Recombinant Human Class A basic helix-loop-helix protein 9 (BHLHA9)

What is the basic structure and molecular characteristics of human BHLHA9?

BHLHA9 is a class II basic helix-loop-helix (bHLH) transcription factor encoded by a single exon. The protein has a molecular weight of approximately 24 kDa and contains the characteristic structural motif of bHLH transcription factors . The full amino acid sequence includes a DNA-binding domain that is critical for its function in regulating gene expression.

BHLHA9 possesses:

• A single exon open reading frame

• bHLH structural motif necessary for DNA binding and protein dimerization

• Nuclear localization, consistent with its transcriptional regulatory function

• Specific expression patterns in developing limbs

What are the primary biological functions of BHLHA9?

BHLHA9 functions as a transcriptional regulator involved in limb development. It plays a crucial role in:

• Regulation of limb morphogenesis, particularly in the development of autopod structures

• Fine-tuning the expression of regulatory factors governing determination of central limb mesenchyme cells

• Modulation of transcriptional activity through dimerization with other bHLH proteins

• Influence on distal limb development, with mutations or duplications associated with limb malformations

In model organisms, BHLHA9 expression is restricted to the distal limb bud mesenchyme underlying the apical ectodermal ridge (AER), supporting its specialized role in limb development .

How should I design expression studies to accurately detect BHLHA9 in developing tissues?

When designing expression studies for BHLHA9, consider the following methodological approach:

• Tissue selection and timing: Focus on distal limb bud mesenchyme underlying the apical ectodermal ridge (AER), as this is the primary expression domain of BHLHA9 in model organisms . For developmental timing, studies in mouse and zebrafish have shown specific expression windows during embryonic limb formation.

• Detection methodology:

  • Whole mount in situ hybridization is effective for spatial expression pattern analysis in embryonic tissues

  • For mouse studies, use transcript BCO48728

  • For zebrafish studies, use transcript wu:fb99e06

• Controls and validation:

  • Include positive controls for known expression domains

  • Perform parallel studies in multiple model organisms (mouse, zebrafish) to confirm conserved expression patterns

  • Validate with complementary techniques (qRT-PCR, immunohistochemistry)

• Experimental blocking: Group similar experimental units to reduce variability and increase detection power . This is particularly important when studying developmentally regulated genes with temporally restricted expression windows.

What are the optimal conditions for handling and storing recombinant BHLHA9 protein?

Recombinant BHLHA9 protein requires specific handling and storage conditions to maintain stability and functionality:

• Storage conditions:

  • Store at -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles which can degrade protein activity

  • Ship on dry ice to maintain integrity during transport

• Buffer composition:

  • Optimal formulation: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol

  • This formulation helps maintain protein stability and activity

• Working concentrations:

  • Typical preparations should yield >50 μg/mL as determined by microplate BCA method

  • Purity should be >80% as determined by SDS-PAGE and Coomassie blue staining

• Handling precautions:

  • Minimize exposure to room temperature

  • Use appropriate protein handling techniques to avoid contamination

  • Consider adding protease inhibitors for sensitive applications

How does BHLHA9 interact with other transcription factors to regulate gene expression?

BHLHA9 functions through protein-protein interactions with other transcription factors, particularly other members of the bHLH family:

• Dimerization partners:

  • Yeast two-hybrid analysis has identified transcription factors 3, 4, and 12 (members of the E protein/class I family of bHLH transcription factors) as potential dimerization partners for BHLHA9

  • These interactions are critical for the protein's regulatory function

• Transcriptional modulation mechanism:

  • BHLHA9 appears to function as a negative regulator in some contexts

  • In the presence of BHLHA9, the potential of class I bHLH proteins (transcription factors 3, 4, and 12) to activate expression of E-box-regulated target genes is reduced considerably

  • This suggests BHLHA9 may function as a competitive or inhibitory dimerization partner

• DNA binding specificity:

  • The DNA-binding domain is critical for function

  • Mutations affecting the DNA-binding domain eliminate the ability of BHLHA9 to modulate transcription activation by class I bHLH proteins

  • This suggests that proper DNA binding is essential for BHLHA9's regulatory activity

• Regulatory network position:

  • BHLHA9 appears to fine-tune expression of regulatory factors governing determination of central limb mesenchyme cells

  • It functions as an essential player in the regulatory network governing limb morphogenesis in humans

What functional assays can be used to study the effects of BHLHA9 mutations on protein activity?

Several functional assays can effectively evaluate the impact of BHLHA9 mutations on protein activity:

• Transcriptional reporter assays:

  • Use E-box-regulated reporter genes to measure transcriptional activity

  • Compare wild-type BHLHA9 with mutated variants, particularly those affecting the DNA-binding domain

  • This approach has demonstrated that mutations in BHLHA9's DNA-binding domain can completely eliminate its ability to modulate transcription activation by class I bHLH proteins

• Protein-protein interaction studies:

  • Yeast two-hybrid analysis to identify dimerization partners and how mutations affect these interactions

  • Co-immunoprecipitation to validate interactions in cellular contexts

  • Bimolecular fluorescence complementation to visualize interactions in living cells

• In vivo functional studies:

  • Morpholino knockdown in zebrafish embryos followed by phenotypic analysis

  • Studies have shown that morpholino knockdown of bhlha9 in zebrafish results in severely truncated pectoral fins compared to controls at 72 hours post-fertilization (hpf)

  • This approach can be used to test the ability of mutant variants to rescue the phenotype

• DNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) to measure binding to target DNA sequences

  • Chromatin immunoprecipitation (ChIP) to identify genomic binding sites

  • Compare wild-type and mutant BHLHA9 proteins for differences in DNA binding specificity or affinity

How are duplications of BHLHA9 associated with limb malformations?

BHLHA9 duplications have been linked to specific limb malformations through several lines of evidence:

• Clinical associations:

  • Duplications encompassing BHLHA9 are associated with split-hand/foot malformation with long bone deficiency (SHFLD)

  • The phenotype is characterized by ectrodactyly (split-hand/foot malformation) and tibia hemimelia (underdevelopment of the tibia)

• Inheritance pattern:

  • BHLHA9 duplications exhibit non-Mendelian inheritance characterized by:

    • High degree of non-penetrance (not all individuals with the duplication show symptoms)

    • Sex bias in expression (different rates of manifestation between males and females)

    • Variable expressivity within families and even between limbs of a single patient

• Smallest region of overlap (SRO):

  • Genomic analysis of affected families has identified the SRO of pathogenic duplications

  • This region is located between positions 1,117,153-1,128,916 on chromosome 17p13.3

  • BHLHA9 is the only putative gene within this critical interval

• Experimental evidence:

  • Knockdown of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, supporting its role in limb development

  • All morpholino-injected embryos (n=83) showed severely truncated pectoral fins compared to controls at 72 hours post-fertilization

What experimental approaches are best for studying the dose-dependent effects of BHLHA9 in limb development?

To effectively study dose-dependent effects of BHLHA9 on limb development, consider these methodological approaches:

• Gene dosage modulation in animal models:

  • Morpholino knockdown with titrated concentrations in zebrafish embryos

    • Standard effective dose: 5 mM per embryo of 1 mM solution at the one-cell stage

    • Monitor pectoral fin development at 72 hours post-fertilization

  • CRISPR/Cas9-mediated genomic engineering to create precise duplications or deletions

  • Conditional gene expression systems to control BHLHA9 levels spatiotemporally

• Blocking experimental design principles:

  • Group experimental units with similar characteristics to reduce variability

  • This approach enhances the ability to detect dose-dependent responses with fewer samples

  • Standardize developmental staging to minimize variability in temporal gene expression

• Molecular readouts for dose-dependent effects:

  • Quantitative analysis of downstream target gene expression

  • Immunohistochemistry to assess protein gradients in developing limb buds

  • RNA-seq to identify genes whose expression correlates with BHLHA9 levels

• Cellular assays:

  • Measure proliferation rates in the progress zone mesenchyme

  • Assess apoptosis in the AER and underlying mesenchyme

  • These processes are critical for normal limb development and disrupted in BHLHA9-related malformations

How can I optimize my experimental design when studying the effects of BHLHA9 mutations?

To optimize experimental design when studying BHLHA9 mutations, implement these research-proven strategies:

• Statistical power optimization:

  • Reduce experimental variability through appropriate blocking techniques

  • Group similar experimental units together to minimize within-block variability

  • This approach allows for more precise estimates of treatment effects and improves power to detect responses

• Controls selection:

  • Include multiple control types:

    • Wild-type BHLHA9 as positive control

    • Known pathogenic mutations as reference controls

    • Conservative mutations (same amino acid class substitutions) as specificity controls

    • For morpholino experiments, include both uninjected embryos (n≥90) and standard control oligonucleotide-injected embryos (n≥40)

• Phenotypic analysis standardization:

  • Establish clear, quantitative criteria for phenotypic assessment

  • Use blinded scoring to prevent observer bias

  • Implement consistent developmental staging across all experimental groups

• Molecular characterization depth:

  • Analyze multiple molecular pathways potentially affected by BHLHA9 mutations

  • Assess both direct DNA binding alterations and protein-protein interaction changes

  • Evaluate downstream transcriptional effects through RNA-seq or targeted gene expression analysis

What are the critical quality control parameters for producing and validating recombinant BHLHA9 protein?

To ensure high-quality recombinant BHLHA9 protein for research applications, implement these critical quality control parameters:

• Expression system selection:

  • Human cell lines (e.g., HEK293T) provide appropriate post-translational modifications

  • Bacterial systems may be suitable for applications not requiring mammalian modifications

  • Consider the addition of purification and/or epitope tags (e.g., C-Myc/DDK tags)

• Purity assessment:

  • Minimum acceptable purity: >80% as determined by SDS-PAGE and Coomassie blue staining

  • Higher purity requirements (>95%) for structural or interaction studies

  • Validation by mass spectrometry for confirming protein identity

• Functional validation:

  • DNA binding activity assessment through EMSA or similar techniques

  • Protein-protein interaction verification with known binding partners

  • Ability to modulate transcription in reporter assays

• Physical characterization:

  • Confirm predicted molecular weight (24 kDa)

  • Assess proper folding through circular dichroism or thermal shift assays

  • Evaluate oligomeric state through size exclusion chromatography

What are the most promising emerging research directions for understanding BHLHA9's role in developmental regulatory networks?

Several promising research directions are emerging for understanding BHLHA9's role in developmental regulatory networks:

• Genome-wide binding site identification:

  • ChIP-seq studies to identify direct BHLHA9 targets during limb development

  • Integration with existing developmental transcriptomics data

  • Comparative analysis across species to identify evolutionarily conserved targets

• Protein interaction network mapping:

  • Beyond the identified interactions with transcription factors 3, 4, and 12

  • Systematic identification of co-factors that modulate BHLHA9 activity

  • Analysis of how interaction networks change during developmental progression

• Integration with signaling pathways:

  • Exploration of how BHLHA9 interacts with established limb development pathways:

    • Sonic hedgehog (Shh) signaling

    • Fibroblast growth factor (FGF) signaling from the AER

    • Bone morphogenetic protein (BMP) signaling

  • Investigation of how these pathways regulate BHLHA9 expression and activity

• Single-cell approaches:

  • Single-cell RNA-seq to identify cell populations expressing BHLHA9

  • Trajectory analysis to understand how BHLHA9-expressing cells contribute to limb morphogenesis

  • Spatial transcriptomics to map BHLHA9 activity in intact developing limbs

How can contradictory results in BHLHA9 functional studies be reconciled?

When faced with contradictory results in BHLHA9 functional studies, consider these methodological approaches to reconciliation:

• Context-dependent function analysis:

  • BHLHA9 may have different effects depending on:

    • Developmental stage

    • Cell type

    • Presence of specific cofactors

    • Species-specific differences

  • Explicitly test function across different contexts to identify conditional effects

• Dose-dependent effects evaluation:

  • Contradictory results may reflect different expression levels

  • Use titrated expression systems to systematically test dose-dependent effects

  • Consider the possibility of biphasic responses (different effects at low vs. high concentrations)

• Experimental design evaluation:

  • Assess whether contradictory results may stem from experimental design differences

  • Implement blocking strategies to reduce experimental variability

  • Standardize key parameters across studies:

    • Developmental staging

    • Readout methodologies

    • Analysis techniques

• Functional redundancy assessment:

  • Test for compensatory mechanisms involving related bHLH factors

  • Consider combinatorial knockdown/knockout approaches

  • Evaluate whether contradictory results might reflect successful vs. failed compensation