Recombinant Chicken Class E basic helix-loop-helix protein 22 (BHLHE22)

What is the structural organization of chicken BHLHE22 and how does it compare to mammalian orthologs?

The BHLHE22 protein (previously called BHLHB5) belongs to the Class II basic helix-loop-helix family of transcription factors. In mammals, it contains approximately 381 amino acids with a molecular weight of 36.9 kD, encoded by a single exon . The protein features several distinct domains including an N-terminal proline-rich domain, a glycine-rich domain, a polyglycine-serine region, the highly conserved helix-loop-helix (HLH) domain, and a C-terminal alanine-rich region .

For investigating chicken BHLHE22 structure, researchers should:

• Perform sequence alignment between chicken and mammalian BHLHE22 to identify conserved domains

• Focus particular attention on the HLH domain, which shows remarkable conservation across vertebrates

• Generate structural predictions using homology modeling based on crystallized mammalian bHLH proteins

• Use circular dichroism spectroscopy to analyze secondary structure elements in recombinant protein

• Compare predicted binding interfaces by examining the basic region preceding the first helix

The HLH domain warrants special attention as it contains two alpha-helices separated by a loop region that mediates dimerization with other proteins, along with a basic region that enables DNA binding to E-box sequences . Evidence from human studies indicates that this domain is highly intolerant to missense variations, suggesting critical functional importance.

What expression patterns does BHLHE22 exhibit in chicken neural tissues during development?

In mammals, BHLHE22 shows highly specific expression, being found exclusively in the retina and central nervous system (CNS) . When investigating chicken BHLHE22 expression, researchers should employ multiple complementary techniques:

• Temporal expression analysis:

  • Quantitative PCR using chicken-specific primers across developmental stages

  • Western blotting across embryonic and post-hatch timepoints

  • RNA-sequencing of neural tissues with temporal resolution

• Spatial expression mapping:

  • In situ hybridization with chicken-specific probes

  • Immunohistochemistry using validated antibodies for chicken BHLHE22

  • Single-cell RNA sequencing to identify cell type-specific expression

• Comparative analysis:

  • Side-by-side comparison with mammalian expression patterns at equivalent developmental stages

  • Special focus on commissural structures, retina, and regions involved in neuronal differentiation

Based on mammalian studies, researchers should examine chicken brain regions equivalent to those where BHLHE22 functions in mice, including structures analogous to the dorsal telencephalon, dorsal horn of the spinal cord, dorsal cochlear nucleus, and retinal amacrine cells .

What are the recommended methods for expressing and purifying recombinant chicken BHLHE22?

For successful expression and purification of recombinant chicken BHLHE22, researchers should consider the following methodological approaches:

Recommended purification strategy:

• Construct design considerations:

  • Include a cleavable affinity tag (His₆, GST, or MBP)

  • Consider expressing the HLH domain separately if full-length protein proves insoluble

  • Codon-optimize for the expression system

• Purification protocol:

  • Initial capture via affinity chromatography (IMAC for His-tagged protein)

  • Intermediate purification using ion exchange chromatography

  • Final polishing with size exclusion chromatography

  • Include reducing agents throughout to maintain disulfide state

• Quality control assessments:

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

  • Circular dichroism to verify secondary structure

  • DNA binding assay (EMSA) to confirm functional activity

  • Mass spectrometry to verify molecular weight

Special attention should be given to buffer optimization, as the stability of bHLH proteins can be significantly affected by pH, salt concentration, and additives such as glycerol or detergents.

How should researchers design experiments to study BHLHE22 DNA binding specificity?

Characterizing the DNA binding preferences of chicken BHLHE22 requires systematic experimental approaches:

• Identification of binding motifs:

  • Electrophoretic Mobility Shift Assays (EMSA) with consensus E-box sequences and variants

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to determine preferred binding sequences

  • Protein-Binding Microarrays (PBMs) to assess binding to diverse DNA sequences

  • Filter-binding assays to determine binding affinities for different motifs

• Genome-wide binding analysis:

  • Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) in relevant chicken neural tissues

  • CUT&RUN or CUT&Tag as alternatives requiring less input material

  • Motif enrichment analysis to identify overrepresented sequences

  • Comparative analysis with mammalian ChIP-seq datasets

• Functional validation:

  • Luciferase reporter assays using identified binding sites

  • Site-directed mutagenesis of binding motifs to confirm specificity

  • CRISPR-mediated deletion of binding sites in chicken cell models

Based on mammalian studies, focus should be placed on E-box sequences, as BHLHE22 forms a repressor complex by binding to sequence-specific DNA elements and recruiting PRDM8 . Special attention should be given to genes involved in neural development, particularly those related to axonal guidance and commissure formation.

What are appropriate model systems for studying chicken BHLHE22 function in neural development?

Several experimental systems are suitable for investigating the role of chicken BHLHE22 in neural development:

• In ovo electroporation:

  • Direct introduction of expression vectors or CRISPR constructs into developing chicken embryos

  • Enables spatiotemporal control of gene manipulation

  • Allows for rapid assessment of neural development phenotypes

  • Compatible with subsequent live imaging or histological analysis

• Ex ovo embryo culture:

  • Provides direct visualization of development in real-time

  • Enables application of pharmacological agents

  • Allows for easier manipulation and imaging of developing structures

• Primary neural culture systems:

  • Chicken neural progenitor cells cultured in vitro

  • Retinal explant cultures for studying retinal development

  • Enables detailed cellular analysis of neuronal differentiation

• Comparison with established mammalian models:

  • Parallel experiments with mouse models where BHLHE22 function is well-characterized

  • Focus on comparative aspects of commissure formation and neural circuit development

When designing functional studies, researchers should note that mice lacking BHLHE22 show nearly complete loss of three brain commissural structures (corpus callosum, hippocampal commissure, and anterior commissure) . While avian brains lack a true corpus callosum, researchers should focus on equivalent commissural structures and region-specific neuronal differentiation.

What are the methodological approaches for investigating the interaction between chicken BHLHE22 and PRDM8?

Based on mammalian studies, BHLHE22 forms a critical repressor complex with PRDM8 that regulates neural development . Investigating this interaction in chicken requires sophisticated methodological approaches:

• Confirmation of the interaction:

  • Co-immunoprecipitation (Co-IP) using chicken neural tissue or transfected cells

  • Proximity ligation assay (PLA) for in situ detection of the interaction

  • Förster resonance energy transfer (FRET) or Bioluminescence resonance energy transfer (BRET) for live-cell interaction studies

  • Split complementation assays (BiFC, split luciferase) to visualize interaction dynamics

• Mapping interaction domains:

  • Generation of truncation and deletion mutants to identify critical regions

  • Site-directed mutagenesis of conserved residues within the HLH domain

  • Peptide array analysis to identify specific binding sequences

  • Structural studies using X-ray crystallography or NMR spectroscopy

• Functional significance assessment:

  • Chromatin immunoprecipitation sequencing (ChIP-seq) for both factors to identify co-occupied genomic sites

  • Sequential ChIP (Re-ChIP) to confirm simultaneous binding

  • Transcriptome analysis after disruption of the interaction

  • Rescue experiments testing if chicken PRDM8 can restore function in BHLHE22-depleted cells

The development of chicken-specific antibodies or validation of cross-reactive antibodies is essential for many of these approaches. Since mice lacking either BHLHE22 or PRDM8 show similar phenotypes (loss of commissures) , researchers should focus on equivalent developmental processes in chicken models.

How can researchers effectively analyze the impact of BHLHE22 variants on protein function?

To systematically evaluate how variants affect chicken BHLHE22 function, researchers should employ:

• Design of relevant variants:

  • Create equivalent mutations to human pathogenic variants (e.g., p.Glu251Gln, p.Met255Arg, p.Leu262Pro) in conserved regions of chicken BHLHE22

  • Focus on the highly conserved HLH domain, which is crucial for dimerization and DNA binding

  • Include both missense variants and truncating mutations analogous to the p.Gly74Alafs*18 frameshift

• Biochemical characterization:

  • Circular dichroism to assess secondary structure alterations

  • Thermal shift assays to evaluate protein stability changes

  • Size exclusion chromatography to determine oligomerization state

  • Surface plasmon resonance to measure binding kinetics with DNA and protein partners

• Cellular functional assays:

  • Subcellular localization studies using fluorescently-tagged constructs

  • Luciferase reporter assays to assess transcriptional regulatory capacity

  • Co-immunoprecipitation to evaluate protein-protein interactions

  • Chromatin binding capability using ChIP-qPCR

• Developmental impact assessment:

  • In ovo electroporation of variant constructs

  • Rescue experiments in BHLHE22-depleted systems

  • Analysis of neuronal differentiation and axon guidance

  • Evaluation of commissure formation in embryos

Human studies have identified both dominant (missense) and recessive (frameshift) inheritance patterns for BHLHE22-related disorders , suggesting different pathogenic mechanisms that could be explored in chicken models.

What strategies should be employed to decipher the transcriptional regulatory network controlled by chicken BHLHE22?

Elucidating the BHLHE22-regulated transcriptional network requires integrative approaches:

• Genome-wide binding site identification:

  • ChIP-seq in relevant chicken neural tissues at critical developmental timepoints

  • CUT&RUN or CUT&Tag for improved resolution with limited material

  • ATAC-seq to correlate binding with chromatin accessibility

  • Motif analysis to identify direct binding sequences

• Target gene identification:

  • RNA-seq after BHLHE22 knockdown or overexpression

  • PRO-seq to capture nascent transcription changes

  • Time-course experiments to distinguish primary from secondary effects

  • Single-cell RNA-seq to identify cell type-specific regulatory effects

• Integrative network analysis:

  • Combined analysis of binding data and expression changes

  • Network inference algorithms to construct regulatory circuits

  • Comparison with mammalian BHLHE22 networks

  • Pathway enrichment analysis to identify biological processes

• Validation strategies:

  • CRISPR interference/activation at enhancer elements

  • Reporter assays for candidate regulatory regions

  • Direct target gene perturbation and phenotypic assessment

  • Analysis of evolutionary conservation of regulatory interactions

Based on mammalian studies, researchers should focus on genes involved in neural development, particularly Cadherin-11 (CDH11), which is regulated by the BHLHE22-PRDM8 repressor complex and controls the assembly of neural circuitry . Additional targets may include genes involved in axonal guidance in dorsal telencephalic neurons and the control of inhibitory synaptic interneurons .

How can researchers effectively study the role of chicken BHLHE22 in retinal development?

BHLHE22 is expressed in the retina and functions as an important regulator of retinogenesis . To study its role in chicken retinal development:

• Expression analysis in developing retina:

  • Temporal profiling across key developmental stages

  • Single-cell RNA-seq to identify retinal cell populations expressing BHLHE22

  • Spatial mapping using in situ hybridization and immunohistochemistry

  • Co-localization with retinal cell type markers

• Functional manipulation approaches:

  • Retina-specific CRISPR/Cas9-mediated knockout

  • Conditional overexpression using retina-specific promoters

  • Temporal control using inducible systems

  • Mosaic analysis with retroviral vectors

• Phenotypic assessment:

  • Quantification of retinal cell types and proportions

  • Analysis of retinal lamination and organization

  • Evaluation of neuronal morphology and connectivity

  • Electrophysiological recording of retinal activity

• Molecular mechanism investigation:

  • ChIP-seq in developing retinal tissue

  • Identification of retina-specific target genes

  • Analysis of interactions with retinal development regulators

  • Comparison with mammalian retinal development programs

The chicken retina offers several advantages for developmental studies, including accessibility for manipulation, rapid development, and well-characterized cell types. Based on mammalian studies, particular attention should be given to amacrine cell differentiation, as BHLHE22 has been implicated in this process .

What experimental approaches can assess the evolutionary conservation of BHLHE22 function between chickens and mammals?

To systematically evaluate the conservation of BHLHE22 function across vertebrates:

• Comparative sequence and structure analysis:

  • Multiple sequence alignment across diverse vertebrate species

  • Identification of conserved domains and motifs

  • Evolutionary rate analysis to detect signatures of selection

  • Structural modeling to compare predicted three-dimensional conformations

• Expression pattern comparison:

  • Side-by-side analysis of expression domains at equivalent developmental stages

  • Cross-species antibody validation for immunohistochemistry

  • Comparative single-cell transcriptomics of neural tissues

  • Reporter assays testing regulatory elements across species

• Functional conservation testing:

  • Cross-species rescue experiments (e.g., can chicken BHLHE22 rescue mouse knockout phenotypes?)

  • Domain swapping between chicken and mammalian BHLHE22

  • Comparative ChIP-seq to identify conserved and divergent binding sites

  • Analysis of conserved protein-protein interactions (especially PRDM8)

• Developmental processes assessment:

  • Comparative analysis of commissure formation

  • Evaluation of neuronal differentiation in equivalent regions

  • Assessment of retinal development across species

  • Analysis of target gene regulation in parallel systems

• Neuroanatomical comparison:

  • Detailed analysis of axon tract formation

  • Investigation of chicken brain structures equivalent to mammalian commissures

  • Comparative connectivity mapping between brain hemispheres

  • Analysis of neural circuit formation in regions expressing BHLHE22

Mice lacking BHLHE22 show nearly complete loss of three brain commissures: the corpus callosum, hippocampal commissure, and anterior commissure . While avian brains have different commissural organization than mammals, identifying equivalent structures and developmental processes will provide insights into functional conservation.

What methods should be used to study how post-translational modifications affect chicken BHLHE22 function?

Post-translational modifications (PTMs) can significantly impact transcription factor function. To study PTMs of chicken BHLHE22:

• Identification of modifications:

  • Mass spectrometry-based proteomic analysis of BHLHE22 purified from chicken neural tissues

  • Phospho-specific antibodies for detection of conserved phosphorylation sites

  • Western blotting with antibodies against common modifications (phosphorylation, acetylation, ubiquitination)

  • Comparison with known PTMs in mammalian BHLHE22

• Modification site mapping:

  • Site-directed mutagenesis of predicted modification sites

  • Mass spectrometry-based peptide mapping

  • In vitro modification assays with purified kinases or other enzymes

  • Bioinformatic prediction based on sequence conservation

• Functional impact assessment:

  • Comparison of wild-type and modification-mimetic mutants (e.g., S→D for phosphorylation)

  • Analysis of subcellular localization and protein stability

  • DNA binding and protein interaction studies with modified protein

  • Transcriptional activity assays using reporter systems

• Regulatory enzyme identification:

  • Co-immunoprecipitation to identify interacting kinases, phosphatases, or other modifying enzymes

  • Pharmacological inhibition of candidate enzymes

  • Genetic manipulation of modifying enzymes

  • Temporal correlation of modifications with developmental events

PTMs may be particularly relevant for understanding the context-specific activity of BHLHE22 during different stages of neural development. The glycine-rich domain and polyglycine-serine region of BHLHE22 represent potential sites for modifications that could regulate protein function.

How can researchers investigate chicken BHLHE22's role in commissure formation despite anatomical differences from mammals?

While avian brains lack a true corpus callosum, BHLHE22's role in commissure formation can still be investigated:

• Identification of equivalent commissural structures:

  • Detailed anatomical mapping of interhemispheric connections in chicken brain

  • Tracer studies to identify commissural axon tracts

  • Comparison with mammalian commissural development

  • Focus on the anterior commissure and other interhemispheric connections present in both birds and mammals

• BHLHE22 manipulation approaches:

  • Targeted knockdown or knockout in developing commissural neurons

  • Time-controlled manipulation using inducible systems

  • Region-specific perturbation using focal electroporation

  • Rescue experiments with wild-type or variant BHLHE22

• Axon guidance analysis:

  • Ex vivo commissural axon turning assays

  • In vivo axon tracing using lipophilic dyes or genetically encoded fluorescent proteins

  • Live imaging of commissural axon growth dynamics

  • Analysis of growth cone morphology and behavior

• Molecular mechanism investigation:

  • Expression analysis of guidance receptors and ligands

  • ChIP-seq to identify direct targets related to axon guidance

  • Cadherin-11 (CDH11) expression and function analysis

  • Comparison with the BHLHE22-PRDM8 repressor complex function in mammals

Based on mouse studies, BHLHE22 forms a repressor complex with PRDM8 that regulates Cadherin-11 and other genes involved in axon guidance . Despite anatomical differences, the molecular mechanisms governing commissural axon guidance may be conserved between birds and mammals, making the chicken an informative model for studying BHLHE22's fundamental role in neural circuit formation.