown of rbbp7 has demonstrated:

  • Reduced cell proliferation

  • Increased apoptosis

  • Cell cycle arrest

• These findings can guide phenotypic analysis in whole-organism studies

Researchers should be aware that discrepancies have been reported between morpholino-mediated and knockout-mediated phenotypes. Therefore, validation of findings using multiple approaches is strongly recommended. Additionally, since RBBP7 is involved in fundamental cellular processes, complete knockout may cause early lethality, necessitating conditional approaches for studying later developmental stages .

How can researchers accurately assess RBBP7's role in cell cycle regulation in zebrafish?

To methodically investigate RBBP7's impact on cell cycle regulation in zebrafish:

Cell-Based Approaches:

• Flow cytometry analysis of zebrafish cell lines (e.g., ZF4) with RBBP7 knockdown to quantify cell cycle phase distribution

• Incorporation of BrdU or EdU to measure S-phase progression

• Immunostaining for phospho-histone H3 to identify mitotic cells

• Live cell imaging with cell cycle reporters (like FUCCI) to track cell cycle dynamics

In Vivo Approaches:

• Transgenic zebrafish expressing fluorescent cell cycle markers in RBBP7 mutant backgrounds

• EdU pulse-chase experiments in embryos to track proliferation rates

• Whole-mount immunostaining for cell cycle markers at different developmental stages

• Lineage tracing to determine if specific cell populations are more affected by RBBP7 loss

Molecular Analysis:

• qRT-PCR and Western blotting to assess expression of key cell cycle regulators

• ChIP-seq to identify genomic regions bound by RBBP7 in relation to cell cycle genes

• RNA-seq to determine transcriptional changes affecting cell cycle in RBBP7-deficient cells

Research has shown that RBBP7 depletion leads to cell cycle arrest and increased apoptosis in various cell types. In mouse spermatogonial and pachytene spermatocyte-derived cells, RBBP7 knockdown resulted in reduced proliferation, higher apoptosis rates, and decreased percentage of cells in S phase. Similar approaches can be applied to zebrafish models to determine conservation of these functions .

How does RBBP7 interact with Polycomb Repressive Complexes during zebrafish development?

RBBP7's role in Polycomb Repressive Complexes (PRCs) during zebrafish development involves complex interactions that regulate gene expression patterns:

RBBP7 in PRC2 Context:

• RBBP7 serves as an auxiliary component of PRC2, contributing to H3K27 trimethylation

• The absence of RBBP7 in zebrafish (if confirmed) may be compensated by RBBP4, which shares approximately 90% amino acid identity

• Functional studies suggest no known differences between RBBP4 and RBBP7 within PRC2, making the potential absence of RBBP7 orthologs in zebrafish unlikely to significantly affect PRC2 function

Developmental Significance:

• PRC2 components regulate cell fate decisions during zebrafish embryogenesis

• The PRC2 complex mediates epigenetic silencing of developmental genes

• Temporal analysis of H3K27me3 patterns can reveal the impact of RBBP7/RBBP4 on developmental gene regulation

Experimental Design Considerations:

• ChIP-seq analysis of H3K27me3 distribution in wild-type versus RBBP4-depleted zebrafish embryos

• RNA-seq to identify genes derepressed in the absence of normal PRC2 function

• Time-course studies to track changes in chromatin modifications during development

• Rescue experiments to determine if overexpression of RBBP4 can compensate for RBBP7 deficiency

The zebrafish model provides unique advantages for studying these interactions, including transparent embryos allowing in vivo imaging of reporter-tagged proteins and rapid development facilitating time-course studies. Researchers should note that the diversity and complexity of PRC complexes appear conserved in zebrafish despite the potential loss of certain components, making it a valid model for studying Polycomb repression during development .

What is the relationship between RBBP7 and DNA damage responses in zebrafish models of cancer?

RBBP7 plays a crucial role in DNA damage response pathways that have significant implications for cancer development and treatment responses in zebrafish models:

Molecular Mechanisms:

• RBBP7 interacts with BRCA1, facilitating DNA repair processes

• Disruption of RBBP7 function leads to decreased BRCA1 levels and increased γH2AX, indicating accumulated DNA damage

• RBBP7 may influence expression of DNA damage-responsive genes such as GADD45

Experimental Approaches:

• Generate zebrafish with tissue-specific RBBP7 depletion using CRISPR/Cas9

• Expose embryos to DNA-damaging agents (e.g., radiation, chemical mutagens)

• Assess DNA repair capacity using comet assays or immunostaining for repair factors

• Monitor tumor development in RBBP7-deficient zebrafish with cancer-predisposing mutations

Cancer Treatment Implications:

• Research in other systems shows that RBBP4 disruption results in heightened DNA damage and apoptosis in glioblastoma cells following temozolomide treatment and radiotherapy

• Similar mechanisms may operate in zebrafish RBBP7, suggesting potential therapeutic vulnerabilities

• Drug sensitivity assays in RBBP7-deficient versus wild-type zebrafish cancer models can reveal treatment-response differences

Data Table: Expected Changes in DNA Damage Response Markers with RBBP7 Depletion

These findings suggest that RBBP7 plays a multifaceted role in maintaining genomic integrity in zebrafish cells. Its aberrant expression could lead to accumulated DNA damage, cell cycle disruption, and increased cancer susceptibility, making it a potential therapeutic target .

How can structural studies of zebrafish RBBP7 inform drug discovery targeting epigenetic regulators?

Structural characterization of zebrafish RBBP7 can provide valuable insights for developing compounds targeting epigenetic regulatory complexes:

Structural Analysis Approaches:

• X-ray crystallography of recombinant zebrafish RBBP7 alone and in complex with binding partners

• Cryo-EM studies of RBBP7 within larger complexes like PRC2

• NMR spectroscopy to analyze protein dynamics and interaction interfaces

• Molecular dynamics simulations to identify druggable pockets

Comparative Structural Biology:

• Analysis of the 3D structure reveals that wild-type RBBP7 contains 23 hydrogen bonds in its C-terminal region

• The WD40 domain creates a characteristic 7-bladed β-propeller structure with key interaction surfaces

• Structure-based comparison between zebrafish and human RBBP7 can identify conserved binding sites versus species-specific features

Drug Development Applications:

• Virtual screening against the histone H4 binding pocket of RBBP7

• Fragment-based drug discovery targeting the interface between RBBP7 and PRC2 components

• Development of protein-protein interaction inhibitors disrupting RBBP7-BRCA1 binding

• Allosteric modulators affecting RBBP7's incorporation into chromatin-modifying complexes

Zebrafish Advantages for Drug Validation:

• Rapid assessment of compound toxicity and efficacy in developing embryos

• Live imaging of fluorescently tagged RBBP7 to monitor drug-induced changes in localization

• High-throughput screening platforms using zebrafish embryos with RBBP7-related phenotypes

Researchers should focus on the critical C-terminal region and WD40 domains when designing potential inhibitors, as these regions mediate key protein-protein interactions. The hydrophobicity/Kyte-Doolittle scale analysis has shown that the C-terminal region contains important hydrophilic residues critical for function .

What comparative genomics approaches can resolve the conflicting reports about RBBP7's presence in the zebrafish genome?

To address contradictions regarding the presence of RBBP7 in zebrafish, researchers should employ comprehensive comparative genomics approaches:

Genomic Analysis Methods:

• Reciprocal BLAST searches using human and mouse RBBP7 sequences against the latest zebrafish genome assembly

• Synteny analysis examining gene order conservation around the putative RBBP7 locus

• Phylogenetic tree construction including RBBP7 and RBBP4 sequences from multiple vertebrate species

• Analysis of conserved non-coding elements that may regulate expression

Experimental Verification:

• PCR amplification and sequencing of candidate RBBP7 genes from zebrafish genomic DNA

• RNA-seq analysis to identify RBBP7 transcripts in different tissues and developmental stages

• Targeted genome sequencing of regions likely to contain RBBP7 genes

• CRISPR-Cas9 targeting of candidate loci followed by phenotypic analysis

Evolutionary Considerations:

• Account for the teleost-specific genome duplication event that may have affected RBBP7 retention

• Examine potential subfunctionalization or neofunctionalization of RBBP4/RBBP7 paralogs

• Consider different naming conventions that may have led to annotation discrepancies

Data Integration:

• Compare experimental findings with latest genome annotations from multiple databases

• Use proteomics data to confirm protein expression

• Analyze functional conservation through rescue experiments with human RBBP7

The conflict in the literature may stem from incomplete genome annotation, as some studies indicate RBBP7 is absent in zebrafish while others report X-linked RBBP7 mutations causing phenotypes similar to those in other vertebrates. Close examination of the relationship between RBBP4 and RBBP7 is crucial, as they share approximately 90% amino acid identity and may have overlapping functions .

How can researchers leverage CRISPR/Cas9 technology to create precise zebrafish models for studying RBBP7 function?

CRISPR/Cas9 technology offers sophisticated approaches for generating zebrafish models to study RBBP7 function:

Strategic Gene Editing Approaches:

• Knockout strategies targeting early exons to ensure complete loss-of-function

• Knock-in of specific point mutations mirroring human disease variants (e.g., p.W401Mfs*5)

• Insertion of epitope tags for tracking endogenous RBBP7 localization and interactions

• Creation of conditional alleles using loxP sites for tissue-specific or temporal control

Guide RNA Design Considerations:

• Target conserved functional domains, particularly WD40 repeats

• Use multiple guide RNAs to increase editing efficiency

• Implement in silico prediction tools to minimize off-target effects

• Design homology-directed repair templates for precise mutations

Validation Methods:

• T7 endonuclease assays to detect CRISPR-induced mutations

• Targeted sequencing to confirm specific edits

• Western blotting to verify protein expression changes

• Functional assays examining histone binding and chromatin modifications

Advanced Applications:

• Prime editing for introducing specific mutations without double-strand breaks

• Base editing for creating specific amino acid changes

• CRISPR activation/interference (CRISPRa/CRISPRi) for modulating RBBP7 expression

• CRISPR screens to identify genetic interactions with RBBP7

Data Table: CRISPR/Cas9 Strategies for RBBP7 Functional Studies

These approaches can help resolve contradictions in the literature regarding RBBP7's presence and function in zebrafish while providing valuable insights into its role in development, epigenetic regulation, and disease processes .

What are the key unresolved questions about RBBP7 in zebrafish that require further investigation?

Despite significant advances in understanding RBBP7 function, several critical questions remain unresolved that warrant further investigation:

• The precise genomic status of RBBP7 in zebrafish requires definitive clarification through comprehensive genomic and transcriptomic analyses to resolve contradictory reports in the literature .

• The extent of functional overlap between RBBP4 and RBBP7 in zebrafish needs to be determined, particularly whether RBBP4 can fully compensate for RBBP7 in epigenetic regulation and development .

• The tissue-specific roles of RBBP7 during zebrafish development remain to be fully characterized, particularly in germ cell development and fertility where mutations have significant impacts in other organisms .

• The contribution of RBBP7 to zebrafish epigenetic landscapes throughout development needs systematic mapping using ChIP-seq and other genome-wide approaches .

• The potential of zebrafish RBBP7 as a therapeutic target for epigenetic modulation requires further exploration, building on structural insights and comparative analyses with human RBBP7 .