Recombinant Chicken Transcription elongation factor SPT5 (SUPT5H), partial

What is the primary function of SUPT5H in transcriptional regulation?

SUPT5H functions as a critical transcription elongation factor that, together with SUPT4H, forms the DSIF (DRB Sensitivity Inducing Factor) complex. This complex interacts directly with RNA polymerase II (RNAPII) to regulate transcriptional elongation. SUPT5H essentially "clamps" RNAPII onto DNA templates, facilitating polymerase processivity during transcript elongation. Research has demonstrated that SUPT5H occupancy on DNA templates varies according to G+C content, with more pronounced effects observed during transcription of G+C-rich sequences . This differential dependency suggests that SUPT5H plays a crucial role in modulating transcription across varied genomic contexts, particularly affecting transcription through repetitive or structurally challenging regions.

What domains are present in chicken SUPT5H and how do they compare to other species?

Chicken SUPT5H contains several conserved functional domains, including an N-terminal domain (Spt5N) with histone-binding capacity, a NusG N-terminal (NGN) domain that interacts with SUPT4H, and multiple KOW (Kyrpides-Ouzounis-Woese) motifs that facilitate interaction with nucleic acids and other proteins. Comparative analyses across species show significant evolutionary conservation, particularly in the NGN and KOW domains. For example, studies in yeast have demonstrated that the Spt5N domain contains a conserved histone binding motif that is essential for cell viability and preserves the integrity of nucleosomes during transcription . This functional conservation extends to zebrafish and Drosophila homologs, where SUPT5H plays critical roles in embryonic development and neural function .

How does SUPT5H interact with chromatin during transcription?

SUPT5H contains a histone-binding motif in its N-terminal domain (Spt5N) that enables it to capture and retain nucleosomal histones released from DNA during transcription. This activity is crucial for maintaining chromatin integrity during active transcription. Research has shown that Spt5N is positioned to contribute to the capture and local retention of nucleosomal histones released during transcription . Mutations in the histone-binding motif (e.g., spt5-3A in yeast) disrupt this function and can be lethal, highlighting its critical importance . By preserving nucleosome positioning, SUPT5H helps prevent cryptic transcription within gene bodies across the genome and ensures proper gene regulation.

What expression systems are optimal for producing functional recombinant chicken SUPT5H?

Multiple expression systems have been successfully employed for producing recombinant chicken SUPT5H, including E. coli, yeast, baculovirus, and mammalian cell systems . The choice of expression system should be guided by experimental requirements:

For functional studies requiring post-translational modifications similar to those in avian systems, insect or mammalian expression systems are preferable despite their higher cost and complexity. For structural studies requiring large protein quantities where modifications are less critical, bacterial systems may be sufficient. All recombinant proteins typically achieve ≥85% purity as determined by SDS-PAGE .

What are the most effective methods for studying SUPT5H-RNAPII interactions in vitro?

Studying SUPT5H-RNAPII interactions requires specialized approaches that maintain native protein conformations and complex integrity. Recommended methodologies include:

• Co-immunoprecipitation (Co-IP): Using antibodies against either SUPT5H or RNAPII components to pull down protein complexes. This approach has successfully demonstrated that wild-type SUPT5H and mutant variants (e.g., SPT5-3A) associate equivalently with RNAPII, while isolated Spt5N domain does not bind RNAPII .

• Surface Plasmon Resonance (SPR): For determining binding kinetics and affinity constants between purified SUPT5H and RNAPII components.

• Chromatin Immunoprecipitation followed by sequencing (ChIP-seq): For genome-wide occupancy analysis. Ultra-deep ChIP-seq analysis has revealed that RNAPII dependence on SUPT4H/SUPT5H varies according to G+C content of template DNA .

• Fluorescence Resonance Energy Transfer (FRET): For visualizing dynamic interactions in real-time, using fluorescently labeled SUPT5H and RNAPII.

When designing these experiments, it's crucial to include appropriate controls, such as truncated SUPT5H variants that lack specific domains, to validate interaction specificity.

How can I effectively analyze the histone-binding activity of chicken SUPT5H?

To analyze the histone-binding activity of chicken SUPT5H, employ the following methodological approaches:

• Histone pull-down assays: Using recombinant or purified histones immobilized on a matrix to capture SUPT5H. Compare wild-type SUPT5H with mutants in the histone-binding motif to quantify binding efficiency differences.

• Electrophoretic Mobility Shift Assays (EMSA): Mix purified SUPT5H with nucleosomes or histone octamers and analyze complex formation by native gel electrophoresis.

• Fluorescence Anisotropy: Using fluorescently labeled histones to measure binding kinetics with SUPT5H in solution.

• Site-directed mutagenesis: Based on research in yeast systems, mutating conserved residues in the histone-binding motif (similar to the spt5-3A mutations) can provide valuable insights into binding specificity .

• Proximity ligation assays: For detecting SUPT5H-histone interactions in cellular contexts.

When interpreting results, consider that histone binding may be modulated by post-translational modifications on both histones and SUPT5H, which may require additional analytical methods such as mass spectrometry.

How should I interpret changes in gene expression following SUPT5H knockdown or mutation?

When analyzing gene expression changes following SUPT5H manipulation, consider these interpretation guidelines:

• Differential effects on gene classes: Research has shown that SUPT5H knockdown does not affect all genes equally. Effects are more pronounced on genes with high G+C content and are particularly evident for G+C-rich segments of long genes . Therefore, genome-wide expression analysis should be stratified by G+C content for proper interpretation.

• Specific vs. global effects: While SUPT5H is a general transcription factor, its knockdown typically affects a limited subset of genes. In zebrafish, comparative expression profiling showed that only about 5% of genes are differentially expressed in Spt5 mutants .

• Direct vs. indirect effects: Primary effects of SUPT5H alteration would be expected on genes with complex regulatory regions or repetitive elements. Secondary expression changes may reflect downstream consequences of altered cellular physiology.

• Temporal dynamics: Gene expression changes may follow different kinetics; some genes respond rapidly to SUPT5H inhibition while others show delayed responses .

For robust data interpretation, combine RNA-seq with ChIP-seq to correlate expression changes with alterations in RNAPII and SUPT5H occupancy across the genome.

What are common challenges in purifying active recombinant SUPT5H and how can they be addressed?

Researchers frequently encounter several challenges when purifying active recombinant SUPT5H:

For optimal results, verify protein activity using functional assays that assess transcription elongation or histone binding. The purified protein should achieve ≥85% purity as determined by SDS-PAGE and maintain its expected molecular interactions with RNA polymerase II components.

How do I resolve contradictory findings regarding SUPT5H function in different experimental systems?

When facing contradictory findings across experimental systems:

• Consider species-specific differences: While SUPT5H is evolutionarily conserved, subtle functional differences exist between species. For example, the effects of SUPT5H mutation in zebrafish development may not directly translate to chicken or mammalian systems .

• Evaluate experimental context: Different cell types or developmental stages may show varying dependencies on SUPT5H function. In Drosophila, Spt5 function is particularly critical in neural progenitor cells during mushroom body development .

• Assess protein domains used: Studies using truncated versions may yield different results than those using full-length protein. Research has shown that the isolated Spt5N domain exhibits different properties than full-length Spt5 .

• Examine interaction partners: The function of SUPT5H depends on its association with SUPT4H and other factors. Variation in expression levels of these partners across experimental systems may affect outcomes.

• Consider post-translational modifications: Different expression systems produce proteins with varying modification patterns that may affect function.

To resolve contradictions, conduct comparative studies using identical experimental conditions and readouts across systems, and explicitly test hypotheses that might explain discrepancies.

How can chicken SUPT5H be utilized as a tool for studying transcription-coupled chromatin remodeling?

Chicken SUPT5H serves as an excellent model for investigating transcription-coupled chromatin remodeling due to several advantageous properties:

• Histone interaction domain: The N-terminal domain of SUPT5H (Spt5N) contains a histone-binding motif essential for capturing and retaining nucleosomal histones during transcription . This property makes it an ideal probe for studying how transcription machinery interacts with chromatin.

• Experimental applications:

  • Use ChIP-seq with antibodies against chicken SUPT5H to map regions of active transcription coupled with chromatin remodeling

  • Deploy SUPT5H mutants defective in histone binding as dominant-negative tools to disrupt normal chromatin maintenance

  • Utilize SUPT5H as bait in proximity labeling experiments (BioID or APEX) to identify novel factors involved in transcription-coupled chromatin remodeling

• Comparative approach: Chicken SUPT5H can be compared with mammalian homologs to identify conserved and divergent aspects of chromatin regulation during transcription.

• Visualizing dynamics: Fluorescently tagged SUPT5H can be used in live-cell imaging to visualize the dynamics of transcription-coupled chromatin remodeling in real-time.

Research has demonstrated that the histone-binding activity of Spt5N is essential for repressing cryptic transcription within gene bodies , suggesting it maintains proper nucleosome organization during active transcription.

What insights can SUPT5H studies provide for understanding transcriptional regulation in neurodevelopmental disorders?

SUPT5H studies offer significant insights into neurodevelopmental disorders through several research avenues:

• Neural development regulation: Research in model organisms has demonstrated that SUPT5H plays critical roles in neural development. In zebrafish, a single amino acid substitution in Spt5 resulted in alteration of dopaminergic and serotonergic neuron numbers , suggesting SUPT5H may influence neurotransmitter system development relevant to numerous neurological conditions.

• Control of neural progenitor proliferation: Studies in Drosophila revealed that Spt4 and Spt5 regulate mushroom body neuroblast (MBNB) proliferation and control remodeling of γ-Kenyon cell axons . This suggests SUPT5H may influence neural circuit formation during development.

• Transcriptional regulation of repeat expansions: SUPT5H and SUPT4H form the DSIF complex that facilitates transcription through nucleotide repeat regions. Chemical interference with this complex formation can differentially affect expression of mutant alleles containing repeat expansions in genes like HTT (Huntington's Disease) . Similar mechanisms may apply to other repeat expansion disorders affecting neurological function.

• Therapeutic targeting potential: Inhibition of the SUPT4H-SUPT5H interaction reduced mutant huntingtin protein in neuronal cells and decreased its aggregation and toxicity , suggesting potential therapeutic approaches for related disorders.

These findings indicate that SUPT5H-focused research may provide valuable insights into the transcriptional mechanisms underlying neurodevelopmental and neurodegenerative conditions, potentially leading to novel therapeutic strategies.

How might SUPT5H manipulation be utilized in targeted gene expression studies?

SUPT5H manipulation offers several sophisticated approaches for targeted gene expression studies:

• G+C content-dependent targeting: Given that SUPT5H dependency varies according to template G+C content , targeted manipulation could selectively affect high G+C regions while minimally impacting low G+C regions. This property could be exploited to:

  • Selectively modulate expression of G+C-rich genes

  • Target regulation of specific gene classes with distinctive G+C signatures

  • Develop tools for studying genes with complex regulatory regions

• Repeat expansion targeting: The DSIF complex (SUPT4H/SUPT5H) is particularly important for transcription through nucleotide repeat expansions. Chemical compounds that interfere with SUPT4H-SUPT5H interaction have shown selective effects on expression of mutant HTT alleles containing CAG expansions . Similar approaches could be developed for:

  • Selective modulation of genes containing repeat expansions

  • Studying the role of repeat-containing regulatory elements

  • Therapeutic development for repeat expansion disorders

• Domain-specific engineering: Creating chimeric SUPT5H proteins with modified domains could generate tools with novel properties:

  • Modified histone-binding domains could alter chromatin interactions

  • Engineered KOW domains might redirect SUPT5H to specific genomic regions

  • Fusion with programmable DNA-binding domains could create targeted transcriptional modulators

These approaches require careful validation of specificity and efficiency but offer promising directions for precise manipulation of gene expression in research and potential therapeutic applications.

How conserved is SUPT5H structure and function across different species, and what can this tell us about its essential roles?

SUPT5H shows remarkable evolutionary conservation across species, providing insights into its fundamental roles in transcription:

What experimental approaches are most effective for comparative studies of SUPT5H function across species?

For rigorous comparative studies of SUPT5H function across species, consider these methodological approaches:

• Complementation assays: Test whether chicken SUPT5H can rescue phenotypes in other species with SUPT5H mutations or knockdowns. This approach can identify functionally conserved domains and species-specific requirements.

• Domain swap experiments: Create chimeric proteins combining domains from chicken SUPT5H with those from other species to pinpoint regions responsible for functional differences. For example, exchanging the histone-binding domain of chicken SUPT5H with that of yeast Spt5 could reveal evolutionary differences in chromatin interaction.

• Parallel genomic approaches: Conduct ChIP-seq and RNA-seq analyses under comparable conditions across species to identify:

  • Conserved vs. species-specific binding patterns

  • Differentially regulated gene sets

  • G+C content dependencies that may vary between species

• Structural biology approaches: Compare protein structures across species using techniques such as X-ray crystallography or cryo-EM to identify structural conservation and divergence.

• Developmental phenotyping: Compare phenotypic effects of SUPT5H manipulation in embryonic development across species, as developmental roles have been documented in both zebrafish and Drosophila .

When implementing these approaches, standardize experimental conditions as much as possible to ensure valid cross-species comparisons, and use evolutionary distance measures to contextualize functional differences.

How do the interaction partners of SUPT5H differ between avian and mammalian systems?

The interaction landscape of SUPT5H shows both conservation and divergence between avian and mammalian systems:

• Core conserved interactions:

  • SUPT4H: Forms the DSIF complex with SUPT5H in both avian and mammalian systems

  • RNA polymerase II: The interaction with the transcription machinery is evolutionarily conserved

  • Histones: The histone-binding function of the Spt5N domain appears conserved across eukaryotes

  • Nucleic acids: KOW domains facilitate interaction with RNA and DNA in all systems

• Potentially divergent interactions:

  • Species-specific transcription factors: Chicken SUPT5H may interact with avian-specific transcription regulators

  • Chromatin remodelers: The repertoire and regulatory patterns of chromatin modifiers show some evolutionary divergence

  • Post-translational modifiers: Enzymes that modify SUPT5H may target different residues or respond to different signals

• Experimental approaches to identify differences:

  • Comparative proteomics using immunoprecipitation followed by mass spectrometry to identify species-specific binding partners

  • Yeast two-hybrid screening with chicken versus mammalian SUPT5H as bait

  • Cross-species protein-protein interaction mapping

• Functional implications:

  • Different interaction partners may contribute to species-specific aspects of gene regulation

  • Convergent interactions highlight evolutionarily critical functions

  • Divergent protein associations may reflect adaptation to different cellular environments or regulatory requirements

Understanding these similarities and differences can provide insights into the core functions of SUPT5H that have been maintained through evolution versus those that have adapted to species-specific requirements.

What quality control measures are critical when working with recombinant chicken SUPT5H?

Rigorous quality control is essential when working with recombinant chicken SUPT5H to ensure reliable experimental outcomes:

• Purity assessment:

  • SDS-PAGE analysis should confirm ≥85% purity

  • Western blotting with specific antibodies to verify protein identity

  • Mass spectrometry to confirm sequence integrity and detect post-translational modifications

• Functional validation:

  • RNAPII binding assays to confirm retention of interaction capacity, as seen in studies with yeast Spt5

  • Histone binding assays to verify the activity of the Spt5N domain

  • In vitro transcription elongation assays to demonstrate functional activity

• Structural integrity:

  • Circular dichroism spectroscopy to assess secondary structure content

  • Limited proteolysis to verify proper domain folding

  • Size exclusion chromatography to confirm monodispersity and appropriate oligomeric state

• Storage stability monitoring:

  • Functional assays before and after storage to assess activity retention

  • Freeze-thaw testing to establish optimal handling protocols

  • Temperature sensitivity analysis to determine appropriate storage conditions

• Batch consistency:

  • Comparative analysis between production batches to ensure reproducibility

  • Reference standards for each quality parameter

  • Detailed documentation of expression and purification conditions

Implementing these quality control measures will minimize experimental variability and ensure that observed effects genuinely reflect SUPT5H biology rather than artifacts of protein preparation.

How can ChIP-seq approaches be optimized for studying chicken SUPT5H genomic occupancy?

Optimizing ChIP-seq for chicken SUPT5H requires careful consideration of several technical aspects:

• Antibody selection and validation:

  • Test multiple antibodies against different epitopes of chicken SUPT5H

  • Validate specificity using western blotting and immunoprecipitation

  • Consider developing custom antibodies if commercial options lack specificity

  • Perform ChIP-qPCR on known targets before proceeding to sequencing

• Chromatin preparation optimization:

  • Adjust formaldehyde fixation time (typically 10-15 minutes) to avoid over-crosslinking

  • Optimize sonication conditions to achieve 200-300 bp fragments

  • Implement dual crosslinking with DSG followed by formaldehyde for improved capture of protein-protein interactions

  • Verify fragment size distribution using Bioanalyzer or gel electrophoresis

• Sequencing considerations:

  • Use ultra-deep sequencing (>50 million reads) for comprehensive coverage, as implemented in studies of DSIF complex

  • Implement paired-end sequencing to improve mapping accuracy

  • Consider ChIP-exo or ChIP-nexus for higher resolution binding site determination

  • Include input controls and IgG controls for proper background normalization

• Bioinformatic analysis strategies:

  • Stratify analysis by G+C content of genomic regions

  • Integrate with RNA-seq data to correlate occupancy with expression

  • Analyze co-occupancy with RNA polymerase II and other transcription factors

  • Implement metagene analysis to examine SUPT5H distribution relative to gene features

• Validation approaches:

  • Confirm key findings with ChIP-qPCR

  • Use orthogonal methods such as CUT&RUN for validation

  • Compare results with publicly available datasets from related species

These optimizations will enhance data quality and facilitate more robust insights into the genomic occupancy patterns of chicken SUPT5H.

What are the most promising therapeutic applications of targeting SUPT5H function?

Several compelling therapeutic directions emerge from SUPT5H research:

• Repeat expansion disorders:

  • Research has shown that chemical compounds interfering with SUPT4H-SUPT5H interaction can selectively decrease expression of mutant huntingtin alleles containing CAG expansions

  • Similar approaches could potentially target other repeat expansion disorders like myotonic dystrophy, fragile X syndrome, and various spinocerebellar ataxias

  • Advantages include selectivity for mutant alleles with minimal effects on normal gene expression

• Cancer therapeutics:

  • SUPT5H dependency of G+C-rich genes could be exploited to target oncogenes with high G+C content

  • Modulation of SUPT5H function might disrupt the transcriptional programs of cancer cells

  • Combinatorial approaches with existing chemotherapeutics might enhance efficacy

• Neurodevelopmental disorders:

  • Given SUPT5H's role in neural development , therapeutic modulation might address specific neurodevelopmental conditions

  • Targeting could be refined to specific developmental windows

  • Potential for cell type-specific delivery to affected neural populations

• Viral infection:

  • SUPT5H plays roles in viral gene expression for several pathogens

  • Targeted inhibition might disrupt viral replication with minimal host toxicity

• Delivery approaches:

  • Antisense oligonucleotides targeting SUPT4H or SUPT5H

  • Small molecule inhibitors of the SUPT4H-SUPT5H interaction

  • PROTAC-based approaches for targeted degradation

These therapeutic directions warrant further investigation, with particular attention to specificity, delivery mechanisms, and potential long-term effects of SUPT5H modulation.

What emerging technologies might enhance our understanding of SUPT5H function in real-time?

Cutting-edge technologies are poised to revolutionize our understanding of SUPT5H dynamics:

• Live-cell imaging techniques:

  • CRISPR-mediated endogenous tagging of SUPT5H with fluorescent proteins for real-time tracking

  • Single-molecule tracking to observe SUPT5H movement along DNA templates

  • Lattice light-sheet microscopy for high-resolution 3D imaging with reduced phototoxicity

  • FRAP (Fluorescence Recovery After Photobleaching) to measure SUPT5H kinetics at active transcription sites

• Proximity labeling approaches:

  • TurboID or miniTurbo fusion with SUPT5H to identify dynamic interaction partners

  • Split-BioID systems to detect conditional interactions dependent on specific cellular states

  • Domain-specific labeling to map interactions of different SUPT5H regions

• Single-cell technologies:

  • Single-cell ChIP-seq to examine cell-to-cell variation in SUPT5H occupancy

  • Single-cell RNA-seq following SUPT5H perturbation to identify cell type-specific responses

  • Combined single-cell multi-omics approaches to correlate SUPT5H binding with expression outcomes

• Structural biology innovations:

  • Cryo-electron microscopy of SUPT5H-containing complexes at near-atomic resolution

  • Single-particle tracking of SUPT5H during elongation complex assembly and progression

  • Integrative structural biology combining multiple data types for complete structural models

• Massively parallel reporter assays:

  • Testing thousands of SUPT5H variants simultaneously to map structure-function relationships

  • High-throughput assessment of sequence-specific effects on SUPT5H activity

These technologies promise to transform our static models of SUPT5H function into dynamic understanding of its activities in living cells.

How might studies of SUPT5H contribute to our understanding of non-coding RNA regulation?

SUPT5H research offers several avenues to illuminate non-coding RNA regulation:

• Long non-coding RNA (lncRNA) transcription:

  • SUPT5H's role in transcription elongation likely extends to lncRNAs

  • ChIP-seq studies could reveal differential SUPT5H occupancy patterns on protein-coding versus lncRNA genes

  • SUPT5H dependency may vary between coding and non-coding transcripts based on sequence composition

• Enhancer RNA (eRNA) production:

  • SUPT5H may facilitate the transcription of enhancer RNAs

  • The protein's histone-binding activity could influence chromatin structure at enhancers

  • Targeted SUPT5H modulation could help distinguish functional versus non-functional eRNAs

• RNA processing and stability:

  • SUPT5H has been implicated in co-transcriptional RNA processing

  • Its influence on elongation rate may affect alternative splicing decisions

  • Potential direct interactions with RNA processing machinery could be investigated

• Repeat-associated non-coding RNAs:

  • Given SUPT5H's involvement in transcription through repeat expansions , it may regulate repeat-associated non-coding RNAs implicated in various diseases

  • Manipulation of SUPT5H function could provide tools to modulate these transcripts selectively

• Chromatin-associated RNAs:

  • SUPT5H's histone-binding capacity suggests potential roles in regulating chromatin-associated RNAs

  • Investigation of SUPT5H interactions with RNA-binding proteins could reveal novel regulatory mechanisms