Butyrly-HIST1H4A (K31) Antibody

What is histone H4 butyrylation and how does it compare to acetylation?

The chemical difference between butyrylation and acetylation (butyryl groups having a longer carbon chain than acetyl groups) results in distinct functional outcomes. For instance, research has demonstrated that butyrylation at H4K5 can abolish the binding of bromodomain-containing proteins like Brdt to H4 tails, whereas acetylation at the same position promotes this interaction .

What applications are supported by Butyryl-HIST1H4A antibodies?

Butyryl-HIST1H4A antibodies have been validated for multiple applications in epigenetics research:

Specifically, the Butyryl-Hist1H4A (K8) Polyclonal Antibody has been validated for ELISA, WB, ICC, IF, and ChIP applications, providing researchers with versatile tools for investigating this modification across different experimental contexts .

How are Butyryl-HIST1H4A antibodies generated and characterized?

Butyryl-HIST1H4A antibodies are typically generated using synthetic peptides corresponding to the sequence surrounding the butyrylated lysine residue of interest. For example, the Butyryl-Hist1H4A (K8) Polyclonal Antibody is produced using a peptide sequence around the site of Butyryl-Lys (8) derived from human Histone H4 . These antibodies are often produced in rabbits as host animals and purified to enhance specificity.

Characterization of these antibodies involves multiple validation steps:

• Surface plasmon resonance (SPR) to measure antibody affinity for modified peptides

• Competition assays with differentially modified peptides or proteins

• ChIP-qPCR to confirm genomic enrichment at target sites

• Cross-reactivity testing against other histone modifications

How can I validate the specificity of butyryl-histone antibodies in my experiments?

Validating antibody specificity is crucial due to the cross-reactivity issues identified with pan-K-acyl antibodies . A comprehensive validation approach includes:

• Competition assays: Perform western blot or immunofluorescence experiments with competitors such as acetyl-BSA, butyryl-BSA, and crotonyl-BSA. This helps determine if your antibody cross-reacts with other acyl modifications. Research has shown that many pan-K-butyryl antibodies significantly cross-react with acetylation marks .

• Dot blot analysis: Test antibody specificity against a panel of modified peptides to quantify the degree of cross-reactivity.

• Surface plasmon resonance: Measure the actual binding affinity of your antibody to various modified peptides. This provides quantitative data on antibody specificity .

• Mass spectrometry validation: Confirm the presence of butyrylation at your sites of interest using mass spectrometry as an antibody-independent method.

• Enzymatic manipulation: Use histone deacylases or inhibitors to modulate butyrylation levels and confirm corresponding changes in antibody signals .

Research has demonstrated that pan-K-crotonyl and pan-K-butyryl antibodies can recognize histone acetylation generated by histone acetyltransferases such as Gcn5 and Esa1, highlighting the importance of rigorous validation .

What factors influence the cross-reactivity of butyryl-histone antibodies with acetylation marks?

Cross-reactivity between butyryl-histone antibodies and acetylation marks represents a significant methodological challenge. Several factors influence this cross-reactivity:

• Structural similarity: The chemical structures of butyryl and acetyl groups share common features, differing primarily in carbon chain length, which can lead to recognition of both modifications by the same antibody .

• Antibody generation methods: Polyclonal antibodies contain a heterogeneous mixture of immunoglobulins that may recognize different epitopes, including those shared between butyrylation and acetylation .

• Epitope context: The amino acid sequence surrounding the modified lysine can influence antibody binding specificity.

• Modification abundance: Acetylation is typically more abundant than butyrylation in most cellular contexts, potentially leading to stronger signals from acetylated histones even with relatively weak cross-reactivity .

Experimental evidence demonstrates that pan-K-butyryl antibody signals can be fully outcompeted by acetyl-BSA in western blot analysis, suggesting strong cross-reactivity with acetylation . In immunofluorescence experiments, pan-nuclear staining with butyrylation antibodies is reduced by acetyl-BSA competitors, further confirming this cross-reactivity .

How do butyrylation and acetylation compete at histones H4K5 and H4K8?

The dynamic competition between butyrylation and acetylation at histone H4K5 and H4K8 positions creates a complex regulatory landscape. Research has revealed several key aspects of this competition:

• Differential protein binding: Butyrylation at H4K5 abolishes the binding of bromodomain-containing protein Brdt to H4 tails, whereas acetylation promotes this interaction. This was confirmed through pull-down assays with nuclear extracts from mouse testis and validated by mass spectrometry analysis .

• Genomic co-occurrence: ChIP-seq analysis shows that many transcription start sites (TSSs) bear both acetylation and butyrylation marks at H4K5K8, suggesting potential dynamic exchange between these modifications .

• Functional outcomes: The BD1 domain of Brdt plays a major role in targeting gene TSSs bearing H4K5K8 acetylation, but this targeting is disrupted when these sites are butyrylated. This suggests functional consequences for chromatin remodeling and gene expression .

• Tissue-specific regulation: The competition between these modifications appears particularly important in cells undergoing dramatic chromatin remodeling, such as during spermatogenesis .

What controls should I include when performing ChIP experiments with butyryl-histone antibodies?

When designing ChIP experiments with butyryl-histone antibodies, the following controls are essential:

• Input control: Always compare your ChIP-enriched DNA to input DNA to account for biases in chromatin preparation and DNA abundance.

• Antibody specificity controls: Include immunoprecipitations with:

  • Pre-immune serum or IgG negative controls

  • Competitive peptide controls (butyrylated vs. unmodified or differently modified peptides)

  • Validation with independent antibodies recognizing the same modification

• Biological controls:

  • Conditions that increase histone butyrylation (e.g., butyrate treatment)

  • Comparison with acetylation patterns at the same sites

  • Tissues or cell types with different expected levels of butyrylation

• Technical controls:

  • qPCR validation of selected ChIP-seq peaks

  • Spike-in normalization standards

  • Sequential ChIP (re-ChIP) to confirm co-occurrence of modifications

For rigorous experimental design, researchers should perform antibody validation using surface plasmon resonance to confirm similar affinity ranges across antibodies being compared . Additionally, ChIP-qPCR at well-characterized genomic regions can validate enrichment before proceeding to genome-wide analyses .

How can I optimize immunoprecipitation protocols for butyrylated histones?

Optimizing immunoprecipitation protocols for butyrylated histones requires several considerations:

• Preservation of modifications: Include deacetylase/debutyrylase inhibitors (such as sodium butyrate) in all buffers to prevent loss of modifications during sample preparation .

• Crosslinking conditions: Optimization of formaldehyde concentration and crosslinking time is crucial for preserving histone modifications while ensuring efficient chromatin fragmentation.

• Antibody concentration: Titrate antibody concentrations to determine optimal amounts. Too little leads to poor enrichment, while excess can increase non-specific binding .

• Washing stringency: Balance between removing non-specific interactions and preserving specific antibody-epitope binding. This may require optimization of salt concentrations and detergent types.

• Enrichment validation: Implement ChIP-qPCR at known butyrylated regions to assess enrichment before proceeding to genome-wide analyses .

In one successful approach, researchers labeled newly replicated HeLa cell chromatin for 5-30 minutes with [3H]thymidine in the presence of sodium butyrate (to inhibit deacetylation of newly deposited H4), which allowed for effective immunoprecipitation of nucleosomes with specific modification patterns .

What are the limitations of using pan-K-butyryl antibodies in epigenetics research?

Several important limitations affect the use of pan-K-butyryl antibodies in epigenetics research:

• Cross-reactivity: Western blot competition assays demonstrate that pan-K-butyryl antibody signals can be fully outcompeted by acetyl-BSA, indicating significant cross-reactivity with acetylation marks .

• Variable specificity across applications: An antibody's specificity may differ between applications (western blot, ChIP, immunofluorescence), requiring validation in each experimental context .

• Background signal issues: In western blot analysis with butyryl antibodies, competition with modified BSA can result in the appearance of background bands, complicating data interpretation .

• Recognition of enzymatically generated acetylation: Pan-K-butyryl antibodies can recognize histone acetylation generated by acetyltransferases like Gcn5 and Esa1, even when only acetyl-CoA is provided as a cofactor .

• Immunofluorescence limitations: In cellular staining, pan-nuclear signals from butyrylation antibodies can be reduced by acetyl-BSA, crotonyl-BSA, and butyryl-BSA competitors, indicating multiple cross-reactivities .

These limitations necessitate careful experimental design and interpretation of results when studying histone butyrylation, with particular attention to appropriate controls and validation steps.

How does histone butyrylation contribute to gene expression regulation?

Histone butyrylation contributes to gene expression regulation through several mechanisms:

• Association with active transcription: ChIP-seq data shows that histone butyrylation, like acetylation, is associated with high levels of gene expression, suggesting a role in transcriptional activation .

• Protein reader interactions: Butyrylation can modulate interactions with chromatin reader proteins. For example, butyrylation at H4K5 abolishes the binding of Brdt to H4 tails, potentially altering chromatin remodeling activities .

• Competitive regulation: The dynamic competition between butyrylation and acetylation at the same lysine residues (particularly H4K5 and H4K8) creates a complex regulatory landscape where these modifications may serve as a molecular switch for different functional outcomes .

• Chromatin structure effects: As an acylation mark, butyrylation neutralizes the positive charge of lysine residues, potentially affecting histone-DNA interactions and higher-order chromatin structure.

• Tissue-specific functions: The functional impact of butyrylation appears particularly important in specific contexts such as spermatogenesis, where dramatic chromatin remodeling occurs .

Understanding these mechanisms provides insights into how cells fine-tune gene expression through diverse histone modifications, with butyrylation representing an additional layer of epigenetic control.

What is the relationship between histone butyrylation and other acyl modifications?

Histone butyrylation exists within a complex landscape of acyl modifications that includes acetylation, crotonylation, and succinylation. These relationships are characterized by:

• Competitive modification of the same residues: Multiple acyl modifications can target the same lysine residues, creating competition for modification sites. This is particularly evident for H4K5 and H4K8, which can be both acetylated and butyrylated .

• Varying abundance levels: Acetylation is typically more abundant than butyrylation and other acyl modifications in most cellular contexts, creating a hierarchical relationship .

• Differential recognition by reader proteins: Different acyl modifications are recognized by specific reader proteins with varying affinities. For example, while acetylated H4 tails are recognized by the BD1 domain of Brdt, butyrylated H4 tails are not, creating functional distinctions .

• Cross-talk in signaling pathways: Metabolic states influence the availability of different acyl-CoA donors, potentially allowing metabolic conditions to regulate the balance between different histone acylations.

• Technical challenges in differentiation: Research is complicated by antibody cross-reactivity issues, where antibodies designed to detect one modification often recognize others. This has been demonstrated in competition assays where pan-K-butyryl antibody signals are outcompeted by acetyl-BSA .

These complex relationships suggest that the complete functional understanding of histone butyrylation requires consideration of the broader acylation landscape and careful experimental design to distinguish between closely related modifications.

How can advanced mass spectrometry approaches complement antibody-based detection of histone butyrylation?

Mass spectrometry offers several advantages that complement antibody-based detection of histone butyrylation:

• Unambiguous modification identification: Mass spectrometry can definitively distinguish between different acyl modifications based on their mass differences, avoiding the cross-reactivity issues inherent to antibodies .

• Quantitative analysis: Modern mass spectrometry approaches provide absolute or relative quantification of modification levels, allowing precise comparison between conditions.

• Multi-modification analysis: Mass spectrometry can identify combinations of modifications on the same histone molecule, providing insights into modification cross-talk that are difficult to achieve with antibody-based methods.

• Novel modification discovery: Untargeted mass spectrometry approaches can identify previously unknown modifications or modification sites.

• Validation of antibody specificity: As demonstrated in research, mass spectrometry can validate the specificity of antibody-based approaches. For example, proteins affinity-isolated by acetylated H4 tail peptides but not by butyrylated peptides can be identified through mass spectrometry analysis .

In practical applications, researchers have used mass spectrometry to confirm the identity of proteins pulled down with either fully acetylated or fully butyrylated immobilized H4 tail peptides, identifying Brdt among the proteins isolated by the H4ac-containing peptide but not by the H4bu-containing peptide .