Recombinant Tetraodon nigroviridis Myogenic factor 6 (myf6)

What is Myf6 in Tetraodon nigroviridis and how does it compare to other myogenic regulatory factors?

Myf6 (also known as MRF4) is one of four myogenic regulatory factors (MRFs) that belong to the basic helix-loop-helix (bHLH) transcription factor family, alongside Myf5, MyoD, and myogenin. In Tetraodon nigroviridis, Myf6 consists of 239 amino acids (AA 1-239) and contains a conserved bHLH domain that is crucial for DNA binding and protein dimerization .

Unlike the other MRFs which are transiently expressed during specific stages of myogenesis, Myf6 is the predominant myogenic factor that remains expressed in fully differentiated myofibers under normal physiological conditions . Comparative analysis with other pufferfish species shows similar genomic organization patterns, though T. nigroviridis Myf6 appears to be slightly shorter than its counterparts in other teleosts like Fugu rubripes .

What expression systems are most effective for producing recombinant Tetraodon nigroviridis Myf6?

While several expression systems can be used for producing recombinant Myf6, the most common and effective systems for Tetraodon nigroviridis Myf6 include:

• Yeast expression systems: Consistently yield high purity (>90%) products with preserved functional activity . This system is particularly suitable for proteins requiring post-translational modifications.

• Cell-free protein synthesis (CFPS) systems: Offer rapid production without cell viability constraints. The ALiCE® system based on Nicotiana tabacum lysate has been successfully used for other MRF proteins and could be adapted for T. nigroviridis Myf6 .

• Wheat germ expression systems: Useful for in vitro expression with high yield and proper folding of eukaryotic proteins .

For optimal results with T. nigroviridis Myf6, yeast expression systems have demonstrated the best combination of yield, purity, and preserved functional activity for subsequent applications like ELISA and binding studies .

How can I verify the DNA-binding activity of recombinant Tetraodon nigroviridis Myf6?

To verify the DNA-binding activity of recombinant T. nigroviridis Myf6, several methodological approaches can be employed:

• Chromatin Immunoprecipitation followed by sequencing (ChIP-Seq): This approach can identify genome-wide binding sites for Myf6. Using MACS2 with a P-value threshold of 10^-5, you can identify binding sites similar to the 12,885 sites found for Myf6 in primary myotubes .

• Motif analysis: Using MEME suite to analyze peak-covered sequences to identify the canonical E-box motif that Myf6 binds to. In other species, this is centrally located and juxtaposed by the MEF2A binding motif .

• Electrophoretic Mobility Shift Assay (EMSA): This can confirm direct binding of Myf6 to E-box containing DNA fragments.

• Comparative binding analysis: Compare binding patterns with other MRFs such as MyoD and Myf5 using known binding profiles from databases like CIS-BP .

Expected results should show enrichment of the canonical E-box motif (CASCTGC) and potential co-localization with binding motifs for known MRF cooperative heterodimers such as E-protein (E12/E47) .

What role does Myf6 play in myogenesis and how can this be studied using the T. nigroviridis model?

Myf6 plays multiple roles in myogenesis, particularly in:

• Myofiber maturation and maintenance: Unlike other MRFs, Myf6 is the predominant factor expressed in differentiated myofibers, suggesting a role in maintaining the differentiated state .

• Myokine regulation: Research has shown that Myf6 binds to regulatory elements of genes encoding secreted proteins, including myokines like EGF and VEGFA .

• Muscle stem cell (MuSC) pool maintenance: Through regulation of myokine production, Myf6 indirectly maintains the MuSC niche .

To study these functions using T. nigroviridis as a model:

• Expression pattern analysis: Perform quantitative RT-PCR across different developmental stages and tissues to compare with established patterns in other teleosts .

• Chromatin occupancy studies: Conduct ChIP-Seq in T. nigroviridis myotubes to identify target genes and compare with known targets in other species .

• Loss-of-function studies: Use CRISPR/Cas9 to generate T. nigroviridis Myf6 knockout models and assess effects on muscle development and regeneration .

• Transcriptome analysis: Perform RNA-Seq on wild-type versus Myf6-deficient muscle to identify differentially expressed genes, particularly those involved in myokine signaling .

How does T. nigroviridis Myf6 cistrome compare to that of other vertebrates, and what are the methodological approaches to elucidate this?

The Myf6 cistrome (genome-wide binding profile) exhibits both conserved and species-specific features across vertebrates. To elucidate and compare the T. nigroviridis Myf6 cistrome:

• ChIP-Seq methodology:

  • Express tagged recombinant T. nigroviridis Myf6 in myotubes

  • Perform chromatin immunoprecipitation using anti-tag antibodies

  • Sequence precipitated DNA fragments

  • Use MACS2 with a P-value threshold of 10^-5 for peak calling

  • Use empty vector ChIP-Seq as background control

• Motif analysis and comparative genomics:

  • Apply MEME suite to identify enriched binding motifs

  • Compare with known vertebrate Myf6 binding motifs in databases like CIS-BP

  • The canonical E-box motif (CASCTGC) should be centrally located in binding sites

• Gene Ontology analysis:

  • Use GREAT software to associate peaks with nearest genes (100kb maximum extension)

  • Perform GO term computation using binomial tests

  • Compare enriched terms with those found in mammals

Expected findings would show conservation of binding to muscle structure and differentiation genes, but potentially species-specific binding patterns in myokine genes and other regulatory networks adapted to teleost physiology .

How can recombinant T. nigroviridis Myf6 be used to investigate the evolution of myokine regulatory networks in vertebrates?

Recombinant T. nigroviridis Myf6 provides a valuable tool for evolutionary studies of myokine regulation due to the compact genome of pufferfish and its evolutionary position. Methodological approaches include:

• Comparative ChIP-Seq analysis:

  • Perform ChIP-Seq with recombinant T. nigroviridis Myf6 in homologous and heterologous cellular contexts

  • Compare binding profiles near myokine genes (e.g., EGF, VEGFA) with those of mammals

  • Analyze conservation and divergence of binding sites across species

• Histone mark co-localization:

  • Analyze overlap of Myf6 binding with activating histone marks (H3K4me1, H3K27Ac)

  • Compare enhancer architecture around myokine genes between teleosts and mammals

• Functional validation through cross-species complementation:

  • Express T. nigroviridis Myf6 in mammalian Myf6-knockout myotubes

  • Assess rescue of myokine expression through ELISA and RT-qPCR

  • Identify conserved versus divergent regulatory functions

• Genome synteny analysis:

  • Leverage the compact genome of T. nigroviridis (~350Mb compared to mammalian genomes)

  • Identify conserved non-coding elements (CNEs) near myokine genes

  • Correlate CNEs with Myf6 binding sites

This approach can reveal how myokine regulatory networks evolved across vertebrate lineages, particularly between teleosts and tetrapods, providing insights into the ancestral functions of Myf6 .

What are the methodological challenges in using recombinant T. nigroviridis Myf6 for cross-species transcriptional studies, and how can they be overcome?

Cross-species transcriptional studies using recombinant T. nigroviridis Myf6 face several methodological challenges:

• Heterologous protein-protein interactions:

  • Challenge: T. nigroviridis Myf6 may not optimally interact with cofactors in mammalian cells

  • Solution: Co-express known Myf6 cofactors (e.g., E-proteins, MEF2A) from T. nigroviridis

  • Validation: Use co-immunoprecipitation to verify complex formation

• DNA binding specificity differences:

  • Challenge: Subtle differences in binding site preferences between species

  • Solution: Perform comparative motif analysis using PBM (protein binding microarrays) or HT-SELEX

  • Expected outcome: Identification of core shared motifs versus species-specific extensions

• Chromatin context incompatibilities:

  • Challenge: Chromatin landscape differences between species affecting accessibility

  • Solution: Pre-analyze chromatin accessibility (ATAC-seq) in target cells

  • Implementation: Target studies to regions with comparable accessibility

• Functional readout interpretation:

  • Challenge: Downstream gene activation may differ due to promoter/enhancer divergence

  • Solution: Use reporter constructs containing both species' regulatory elements

  • Analysis: Normalize activity relative to species-matched positive controls

• Post-translational modification differences:

  • Challenge: Species-specific PTMs may affect function

  • Solution: Compare protein modifications using mass spectrometry

  • Application: Engineer critical modifications if necessary

These challenges can be systematically addressed through careful experimental design, appropriate controls, and integration of data from multiple approaches to ensure robust interpretation of cross-species functional studies .

How does T. nigroviridis Myf6 differ structurally from other teleost and mammalian orthologs, and what are the functional implications?

T. nigroviridis Myf6 exhibits both conserved and unique structural features compared to orthologs in other species:

Functional implications of these differences include:

• Conservation of core myogenic functions across vertebrates

• Potential species-specific regulation of target genes via differences in protein-protein interactions

• Differences in temporal expression patterns during myogenesis

• Varied roles in maintenance of muscle stem cell pools via myokine regulation

Methodologically, these differences highlight the value of using recombinant T. nigroviridis Myf6 as a model for understanding the core conserved functions of Myf6 while also revealing evolutionary adaptations in muscle development regulation .

What are the methodological approaches to investigate the role of T. nigroviridis Myf6 in muscle development compared to other MRF factors?

To comprehensively investigate T. nigroviridis Myf6's role in muscle development compared to other MRFs (Myf5, MyoD, myogenin), several methodological approaches can be employed:

• Temporal expression profiling:

  • Perform qPCR analysis of all four MRFs across developmental stages

  • Use in situ hybridization to localize expression in embryos and adult tissues

  • Compare with known expression patterns in zebrafish and other teleosts

• Chromatin occupancy comparison:

  • Conduct parallel ChIP-Seq for all four MRFs in T. nigroviridis myoblasts and myotubes

  • Analyze overlap and unique binding sites using bioinformatic tools

  • Expected finding: Partial yet incomplete overlap between Myf6 and MyoD peaks in myotubes (approximately 50% shared targets)

• Loss-of-function studies:

  • Generate individual and combined knockdowns/knockouts of MRFs

  • Assess phenotypes at multiple developmental stages

  • Compare with findings from zebrafish where myf5 and myf6 mutants show milder defects than myod morphants

• Target gene regulation analysis:

  • Perform RNA-Seq after individual MRF depletion

  • Expected result: Loss of Myf6 would specifically affect genes involved in myofiber maturation and myokine production

  • Compare with MyoD depletion, which would affect broader myogenic program activation

• Muscle regeneration models:

  • Study expression and function of each MRF during muscle injury and regeneration

  • Assess stem cell activation, proliferation, and differentiation

  • Myf6's specific role would likely emerge in late differentiation and maintenance phases

These approaches together would reveal the unique versus overlapping functions of Myf6 compared to other MRFs, highlighting its specialized role in myofiber maturation and maintenance of the muscle stem cell niche through myokine regulation .

What are the optimal conditions for using recombinant T. nigroviridis Myf6 protein in in vitro DNA binding assays?

For optimal results in DNA binding assays with recombinant T. nigroviridis Myf6, the following conditions are recommended:

• Protein preparation:

  • Use freshly purified protein (>90% purity) with His tag or other suitable tag

  • Optimal concentration range: 50-200 nM for EMSA, 100-500 nM for DNA pulldown assays

  • Store protein in buffer containing 20-25% glycerol at -80°C with minimal freeze-thaw cycles

• Buffer composition for binding reactions:

  • 20 mM HEPES or Tris-HCl (pH 7.5-7.9)

  • 100-150 mM NaCl or KCl

  • 5 mM MgCl₂

  • 0.1 mM EDTA

  • 1 mM DTT

  • 0.1% NP-40 or Triton X-100

  • 10% glycerol

  • 50 μg/ml BSA (to prevent non-specific binding)

• DNA target selection:

  • Include canonical E-box motifs (CASCTGC)

  • Optimal oligo length: 25-30 bp with the E-box centrally positioned

  • Include 3-5 bp flanking regions from known binding sites identified in ChIP-Seq data

• Binding partners:

  • For maximum activity, include heterodimeric partners such as E-proteins (E12/E47)

  • Consider including known cooperative factors like MEF2A

  • Control for potential inhibitory factors such as TWIST1 and ID4

• Incubation conditions:

  • Optimal temperature: 25°C for 20-30 minutes

  • For EMSA: 15-30 minutes binding followed by 5% non-denaturing PAGE

  • For DNA pulldown: 1-2 hours binding followed by magnetic or affinity bead capture

These optimized conditions should yield robust and reproducible results for studying the DNA binding properties and specificity of T. nigroviridis Myf6 .

How can researchers use recombinant T. nigroviridis Myf6 to study evolutionary conservation of muscle-specific enhancers?

Recombinant T. nigroviridis Myf6, with its compact genome context, provides an excellent tool for studying the evolutionary conservation of muscle-specific enhancers. A systematic methodological approach includes:

• Identifying conserved enhancer elements:

  • Perform ChIP-Seq with recombinant T. nigroviridis Myf6 to identify binding sites

  • Cross-reference with DNase I hypersensitivity or ATAC-seq data

  • Identify regions enriched for enhancer-associated histone marks (H3K4me1, H3K27ac)

  • Use GREAT algorithm with single nearest gene association rule (100 kb maximum extension)

• Comparative genomic analysis:

  • Align identified enhancer regions with orthologous regions from other species

  • Utilize the compact nature of the T. nigroviridis genome (~350 Mb) as an advantage

  • Identify core conserved motifs versus species-specific regulatory elements

  • Expected finding: Conservation of E-box motifs with variable flanking sequences

• Functional validation through reporter assays:

  • Clone candidate enhancers upstream of minimal promoters driving reporter genes

  • Test activity in heterologous cell systems (zebrafish, mammalian)

  • Compare enhancer activity patterns with native expression of target genes

  • Perform mutagenesis of key motifs to identify critical binding sites

• Cross-species enhancer swapping experiments:

  • Replace mammalian enhancers with orthologous T. nigroviridis sequences

  • Test if T. nigroviridis enhancers can drive appropriate expression in mammalian contexts

  • Methodological consideration: Include both core motifs and surrounding context sequences

• Integration with evolution of myokine regulation:

  • Focus on enhancers controlling genes for secreted factors (myokines)

  • Compare with enhancer architecture of genes like EGF and VEGFA in mammals

  • Assess if Myf6-dependent myokine regulation is a conserved feature across vertebrates