β-hydroxybutyryl-HIST1H3A (K79) Antibody

What is histone β-hydroxybutyrylation and its biological significance?

Histone β-hydroxybutyrylation (Kbhb) is a post-translational modification where β-hydroxybutyrate (BHB) is covalently attached to lysine residues on histone proteins, including histone H3. This modification is particularly significant as it represents a direct link between cellular metabolism and epigenetic regulation of gene expression. β-hydroxybutyric acid (BHB) is a ketone body produced during fatty acid metabolism, particularly during states of fasting, ketogenic diet, or diabetic ketoacidosis . When BHB levels rise in the cell, this metabolite can be enzymatically transferred to specific lysine residues on histone proteins, creating the Kbhb modification. This process effectively converts a metabolic signal into an epigenetic mark that can influence chromatin structure and gene transcription. The modification plays a crucial role in coordinating transcriptional responses to metabolic changes, particularly during starvation or ketogenic states . Understanding this modification is critical for research into metabolic diseases, cancer metabolism, and fundamental epigenetic regulation mechanisms.

What are the validated applications for β-hydroxybutyryl-HIST1H3A antibodies?

The β-hydroxybutyryl-HIST1H3A (K9) Polyclonal Antibody has been validated for several important research applications in epigenetics and molecular biology. These validated applications include:

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative detection of β-hydroxybutyrylated histone H3 in solution .

• Western Blotting (WB): Enables detection of β-hydroxybutyrylated histone H3 in cell and tissue lysates, typically showing bands around 17kDa .

• Immunocytochemistry (ICC): Allows visualization of the cellular and subcellular localization of β-hydroxybutyrylated histones, primarily showing nuclear localization .

• Chromatin Immunoprecipitation (ChIP): Used to identify genomic regions associated with β-hydroxybutyrylated histone H3 .

What protocols can verify the specificity of β-hydroxybutyryl-HIST1H3A antibodies?

Given the documented cross-reactivity of commercially available H3K9bhb antibodies, implementing rigorous validation protocols is essential. The following methodological approach can help verify antibody specificity:

Comprehensive Validation Protocol:

• Parallel Treatment Comparisons: Treat cells with:

  • β-hydroxybutyrate (BHB) to induce β-hydroxybutyrylation

  • Butyrate (structurally similar to BHB)

  • Trichostatin A (TSA, histone deacetylase inhibitor)

  • Vehicle control

  Compare antibody signals across these conditions using western blotting. A truly specific H3K9bhb antibody should show significantly stronger signals in BHB-treated samples compared to other treatments .

• Immunoprecipitation Followed by Mass Spectrometry:

  • Perform immunoprecipitation (IP) with the H3K9bhb antibody

  • Analyze the immunoprecipitated material by mass spectrometry

  • Quantify the percentage of peptides containing β-hydroxybutyrylation versus other modifications

  In a study using this approach, researchers found that only 13.99% of H3 peptides from BHB-treated samples contained Kbhb modifications, while in butyrate-treated samples, only 1.74% contained Kbhb . This indicates substantial non-specific binding.

• Peptide Competition Assays: Pre-incubate the antibody with:

  • Unmodified H3K9 peptides

  • H3K9bhb peptides

  • H3K9ac (acetylated) peptides

  • Other modified H3K9 peptides

  A specific antibody should only have its signal blocked by the H3K9bhb peptide.

• Antibody Validation in Genetic Models: Use cells with mutations in enzymes responsible for β-hydroxybutyrylation or in the H3K9 residue itself (K9R mutation) to confirm antibody specificity.

Implementing these rigorous validation protocols will help researchers determine the true specificity of their antibody and interpret their experimental results with appropriate caution.

How can mass spectrometry complement antibody-based detection of histone β-hydroxybutyrylation?

Mass spectrometry (MS) offers a powerful complementary approach to antibody-based detection of histone β-hydroxybutyrylation, providing higher specificity and the ability to identify novel modification sites. The following methodological workflow integrates MS with antibody-based detection:

Integrated MS-Antibody Workflow:

• Sample Preparation:

  • Isolate histones using acid extraction or high-salt extraction

  • Perform propionylation of unmodified and monomethylated lysines to prevent trypsin digestion at these sites

  • Digest with trypsin to generate peptides suitable for MS analysis

• Mass Spectrometry Approaches:

  • Untargeted LC-MS/MS: For discovery of novel β-hydroxybutyrylation sites

  • Parallel Reaction Monitoring (PRM): For targeted quantification of known modification sites

  • SILAC or TMT Labeling: For comparative quantification across different treatments or conditions

• Data Analysis:

  • Look for mass shifts of +86.04 Da, characteristic of β-hydroxybutyrylation

  • Analyze MS/MS fragmentation patterns to confirm modification site

  • Use specialized software (e.g., MaxQuant, Skyline) for quantification

• Correlation with Antibody-Based Methods:

  • Perform immunoprecipitation with the β-hydroxybutyryl-HIST1H3A antibody

  • Analyze the immunoprecipitated material by MS

  • Calculate the percentage of peptides containing the target modification

In published research, MS analysis of H3K9bhb antibody immunoprecipitations showed that only a small percentage of immunoprecipitated peptides actually contained β-hydroxybutyrylation, even after treatment with high concentrations of BHB . This suggests that current antibodies may be detecting low-abundance modifications or cross-reacting with other histone marks.

By combining antibody-based approaches with MS validation, researchers can achieve more reliable and comprehensive characterization of histone β-hydroxybutyrylation patterns and distinguish true signals from potential antibody cross-reactivity.

What are the enzymatic regulators of histone β-hydroxybutyrylation and how can they be studied?

Understanding the enzymes that regulate histone β-hydroxybutyrylation is crucial for comprehending its biological functions. Current research has identified several key enzymes involved in the addition ("writers") and removal ("erasers") of this modification:

Enzymatic Regulators and Research Methodologies:

• Writers of β-hydroxybutyrylation:
  While specific histone β-hydroxybutyryltransferases have not been definitively characterized, several p300/CBP family acyltransferases may catalyze this reaction. Research approaches include:

  • In vitro enzymatic assays: Incubating recombinant histone H3 with purified p300/CBP in the presence of β-hydroxybutyryl-CoA

  • Cellular enzyme manipulation: Overexpression or knockdown of candidate enzymes followed by western blotting for H3K9bhb

  • Chemical inhibition: Using p300/CBP inhibitors (e.g., C646) and measuring changes in global H3K9bhb levels

• Erasers of β-hydroxybutyrylation:
  Sirtuins, particularly SIRT3, have been identified as de-β-hydroxybutyrylases with class-selective activity for certain histone residues . SIRT3 shows preference for H3K4, K9, K18, K23, K27, and H4K16, but not for H4K5, K8, or K12 . Research approaches include:

  • Systematic profiling: Testing various sirtuin family members against different β-hydroxybutyrylated histone peptides

  • Structural studies: X-ray crystallography or cryo-EM of SIRT3 bound to β-hydroxybutyrylated substrates

  • Molecular basis for specificity: Investigating how SIRT3's hydrogen bond-lined hydrophobic pocket favors S-form Kbhb recognition and catalysis

• Metabolic regulation:
  β-hydroxybutyrylation depends on cellular BHB levels, which are elevated during ketosis, fasting, or diabetic ketoacidosis . Research approaches include:

  • Metabolic manipulation: Treating cells with exogenous BHB, using ketogenic diets in animal models, or inducing ketosis through fasting

  • Metabolic enzyme modulation: Manipulating enzymes in the BHB synthesis pathway and measuring effects on histone modifications

  • Quantification of cellular BHB: Using enzymatic assays or MS to correlate cellular BHB levels with histone β-hydroxybutyrylation

The table below summarizes key enzymes involved in histone β-hydroxybutyrylation regulation:

Understanding these enzymatic regulators provides opportunities for pharmacological intervention and deeper insights into the physiological roles of histone β-hydroxybutyrylation in health and disease.

What experimental controls are essential when using β-hydroxybutyryl-HIST1H3A antibodies in chromatin studies?

Due to the documented cross-reactivity of β-hydroxybutyryl-HIST1H3A antibodies, implementing rigorous controls is imperative for obtaining reliable results in chromatin studies. The following comprehensive control strategy should be employed:

Essential Experimental Controls:

• Antibody Specificity Controls:

  • Peptide Competition: Include samples where the antibody is pre-incubated with excess β-hydroxybutyrylated peptides to block specific binding

  • Cross-Reactivity Assessment: Pre-incubate separate antibody aliquots with acetylated, butyrylated, and other acylated histone peptides to evaluate potential cross-reactivity

  • Knockout/Knockdown Validation: Use cell lines with CRISPR-mediated knockout of histone H3 variants or with K9R mutations that prevent modification at this residue

• Treatment Controls:

  • Metabolic Manipulation: Include parallel samples treated with:

    • β-hydroxybutyrate (BHB)

    • Butyrate

    • Trichostatin A (TSA)

    • Vehicle control

  This approach revealed that H3K9bhb antibodies can produce signals in TSA and butyrate-treated cells comparable to BHB-treated cells, suggesting cross-reactivity

• ChIP-Specific Controls:

  • Input Control: Analyze a portion of chromatin before immunoprecipitation

  • IgG Control: Perform parallel immunoprecipitation with non-specific IgG

  • Positive Control Regions: Include genomic regions known to be enriched for histone marks

  • Negative Control Regions: Include genomic regions known to lack the modification

  • Parallel ChIP: Perform parallel ChIP with antibodies against other histone modifications (especially H3K9ac) to identify potential overlapping signals due to cross-reactivity

• Validation by Orthogonal Methods:

  • Mass Spectrometry Validation: Confirm the presence of β-hydroxybutyrylation by MS analysis of immunoprecipitated material

  • Sequential ChIP: Perform sequential ChIP (re-ChIP) with antibodies against different modifications to determine co-occurrence

  • Correlation with Metabolic State: Correlate ChIP-seq signals with cellular BHB levels measured by metabolomics

By implementing these controls, researchers can better interpret their results and distinguish genuine β-hydroxybutyrylation signals from potential artifacts due to antibody cross-reactivity. This is particularly important given that previous datasets using H3K9bhb antibodies should be interpreted with caution, as they may actually be detecting H3K9ac or other PTMs .

How does histone β-hydroxybutyrylation differ from other acylation modifications in function and detection?

Histone β-hydroxybutyrylation represents a distinct post-translational modification with unique properties compared to other acylation marks. Understanding these differences is crucial for accurate interpretation of experimental results:

Comparative Analysis of Histone Acylation Modifications:

• Structural and Chemical Differences:

  Modification	Chemical Structure	Molecular Weight	Charge at pH 7.4
  Acetylation (Kac)	CH₃CO-	+42 Da	Neutral
  Butyrylation (Kbu)	CH₃CH₂CH₂CO-	+70 Da	Neutral
  β-Hydroxybutyrylation (Kbhb)	CH₃CH(OH)CH₂CO-	+86 Da	Neutral
  Succinylation (Ksucc)	HOOCCH₂CH₂CO-	+100 Da	Negative

  The distinctive feature of β-hydroxybutyrylation is the hydroxy group that creates potential for additional hydrogen bonding interactions .

• Metabolic Origins and Physiological Conditions:

  • Acetylation: Derives from acetyl-CoA, prevalent under normal metabolic conditions

  • Butyrylation: Derives from butyryl-CoA, often from gut microbiota-produced butyrate

  • β-Hydroxybutyrylation: Derives from β-hydroxybutyrate, elevated during ketosis, fasting, or diabetic ketoacidosis

  • Succinylation: Derives from succinyl-CoA, an intermediate in the TCA cycle

• Enzymatic Regulation:

  • Deacylases: While many sirtuins can remove multiple acylation types, they show specificity patterns. SIRT3 shows preference for removing β-hydroxybutyrylation from specific sites (H3K4, K9, K18, K23, K27, and H4K16) but not others (H4K5, K8, K12)

  • Writers: Different acyltransferases may have varying specificities for different acyl-CoA donors

• Detection Challenges:

  • Antibody Cross-Reactivity: H3K9bhb antibodies have demonstrated cross-reactivity with other modifications, particularly acetylation

  • Mass Spectrometry: MS can distinguish these modifications based on their characteristic mass shifts (+42 Da for acetylation vs. +86 Da for β-hydroxybutyrylation)

  • Differential Extraction: Different acylations may affect histone solubility in various extraction buffers

• Functional Significance:

  • Transcriptional Effects: Different acylations may recruit distinct reader proteins

  • Metabolic Signaling: β-hydroxybutyrylation provides a direct link to ketone body metabolism and fasting responses

  • Evolutionary Conservation: While acetylation is highly conserved, newer acylations like β-hydroxybutyrylation may have evolved more recently as metabolic sensors

The unique properties of β-hydroxybutyrylation necessitate specialized approaches for its study, particularly given the cross-reactivity issues with current antibodies. Researchers should combine antibody-based methods with mass spectrometry and consider the metabolic context when interpreting results related to this modification.

What are the current limitations in studying β-hydroxybutyrylation in chromatin research?

Despite the growing interest in histone β-hydroxybutyrylation, several significant limitations challenge researchers in this field. Understanding these limitations is essential for designing robust experiments and interpreting results accurately:

Critical Limitations in β-Hydroxybutyrylation Research:

• Antibody Specificity Issues:

  • The only commercially available H3K9bhb antibody recognizes additional modifications, including acetylation, undermining its reliability for ChIP experiments

  • Immunoprecipitation with H3K9bhb antibody followed by mass spectrometry revealed that β-hydroxybutyrylated peptides constituted only about 14% of enriched peptides, even in BHB-treated samples

  • Previous datasets using H3K9bhb antibodies should be interpreted with caution as they may actually be detecting H3K9ac or other PTMs

• Low Prevalence of Modification:

  • Even after treatment with high concentrations of BHB, the prevalence of H3K9bhb appears to be relatively low

  • This low abundance makes detection challenging and increases the risk of false positives from cross-reactive antibodies

• Limited Knowledge of Enzymatic Machinery:

  • While SIRT3 has been identified as a de-β-hydroxybutyrylase with preference for certain histone sites , the specific "writer" enzymes that catalyze the addition of this modification remain poorly characterized

  • Without a complete understanding of the enzymatic regulation, manipulating this modification for functional studies remains challenging

• Technical Challenges in Chromatin Studies:

  • The cross-reactivity of antibodies complicates ChIP-seq experiments aimed at genome-wide mapping of β-hydroxybutyrylation

  • Traditional histone extraction methods may not be optimized for preserving all acylation modifications equally

  • Distinguishing direct effects of β-hydroxybutyrylation from indirect effects of altered metabolism is difficult

• Biological Complexity:

  • β-hydroxybutyrylation is intimately connected to metabolic state, making it difficult to manipulate independently of other metabolic changes

  • The modification may have context-dependent functions that vary across cell types and physiological conditions

  • Potential interplay with other histone modifications adds another layer of complexity

• Methodological Limitations:

  • Mass spectrometry, while more specific than antibody-based methods, requires specialized equipment and expertise

  • The development of synthetic histone substrates with site-specific β-hydroxybutyrylation for in vitro studies is technically challenging

  • Current cell culture models may not accurately recapitulate the physiological conditions that promote β-hydroxybutyrylation in vivo

To address these limitations, the field requires the development of new reagents with improved specificity, more sensitive detection methods, and integrated approaches that combine genetic, biochemical, and metabolic analyses. Until these advancements are made, researchers should approach studies of histone β-hydroxybutyrylation with appropriate caution and implement rigorous controls.

What emerging technologies could improve the study of histone β-hydroxybutyrylation?

Overcoming the current limitations in studying histone β-hydroxybutyrylation requires innovative technological approaches. The following emerging technologies show promise for advancing this field:

Innovative Technologies for β-Hydroxybutyrylation Research:

• Next-Generation Antibody Development:

  • Synthetic Antibody Libraries: Using phage or yeast display to screen for antibodies with improved specificity for β-hydroxybutyrylated histones

  • Camelid Single-Domain Antibodies (Nanobodies): Developing smaller antibody fragments that may access epitopes with greater specificity

  • Antibody Engineering: Rational design of antibodies using structural information about the β-hydroxybutyryl modification to enhance specificity

  • Proximity Ligation Assays: Combining multiple antibodies to increase specificity through coincidence detection

• Advanced Mass Spectrometry Approaches:

  • Top-Down Proteomics: Analyzing intact histone proteins to preserve combinatorial modification patterns

  • Ion Mobility-Mass Spectrometry: Separating modified peptides based on both mass and shape

  • Targeted Quantitative Proteomics: Developing highly sensitive SRM/MRM methods specifically for β-hydroxybutyrylated peptides

  • Mass Spectrometry Imaging: Visualizing the distribution of histone modifications in tissue sections

• Chemical Biology Strategies:

  • Bioorthogonal Chemistry: Developing chemical reporters for β-hydroxybutyrylation that allow selective labeling and enrichment

  • Clickable β-Hydroxybutyrate Analogs: Synthesizing cell-permeable precursors that can be metabolically incorporated and later detected via click chemistry

  • Photo-crosslinking Probes: Creating probes that can covalently capture proteins interacting with β-hydroxybutyrylated histones

• Genetic and Genome Engineering:

  • Site-Specific Incorporation of β-Hydroxybutyrylated Lysine: Using expanded genetic code systems to incorporate this modification at specific sites

  • CRISPR Screens: Identifying genes involved in regulating histone β-hydroxybutyrylation

  • Histone Mutation Libraries: Systematic analysis of the functional impact of β-hydroxybutyrylation at different histone residues

• Advanced Imaging Technologies:

  • Super-Resolution Microscopy: Visualizing the distribution of β-hydroxybutyrylated histones in chromatin at nanoscale resolution

  • Live-Cell Sensors: Developing fluorescent reporters for real-time monitoring of histone β-hydroxybutyrylation

  • Correlative Light and Electron Microscopy: Linking histone modifications to chromatin ultrastructure

• Computational Approaches:

  • Machine Learning Algorithms: Developing tools to predict β-hydroxybutyrylation sites based on sequence context and chromatin features

  • Molecular Dynamics Simulations: Modeling the structural impact of β-hydroxybutyrylation on chromatin organization

  • Integrative Multi-omics Analysis: Combining epigenomics, transcriptomics, and metabolomics data to understand the relationship between metabolism and histone modifications

These emerging technologies hold promise for overcoming current limitations and advancing our understanding of histone β-hydroxybutyrylation in chromatin biology and metabolic regulation. By integrating multiple approaches, researchers can build a more comprehensive picture of this important epigenetic modification.

How might histone β-hydroxybutyrylation connect to human disease states?

Histone β-hydroxybutyrylation represents a direct link between metabolism and epigenetic regulation, suggesting important roles in various disease states. Emerging research points to several potential connections:

β-Hydroxybutyrylation in Disease Pathophysiology:

• Metabolic Disorders:

  • Type 2 Diabetes: Altered ketone body metabolism in diabetes may lead to abnormal patterns of histone β-hydroxybutyrylation, potentially affecting gene expression related to glucose homeostasis

  • Obesity: Changes in fatty acid metabolism could affect BHB levels and subsequent histone modifications, influencing adipogenesis and fat storage genes

  • Non-alcoholic Fatty Liver Disease (NAFLD): Dysregulated β-hydroxybutyrylation may contribute to altered hepatic gene expression patterns in NAFLD

• Neurodegenerative Diseases:

  • Alzheimer's Disease: The brain can utilize ketone bodies as an alternative energy source, and β-hydroxybutyrylation may mediate neuroprotective gene expression changes

  • Parkinson's Disease: Mitochondrial dysfunction in Parkinson's may affect BHB metabolism and subsequent histone modifications

  • Epilepsy: Ketogenic diets are used to treat epilepsy, potentially acting partly through epigenetic mechanisms involving β-hydroxybutyrylation

• Cancer:

  • Metabolic Reprogramming: Cancer cells often exhibit altered metabolism, which could affect BHB levels and histone β-hydroxybutyrylation patterns

  • Tumor Suppressor Regulation: Changes in β-hydroxybutyrylation may influence the expression of tumor suppressors or oncogenes

  • Therapy Resistance: Metabolic adaptations in cancer cells might use β-hydroxybutyrylation as a mechanism to survive therapy-induced stress

• Inflammatory Conditions:

  • Autoimmune Disorders: β-hydroxybutyrylation may influence the expression of immune-related genes

  • Inflammatory Bowel Disease: Gut microbiota-derived short-chain fatty acids may affect histone modifications in intestinal cells

• Aging-Related Conditions:

  • Cardiovascular Disease: Altered epigenetic patterns including β-hydroxybutyrylation may contribute to vascular aging

  • Sarcopenia: Age-related changes in muscle metabolism might involve altered histone modifications

Research Challenges and Future Directions:

• Specificity of Disease Associations:

  • Current antibody specificity issues complicate the accurate mapping of β-hydroxybutyrylation patterns in disease states

  • More specific detection methods are needed to establish reliable disease associations

• Causality vs. Correlation:

  • Determining whether changes in β-hydroxybutyrylation are causal factors or consequences of disease is challenging

  • Animal models with manipulations of enzymes regulating β-hydroxybutyrylation will be valuable for establishing causality

• Therapeutic Implications:

  • Ketogenic diets and exogenous ketone supplements might exert some effects through modulation of histone β-hydroxybutyrylation

  • Targeting enzymes that regulate β-hydroxybutyrylation could represent a novel therapeutic approach

  • SIRT3 activators or inhibitors might modulate disease-associated β-hydroxybutyrylation patterns

• Biomarker Potential:

  • Patterns of histone β-hydroxybutyrylation might serve as biomarkers for metabolic state and disease progression

  • More sensitive and specific detection methods are needed for biomarker development

Understanding the connections between histone β-hydroxybutyrylation and human disease requires addressing current limitations in detection specificity while exploring disease models that incorporate metabolic perturbations. As methods improve, this emerging field may yield important insights for diagnostic and therapeutic applications.

What are the key considerations for researchers planning experiments with β-hydroxybutyryl-HIST1H3A antibodies?

Researchers considering the use of β-hydroxybutyryl-HIST1H3A antibodies must carefully plan their experiments with awareness of current limitations. Several key considerations should guide experimental design:

• Antibody Validation is Non-negotiable: The documented cross-reactivity of H3K9bhb antibodies with other modifications, particularly acetylation, necessitates rigorous validation before interpretation of results . Researchers should not rely solely on manufacturer specifications but should perform their own validation using the methods described in section 2.1.

• Implement Multiple Orthogonal Approaches: Combine antibody-based detection with mass spectrometry validation whenever possible. This multi-method approach provides higher confidence in the identification of genuine β-hydroxybutyrylation signals.

• Control for Metabolic State: Since β-hydroxybutyrylation is directly linked to ketone body metabolism, researchers should carefully control and document the metabolic conditions of their experimental systems. Fasting, feeding, and culture medium composition can significantly impact results.

• Consider Cross-talk with Other Modifications: Design experiments to account for potential interplay between β-hydroxybutyrylation and other histone modifications, particularly acetylation, which may be recognized by the same antibodies .

• Interpret Published Literature with Caution: Previous studies using H3K9bhb antibodies should be interpreted with awareness of potential cross-reactivity issues. Results attributed to β-hydroxybutyrylation may actually reflect other modifications .

• Report Methodology Transparently: When publishing results, provide detailed methodological information about antibody validation, controls, and potential limitations to allow proper interpretation by the scientific community.

As the field advances, new reagents with improved specificity will likely emerge, addressing many current limitations. Until then, a cautious and rigorous approach combining multiple methodologies represents the most reliable strategy for studying histone β-hydroxybutyrylation.