Recombinant Saguinus labiatus Homeobox protein Hox-D4 (HOXD4)

What is HOXD4 and what is its primary function in biological systems?

HOXD4 is a transcription factor belonging to the homeobox protein family, which plays critical roles in morphogenesis during embryonic development . As a component of HOX gene clusters, HOXD4 contributes to the regulation of cell differentiation and growth along the embryonic anteroposterior axis . HOX genes encode proteins that function as transcription factors, regulating the expression of downstream genes involved in development and cell fate determination. HOXD4 specifically helps establish positional identity during embryogenesis and participates in neural development through complex regulatory mechanisms involving histone modifications and chromatin remodeling .

What is the protein structure of Saguinus labiatus HOXD4?

The Saguinus labiatus (Red-chested mustached Tamarin) HOXD4 protein consists of 255 amino acids . Like other HOX proteins, it contains a highly conserved homeodomain that enables DNA binding and transcriptional regulation. Recombinant forms of this protein are available with a His tag, which facilitates purification without significantly altering its functional properties . While the tamarin-specific HOXD4 structure hasn't been fully elucidated in the provided resources, HOX proteins generally share a common structural organization with N-terminal and C-terminal regions flanking the homeodomain, which is a 60-amino acid DNA-binding motif.

How conserved is HOXD4 across species, and how does Saguinus labiatus HOXD4 compare to human HOXD4?

HOXD4 is highly conserved across species, reflecting its fundamental role in embryonic development. The available recombinant HOXD4 proteins from various species, including human, mouse, chicken, sheep, bonobo, spider monkey, and Saguinus labiatus (Red-chested mustached Tamarin), suggest evolutionary conservation of this protein's structure and function . While the exact sequence homology between tamarin and human HOXD4 isn't specified in the provided information, the conservation of function across primates suggests significant sequence similarity, particularly in the homeodomain region which is crucial for DNA binding and transcriptional activity.

What expression systems are optimal for producing recombinant Saguinus labiatus HOXD4?

For recombinant Saguinus labiatus HOXD4 (AA 1-255) with His tag, yeast expression systems have been successfully employed with purity levels exceeding 90% . This expression system appears optimal for full-length tamarin HOXD4 production. For comparison, human HOXD4 has been expressed in multiple systems: full-length human HOXD4 with N-terminal His tag has been produced in E. coli with >80% purity, while custom variants have been expressed in HEK-293 cells with >90% purity . When selecting an expression system, researchers should consider the intended experimental application, required protein folding, and post-translational modifications needed for functional studies.

What purification strategies are most effective for recombinant HOXD4 proteins?

Recombinant HOXD4 proteins are typically purified using affinity chromatography, leveraging the His tag or GST tag conjugated to the protein . For His-tagged Saguinus labiatus HOXD4, nickel or cobalt-based immobilized metal affinity chromatography (IMAC) would be the primary purification method. Purity levels exceeding 90% have been achieved for recombinant tamarin HOXD4 expressed in yeast . For optimal results, researchers should consider implementing additional purification steps such as size exclusion chromatography to remove potential aggregates or ion exchange chromatography to eliminate host cell proteins with similar affinity to the primary purification resin.

How can researchers verify the activity of purified recombinant HOXD4?

Verification of recombinant HOXD4 activity can be performed through multiple methodologies:

• DNA-binding assays: Electrophoretic mobility shift assays (EMSA) to confirm the protein's ability to bind specific DNA sequences

• Reporter gene assays: Transfection of cells with a HOXD4-responsive promoter driving a reporter gene

• Functional ELISA: As indicated for the recombinant Saguinus labiatus HOXD4, ELISA-based approaches can verify protein activity

• Chromatin immunoprecipitation (ChIP): To demonstrate binding to native genomic targets

• Gene expression analysis: Assessing the impact on downstream target genes after introduction of recombinant HOXD4

For Saguinus labiatus HOXD4 specifically, ELISA-based approaches have been validated and can serve as a primary activity verification method .

How does HOXD4 contribute to embryonic neurodevelopment?

HOXD4 plays essential roles in neurodevelopment, particularly in establishing the hindbrain segmentation pattern. Research shows that HOXD4 (along with other Hox4 paralogue genes) is necessary for the formation of the r6/r7 boundary in the developing hindbrain . During neural induction, HOXD4 expression follows a specific temporal and spatial pattern, with histone modifications typical of transcriptionally active chromatin occurring first at the 3′ neural enhancer and then at the promoter . This sequential activation is critical for proper anteroposterior patterning. HOXD4 can also drive cell segregation in the neuroepithelium through non-cell autonomous mechanisms, contributing to boundary formation and tissue compartmentalization during development .

What is the relationship between chromatin modifications and HOXD4 expression during development?

The relationship between chromatin modifications and HOXD4 expression follows a specific pattern during development:

• Initially, histone modifications typical of transcriptionally active chromatin occur at the 3′ neural enhancer of HOXD4

• Subsequently, these modifications appear at the promoter region

• The sequential distribution of histone modifications is consistent with a spreading of open chromatin, beginning at the enhancer, progressing through intervening sequences, and culminating at the promoter

This pattern mirrors the colinear activation of genes across HOX clusters during development. Neither RNA polymerase II (Pol II) nor CBP associates with the inactive gene. During HOXD4 induction, CBP and RNA Pol II are recruited first to the enhancer and then to the promoter . While CBP association is transient, RNA Pol II remains associated with both regulatory regions, facilitating transcription. This mechanism ensures proper spatial and temporal HOXD4 expression during development.

What are the known target genes and pathways regulated by HOXD4?

While the provided search results don't completely detail the target genes of HOXD4, they offer insights into its regulatory interactions. HOXD4 is involved in the regulation of segmentation in the hindbrain, working in connection with genes like Krox20 and kreisler/Mafb . Research has shown that Hoxb4 and Hoxd4 are not required to regulate these anterior segmentation genes but are essential for suppressing later aspects of segmentation .

The expression pattern of Crabp1 (Cellular retinoic acid-binding protein 1) is affected by HOXD4, as demonstrated in experiments where RA-mediated suppression of boundary stripes of Crabp1 expression was dependent on ectopic expression of Hoxb4 and Hoxd4 . Additionally, given HOXD4's role in cancer progression, it likely regulates genes involved in cell proliferation, apoptosis, and migration pathways, though specific target genes would require further research to identify.

How does HOXD4 expression correlate with ovarian cancer prognosis?

• The mean survival time of low-HOXD4 cases was 98.2 ± 3.5 months

• The mean survival time of high-HOXD4 cases was only 78.5 ± 6.6 months

Multivariate Cox regression analysis confirmed HOXD4 as an independent risk factor for OSC prognosis (P=0.004, HR=3.554, 95% CI: 1.493-8.459) . Cellular and xenograft experiments further validated the oncogenic effect of HOXD4 in OSC. These findings suggest that HOXD4 plays a role in OSC progression and could serve as a prognostic biomarker for this malignancy.

What mechanisms underlie HOXD4's role in cancer progression?

While the exact mechanisms by which HOXD4 contributes to cancer progression are not fully detailed in the provided information, several aspects can be inferred:

• As a transcription factor, HOXD4 likely regulates genes involved in cell proliferation, survival, and invasion

• The correlation between HOXD4 expression and tumor stage in both gliomas and ovarian cancer suggests a role in disease progression rather than just initiation

• Cellular and xenograft experiments have confirmed the oncogenic effect of HOXD4, demonstrating its direct contribution to cancer cell proliferation

• The association with poorer prognosis in multiple cancer types indicates that HOXD4 may promote more aggressive disease phenotypes

Further research is needed to elucidate the specific downstream targets and signaling pathways through which HOXD4 promotes cancer development and progression. Understanding these mechanisms could potentially reveal new therapeutic approaches targeting HOXD4 or its regulated pathways.

What PCR primers are recommended for HOXD4 gene expression analysis?

For HOXD4 gene expression analysis, several validated primer sets targeting different regions of the gene have been documented in research. The table below presents primers that have been successfully used for real-time quantitative PCR:

These primers can be used for various applications including expression analysis, chromatin immunoprecipitation (ChIP) studies, and monitoring HOXD4 expression in different experimental conditions . For Saguinus labiatus-specific HOXD4 expression analysis, researchers would need to design custom primers based on the tamarin HOXD4 sequence, aligning with conserved regions when compared to the well-characterized human or mouse sequences.

What experimental approaches are effective for studying HOXD4's role in cancer?

Based on the research presented in the search results, several experimental approaches have proven effective for studying HOXD4's role in cancer:

• Expression Analysis:

  • Real-time quantitative PCR to measure HOXD4 mRNA levels in tumors versus normal tissues

  • Immunohistochemistry to detect protein expression patterns and localization in tissue samples

• Clinical Correlation Studies:

  • Analysis of HOXD4 expression in patient samples correlated with clinical parameters (stage, grade) and survival outcomes

  • Multivariate analysis to determine if HOXD4 is an independent prognostic factor

• Functional Studies:

  • Cell line models with HOXD4 overexpression or knockdown to study effects on proliferation, migration, and invasion

  • Xenograft models in nude mice to assess the impact of HOXD4 manipulation on tumor growth in vivo

• Mechanistic Studies:

  • Chromatin immunoprecipitation to identify direct genomic targets of HOXD4

  • Transcriptome analysis to identify differentially expressed genes following HOXD4 modulation

  • Protein interaction studies to identify HOXD4 cofactors in cancer cells

These approaches provide a comprehensive framework for investigating HOXD4's role in cancer initiation, progression, and as a potential therapeutic target.

How can researchers effectively use recombinant HOXD4 protein in epigenetic studies?

Recombinant HOXD4 protein can be effectively utilized in epigenetic studies through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP) Assays:

  • Use recombinant HOXD4 as a positive control or for generating antibodies for ChIP experiments

  • Study the sequential histone modifications at HOXD4 regulatory regions during development or in disease states

  • Investigate the recruitment of RNA polymerase II and CBP to enhancer and promoter regions

• DNA-Protein Interaction Studies:

  • Employ electrophoretic mobility shift assays (EMSA) with recombinant HOXD4 to identify DNA binding sites

  • Use DNA-protein pull-down assays to isolate and identify HOXD4-interacting genomic regions

• Histone Modification Analysis:

  • Study how HOXD4 binding affects histone modifications at target genes

  • Investigate the relationship between HOXD4 expression and chromatin state transitions

• 3D Chromatin Structure Analysis:

  • Examine how HOXD4 influences chromatin looping and 3D genome organization

  • Study the role of HOXD4 in establishing or maintaining topologically associating domains (TADs)

The research has demonstrated that during HOXD4 induction, histone modifications typical of transcriptionally active chromatin occur first at the enhancer and then spread to the promoter . This sequential process provides an excellent model system for studying epigenetic regulation of gene expression using recombinant HOXD4 protein.

What are promising therapeutic approaches targeting HOXD4 in cancer?

Given HOXD4's overexpression and correlation with poor prognosis in multiple cancer types, including gliomas and ovarian cancer , several therapeutic approaches targeting HOXD4 could be explored:

• Small Molecule Inhibitors:

  • Development of compounds that disrupt HOXD4 binding to DNA or essential cofactors

  • Targeting the homeodomain-DNA interaction specifically

• Gene Silencing Approaches:

  • siRNA or shRNA delivery systems to downregulate HOXD4 expression

  • CRISPR-Cas9 mediated gene editing to disrupt HOXD4 function in tumors

• Peptide-Based Therapies:

  • Designing peptides that mimic HOXD4 interaction domains and act as competitive inhibitors

  • Developing peptide-drug conjugates for targeted delivery to cancer cells

• Epigenetic Modifiers:

  • Compounds that alter the chromatin state at the HOXD4 locus to repress its expression

  • Targeting the sequential histone modifications observed during HOXD4 activation

• Combination Therapies:

  • Pairing HOXD4 inhibition with standard chemotherapies or other targeted therapies

  • Exploiting synthetic lethality with other genetic alterations common in HOXD4-expressing tumors

The development of such approaches would require further investigation into the precise mechanisms by which HOXD4 promotes cancer progression and identification of its key interaction partners and downstream targets.

How might comparative studies of HOXD4 across species inform human disease research?

Comparative studies of HOXD4 across species, including examination of the Saguinus labiatus variant, could provide valuable insights for human disease research:

• Evolutionary Conservation Analysis:

  • Identifying highly conserved domains across species may highlight functionally critical regions of HOXD4 that could be therapeutic targets

  • Understanding species-specific differences might reveal mechanistic insights into HOXD4 function

• Model System Development:

  • Non-human primate models expressing species-specific HOXD4 could provide insights closer to human physiology than rodent models

  • Cross-species functional complementation experiments could reveal shared and divergent functions

• Regulatory Network Comparison:

  • Comparing HOXD4 regulatory networks across species could identify conserved pathways relevant to human disease

  • Species-specific differences in HOXD4 regulation might explain differential susceptibility to certain diseases

• Structure-Function Relationships:

  • Structural comparisons of HOXD4 proteins from different species could inform protein engineering approaches

  • Identifying differences in protein-protein interactions across species could reveal novel regulatory mechanisms

Such comparative studies would benefit from the availability of recombinant HOXD4 proteins from multiple species, including Saguinus labiatus, human, mouse, and other organisms , allowing direct functional and structural comparisons.

What emerging technologies could advance our understanding of HOXD4 function?

Several emerging technologies hold promise for advancing our understanding of HOXD4 function in development and disease:

• Single-Cell Genomics and Epigenomics:

  • Single-cell RNA-seq to map HOXD4 expression at unprecedented resolution

  • Single-cell ATAC-seq or CUT&Tag to profile chromatin accessibility and HOXD4 binding at the single-cell level

  • Spatial transcriptomics to map HOXD4 expression patterns in tissues with spatial context preserved

• CRISPR Technologies:

  • CRISPR activation/repression systems for precise modulation of HOXD4 expression

  • CRISPR screens to identify synthetic lethal interactions with HOXD4 in cancer cells

  • Base editing to introduce specific mutations for structure-function studies

• Advanced Imaging Techniques:

  • Live-cell imaging of HOXD4 dynamics during development using fluorescent tagging

  • Super-resolution microscopy to visualize HOXD4 interactions with chromatin

  • Cryo-electron microscopy for high-resolution structural studies of HOXD4 complexes

• Organoid and Organ-on-Chip Models:

  • Development of 3D organoid cultures to study HOXD4 function in a physiologically relevant context

  • Microfluidic organ-on-chip systems to examine HOXD4's role in tissue-specific contexts

• Proteomics and Interactomics:

  • Proximity labeling techniques to identify HOXD4 interaction partners in living cells

  • Mass spectrometry-based approaches to map post-translational modifications on HOXD4