Recombinant Danio rerio HORMA domain-containing protein 1 (hormad1)

What is HORMAD1 and what is its primary function in zebrafish?

HORMAD1 (HORMA Domain-Containing Protein 1) is a meiosis-specific protein that contains the evolutionarily conserved HORMA domain. In zebrafish (Danio rerio), as in other organisms, HORMAD1 plays critical roles in meiotic processes, particularly in the formation and regulation of the synaptonemal complex and double-strand break (DSB) formation. The protein is specifically expressed in germ cells and is essential for proper meiotic progression .

The primary function of HORMAD1 in zebrafish appears to be closely related to its role in mammals, where it associates with chromosome axes during early meiotic prophase and interacts with other meiotic proteins like IHO1 to recruit DSB-promoting factors such as REC114 and MEI4 . This recruitment is crucial for proper homologous recombination during meiosis.

How is recombinant zebrafish HORMAD1 typically expressed and purified?

Recombinant zebrafish HORMAD1 protein can be expressed using several host systems including E. coli, yeast, baculovirus, or mammalian cell expression systems . The choice of expression system depends on experimental requirements for protein folding, post-translational modifications, and yield.

For purification, the recombinant protein is typically tagged with affinity markers such as His-tag, which facilitates purification through affinity chromatography . Standard purification protocols yield protein with purity greater than 85-90% as determined by SDS-PAGE analysis . The full-length recombinant protein covers amino acids 1-356 of the native zebrafish HORMAD1 .

A typical purification workflow includes:

• Expression in the selected host system

• Cell lysis and clarification of lysate

• Affinity chromatography using the protein tag (e.g., His-tag)

• Optional further purification by ion exchange or size exclusion chromatography

• Purity assessment by SDS-PAGE and/or Western blotting

How does HORMAD1 localization change during meiotic progression in zebrafish?

HORMAD1 localization in zebrafish follows a dynamic pattern during meiotic progression, similar to what has been observed in mammals. Based on research in model organisms including zebrafish:

• Early Meiotic Prophase: HORMAD1 localizes along unsynapsed chromosome axes during leptotene and zygotene stages.

• Mid-Prophase: As homologous chromosomes begin to synapse, HORMAD1 is progressively removed from synapsed regions while remaining on unsynapsed axes.

• Late Prophase: In pachytene spermatocytes with complete synapsis, HORMAD1 is largely depleted from chromosome axes in wild-type cells, but remains abundant in synapsis-defective mutants (e.g., sycp1 mutants) .

The removal of HORMAD1 from synapsed chromosomes is dependent on the formation of the synaptonemal complex and the activity of the AAA-ATPase TRIP13 . In sycp1 mutant zebrafish spermatocytes, where synapsis fails to occur properly, HORMAD1 shows persistent localization along chromosome axes, indicating that synapsis triggers HORMAD1 removal .

What functional insights can be gained from studying HORMAD1 in zebrafish compared to mammalian models?

Zebrafish (Danio rerio) is emerging as a valuable model for studying meiotic recombination with several characteristics similar to those of humans . Studying HORMAD1 in zebrafish offers several advantages:

• Evolutionary Conservation: The HORMA domain in zebrafish HORMAD1 shares significant homology with mammalian counterparts, allowing for comparative functional studies.

• Visual Accessibility: Zebrafish embryonic development occurs externally and transparently, facilitating visualization of cellular processes.

• Genetic Manipulation: Zebrafish are amenable to various genetic manipulation techniques, including CRISPR/Cas9, enabling the creation of specific mutants.

• Complementary Insights: Research in zebrafish can validate and complement findings from mammalian models:

  • In both zebrafish and mice, HORMAD1 is essential for proper chromosome dynamics during meiosis

  • The relationship between HORMAD1 and synaptonemal complex formation appears conserved

  • DSB formation mechanisms involving HORMAD1 interaction with IHO1 and recruitment of REC114/MEI4 are similar

Unique observations in zebrafish sycp1 mutants have shown that despite synapsis failure, axial elements can still form and pair at chromosome ends between homologs during early to mid-zygonema, providing insights into the hierarchical assembly of meiotic chromosome structures .

What methods are most effective for studying HORMAD1 protein interactions in meiosis?

Several complementary methods have proven effective for studying HORMAD1 protein interactions during meiosis:

• Co-immunoprecipitation (Co-IP): Useful for identifying direct protein-protein interactions. Anti-HORMAD1 antibodies can pull down interaction partners such as IHO1, REC114, and MEI4 .

• Yeast Two-Hybrid (Y2H): Can be used to screen for potential binding partners and map interaction domains within HORMAD1.

• Proximity Ligation Assays (PLA): Provides spatial information about protein interactions in situ on meiotic chromosomes.

• Chromatin Immunoprecipitation (ChIP): Identifies genomic regions where HORMAD1 binds, particularly relevant for studying its role in DSB formation.

• Immunofluorescence Microscopy: Essential for visualizing the localization and co-localization of HORMAD1 with other proteins during meiotic progression. This technique has revealed the dynamics of HORMAD1 localization relative to synaptonemal complex formation .

• Mass Spectrometry Analysis: Following immunoprecipitation, can identify both known and novel interacting proteins and post-translational modifications.

When studying recombinant HORMAD1, in vitro binding assays with purified proteins can confirm direct interactions identified through other methods.

How does HORMAD1 regulate double-strand break (DSB) formation during meiosis?

HORMAD1 plays a critical role in regulating meiotic DSB formation through several mechanisms:

• Recruitment of DSB-Promoting Factors: HORMAD1 interacts with IHO1, which subsequently recruits REC114 and MEI4 onto chromosome axes. These proteins are essential components of the meiotic DSB formation machinery .

• Spatial Regulation: By localizing to unsynapsed chromosome axes, HORMAD1 helps ensure that DSBs form in the correct chromosomal regions. In zebrafish, similar to mammals, γH2AX signals (markers of DSBs) and Dmc1/Rad51 and RPA signals (markers of DSB repair) appear predominantly near telomeres, suggesting a specialized regulation of DSB formation and repair at subtelomeric regions .

• Temporal Regulation: The removal of HORMAD1 from synapsed chromosomes correlates with the cessation of new DSB formation, suggesting that HORMAD1 removal provides a mechanism to prevent excessive DSB formation once homologs have synapsed.

• Feedback Control: In sycp1 mutant zebrafish spermatocytes, HORMAD1 shows persistent localization along chromosome axes, which may influence the processing of DSBs when synapsis fails .

This regulatory role is evolutionarily conserved from yeast (Hop1) to mammals, although specific mechanisms may vary. In yeast, Hop1 appears to bind near or at the sites of DSB formation and may modulate the initial DSB cleavage. Hop1 mutants in yeast have reduced numbers of DSBs, highlighting its positive regulatory role .

What is known about the relationship between HORMAD1 and DNA repair pathways in both meiotic and cancer contexts?

HORMAD1's relationship with DNA repair pathways spans both meiotic and pathological contexts:

In Meiosis:

• HORMAD1 promotes meiotic recombination by recruiting DSB-forming machinery .

• It facilitates loading of the RecA homologs RAD51 and DMC1, which are essential for homologous recombination .

• HORMAD1 ensures interhomolog rather than intersister recombination during meiosis.

In Cancer:

• Aberrant expression of HORMAD1 in somatic cells, particularly in triple-negative breast cancers (TNBCs), interferes with normal DNA repair processes.

• Elevated HORMAD1 expression suppresses RAD51-dependent homologous recombination (HR) and drives the use of alternative forms of DNA repair .

• HORMAD1 mediates these effects by suppressing RAD51-dependent HR and driving 53BP1-dependent non-homologous end-joining (NHEJ) .

• This shift in repair pathway choice contributes to the generation of allelic imbalance-associated copy number alterations (AiCNAs) and genomic instability .

Therapeutic Implications:
HORMAD1 positivity correlates with better response to HR defect-targeting agents in TNBC cell lines and clinical trials . This suggests that HORMAD1 status could potentially serve as a biomarker for sensitivity to platinum-based chemotherapy and PARP inhibitors, similar to BRCA1/2 mutations.

What experimental approaches can be used to study the functional consequences of HORMAD1 mutations in zebrafish?

Several advanced experimental approaches can be employed to study the functional consequences of HORMAD1 mutations in zebrafish:

• CRISPR/Cas9 Genome Editing:

  • Generate precise mutations in the zebrafish hormad1 gene

  • Create domain-specific mutations to dissect the function of different protein regions

  • Develop conditional knockout models for temporal control of HORMAD1 expression

• Cytological Analysis:

  • Immunofluorescence microscopy to analyze chromosome dynamics and protein localization

  • Structured illumination microscopy (SIM) or super-resolution microscopy for detailed structural analysis

  • Live cell imaging of tagged proteins to study real-time dynamics

• Molecular Analysis:

  • ChIP-seq to map genome-wide binding sites of wild-type vs. mutant HORMAD1

  • RNA-seq to identify transcriptional changes in hormad1 mutants

  • Single-cell approaches to capture heterogeneity in meiotic progression

• Functional Assays:

  • Analysis of meiotic DSB formation using γH2AX or DMC1/RAD51 foci quantification

  • Measurement of crossover frequency and distribution using genetic markers

  • Assessment of fertility and gamete quality in mutant fish

• Protein Interaction Studies:

  • Comparative interactome analysis between wild-type and mutant HORMAD1

  • Domain mapping to identify critical interaction regions

  • In vitro reconstitution experiments with purified components

• Rescue Experiments:

  • Complementation with wild-type or mutant recombinant HORMAD1 proteins

  • Structure-function analysis through domain swapping or point mutations

  • Cross-species complementation to test functional conservation

These approaches can reveal how HORMAD1 mutations affect meiotic progression, chromosome dynamics, DSB formation and repair, and ultimately fertility in zebrafish, providing insights that may be applicable across species.

How does HORMAD1 function integrate with other meiotic regulatory networks?

HORMAD1 functions at the intersection of multiple meiotic regulatory networks:

• Integration with Synaptonemal Complex Assembly:

  • HORMAD1 localizes to chromosome axes before synaptonemal complex (SC) formation

  • SC component SYCP1 is involved in the removal of HORMAD1 from synapsed chromosomes

  • In sycp1 mutant zebrafish, HORMAD1 shows persistent localization along chromosome axes

• Coordination with DSB Formation and Repair Machinery:

  • HORMAD1 interacts with IHO1, which recruits REC114 and MEI4 to promote DSB formation

  • DSB patterns in zebrafish show predominance near telomeres, suggesting specialized regulation

  • γH2AX signals (markers of DSBs) and Dmc1/Rad51 and RPA signals (markers of DSB repair) appear with similar patterns in both wild-type and sycp1 mutant spermatocytes, despite the absence of synapsis in the latter

• Intersection with Post-Transcriptional Regulation:

  • Meiotic progression requires both DSB formation (involving HORMAD1) and transcript stabilization

  • MEIOC, another meiosis-specific protein, prevents degradation of meiotic transcripts

  • Complete meiotic program induction requires both retinoic acid-dependent and -independent mechanisms

• Connection to Checkpoint Pathways:

  • HORMAD1 may contribute to meiotic checkpoint activation when synapsis or recombination is defective

  • Persistent HORMAD1 on unsynapsed axes may signal to checkpoint proteins

• Evolutionary Conservation of Regulatory Networks:

  • The basic functions of HORMAD1 appear conserved from yeast Hop1 to zebrafish and mammals

  • In yeast, Hop1 phosphorylation by Mec1/Tel1 kinases is important for interhomologue recombination

  • Post-transcriptional regulation involving proteins like MEIOC likely represents an ancestral mechanism, as MEIOC homologues are conserved throughout multicellular animals

Understanding how these networks interact provides insights into the coordinated regulation of meiotic processes and may reveal how defects in one pathway can be compensated by others or result in meiotic failure.

What are the optimal conditions for functional assays using recombinant zebrafish HORMAD1?

When designing functional assays with recombinant zebrafish HORMAD1, researchers should consider these optimal conditions:

Protein Preparation:

• Express full-length protein (AA 1-356) with appropriate tags (His-tag is commonly used)

• Ensure >90% purity by SDS-PAGE analysis

• Verify protein activity through limited functional tests before comprehensive assays

• Store in appropriate buffer conditions that maintain protein stability

DNA Binding Assays:

• Use physiological salt concentrations (typically 100-150 mM NaCl)

• Include divalent cations (Mg²⁺, Ca²⁺) at 1-5 mM

• Maintain pH between 7.0-7.5

• Include reducing agents to prevent oxidation of cysteine residues

• Consider including DNA substrates that mimic recombination intermediates

Protein Interaction Assays:

• For co-immunoprecipitation, use mild detergents (0.1% NP-40 or Triton X-100)

• For pull-down assays, use recombinant binding partners (IHO1, REC114, MEI4)

• Consider the presence of DNA in your binding reactions, as some interactions may be DNA-dependent

• Include appropriate controls (GST-only or irrelevant proteins)

Enzymatic Assays:

• When testing effects on recombination, include core recombination proteins (RAD51, DMC1)

• Maintain ATP concentrations at 1-5 mM for energy-dependent processes

• Include appropriate cofactors based on the specific activity being measured

How can comparative studies between zebrafish and mammalian HORMAD1 advance our understanding of meiotic processes?

Comparative studies between zebrafish and mammalian HORMAD1 can significantly advance meiotic research through several approaches:

Methodologically, comparative studies should combine genomic, proteomic, and cytological approaches with careful phenotypic analysis to build comprehensive models of HORMAD1 function that span evolutionary distance and reveal conserved principles of meiotic regulation.

What technical challenges exist in studying recombinant HORMAD1 function in vitro and how can they be overcome?

Researchers face several technical challenges when studying recombinant HORMAD1 function in vitro:

• Protein Solubility and Stability Issues:
  Challenge: HORMAD1 may aggregate or misfold during expression and purification.
  Solutions:

  • Use solubility-enhancing tags (MBP, SUMO, etc.)

  • Optimize buffer conditions (pH, salt concentration, additives)

  • Express protein at lower temperatures (16-18°C)

  • Consider co-expression with binding partners

• Post-translational Modifications:
  Challenge: Recombinant protein from bacterial systems lacks meiosis-specific modifications.
  Solutions:

  • Express in eukaryotic systems (yeast, insect cells, mammalian cells)

  • Use phosphomimetic mutations to simulate phosphorylation

  • Employ in vitro modification systems with relevant kinases

• Functional Context:
  Challenge: In vitro systems lack the chromosomal and cellular context of meiosis.
  Solutions:

  • Develop chromatin-based assays with reconstituted nucleosomes

  • Use cell extracts from meiotic cells to provide relevant factors

  • Combine with ex vivo approaches using isolated meiotic nuclei

• Assay Development:
  Challenge: Direct functional readouts for HORMAD1 activity are difficult to establish.
  Solutions:

  • Develop DNA binding assays specific to recombination intermediates

  • Establish protein interaction networks using pull-down or crosslinking approaches

  • Create fluorescence-based sensors for conformational changes

• Reproducibility Issues:
  Challenge: Batch-to-batch variation in protein activity.
  Solutions:

  • Implement rigorous quality control measures

  • Establish activity benchmarks with standardized assays

  • Document and standardize purification protocols

By addressing these challenges through careful experimental design and method optimization, researchers can develop robust in vitro systems to study HORMAD1 function that complement in vivo approaches in model organisms.

How might HORMAD1 functions in zebrafish inform our understanding of human fertility disorders?

Research on HORMAD1 in zebrafish offers several potential insights into human fertility disorders:

• Meiotic Failure Mechanisms:

  • HORMAD1's essential role in proper meiotic progression in zebrafish parallels its function in mammals

  • Understanding how HORMAD1 mutations affect meiosis in zebrafish can inform the molecular basis of certain unexplained infertility cases in humans

  • Zebrafish models can help dissect the relative contributions of synapsis defects versus recombination defects in meiotic arrest

• Diagnostic Applications:

  • Identification of HORMAD1 mutations or expression changes in infertile patients

  • Development of biomarkers based on HORMAD1 function or interacting partners

  • Cytological analyses of patient samples for HORMAD1 localization patterns

• Therapeutic Targets:

  • Zebrafish screens could identify compounds that modulate HORMAD1 function or bypass requirements

  • Potential applications in assisted reproductive technologies

• Cancer Implications:

  • HORMAD1's aberrant expression in cancer contexts, particularly triple-negative breast cancers, suggests connections between meiotic and oncogenic processes

  • Understanding HORMAD1's normal function in zebrafish meiosis may provide insights into its pathological roles

  • HORMAD1 positivity correlates with better response to HR defect-targeting agents, suggesting potential as a biomarker for therapy selection

• Evolutionary Conservation:

  • The conservation of HORMAD1 function across species suggests fundamental mechanisms in meiosis

  • Zebrafish studies can help distinguish between species-specific adaptations and core conserved functions relevant to human fertility

Research approaches should combine detailed phenotypic analysis of zebrafish hormad1 mutants with translational studies examining HORMAD1 variants identified in human infertility patients.

What novel experimental approaches could enhance our understanding of HORMAD1 dynamics during meiosis?

Several cutting-edge approaches could significantly advance our understanding of HORMAD1 dynamics during meiosis:

• Live Cell Imaging Technologies:

  • CRISPR knock-in of fluorescent tags to endogenous HORMAD1 in zebrafish

  • Light-sheet microscopy for 3D visualization of meiotic chromosomes with minimal phototoxicity

  • Single-molecule tracking to follow individual HORMAD1 molecules during meiotic progression

• Spatial Omics Approaches:

  • Proximity labeling (BioID, APEX) to identify proteins near HORMAD1 in specific meiotic stages

  • ChIP-seq with stage-specific isolation of meiotic cells to map genome-wide HORMAD1 binding sites

  • Spatial transcriptomics to correlate HORMAD1 localization with gene expression patterns

• Protein Structure and Interaction Analysis:

  • Cryo-EM structures of HORMAD1 in complex with binding partners or DNA

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Single-molecule FRET to detect conformational changes upon binding to partners or DNA

• Synthetic Biology Approaches:

  • Engineered HORMAD1 variants with optogenetic control of function

  • Reconstitution of minimal HORMAD1-dependent systems in vitro

  • Designed orthogonal systems to test specific mechanistic hypotheses

• Advanced Genetic Manipulation:

  • Inducible degradation systems for temporal control of HORMAD1 levels

  • Base editing for precise mutation of specific residues

  • Combinatorial mutations in HORMAD1 and interacting partners

• Multi-scale Integration:

  • Correlative light and electron microscopy to connect protein localization with ultrastructural features

  • Integration of genomic, proteomic, and imaging data through computational modeling

  • Systems biology approaches to understand HORMAD1 in the context of the entire meiotic program

These approaches, when applied to zebrafish models, can provide unprecedented insights into HORMAD1 dynamics during meiosis, potentially revealing new principles of chromosome biology relevant across species.