β-hydroxybutyryl-HIST1H3A (K4) Antibody

Basic Research Questions

• What is histone β-hydroxybutyrylation and how does it function as an epigenetic mark?

  Histone β-hydroxybutyrylation (Kbhb) is a post-translational modification where a β-hydroxybutyryl group is attached to lysine residues on histone proteins. This modification was identified as a new type of histone mark that is dramatically induced in response to elevated β-hydroxybutyrate levels in cells . β-hydroxybutyryl-CoA serves as the cofactor for this modification, similar to how acetyl-CoA functions for histone acetylation . Functionally, histone Kbhb marks are enriched in active gene promoters and associate with upregulated genes in starvation-responsive metabolic pathways . This represents a direct mechanism by which ketone bodies can regulate cellular physiology and gene expression through metabolite-directed histone modifications . The modification is evolutionarily conserved and has been detected in yeast, Drosophila, mouse, and human cells .

• How does the β-hydroxybutyryl-HIST1H3A (K4) modification differ from other H3K4 modifications?

  The β-hydroxybutyryl modification at H3K4 (H3K4bhb) represents a distinct epigenetic mark compared to other well-studied H3K4 modifications such as methylation (H3K4me2/me3) and acetylation (H3K4ac). While H3K4 methylation generally marks active or poised promoters and enhancers , β-hydroxybutyrylation at H3K4 is specifically induced under conditions of elevated β-hydroxybutyrate, such as fasting or diabetic ketoacidosis . Studies have shown that H3K9bhb distinguishes a set of upregulated genes from others that bear H3K9ac and H3K4me3 marks, suggesting histone Kbhb has different functions from histone acetylation and methylation . Importantly, H3K4bhb levels increase in a dose-dependent manner with elevated β-hydroxybutyrate, whereas acetylation marks show minimal changes under the same conditions .

• What experimental applications are suitable for the β-hydroxybutyryl-HIST1H3A (K4) antibody?

  The β-hydroxybutyryl-HIST1H3A (K4) antibody can be utilized in multiple experimental applications to investigate this histone modification:

  • Western Blotting (WB): For detecting and quantifying global or specific β-hydroxybutyrylation levels (typically using dilutions of 1:100-1:1000)

  • Immunocytochemistry (ICC): For visualizing cellular localization of the modification (recommended dilution 1:200-1:500)

  • Immunofluorescence (IF): For high-resolution imaging of β-hydroxybutyrylated histones (recommended dilution 1:50-1:200)

  • Chromatin Immunoprecipitation (ChIP): For identifying genomic regions associated with this modification

  • ChIP-seq: For genome-wide mapping of β-hydroxybutyrylation patterns

  • ELISA: For quantitative detection of the modification

  These applications enable comprehensive investigation of the presence, distribution, and dynamics of β-hydroxybutyrylation in various experimental contexts.

Advanced Research Applications

• How should experiments be designed to study the dynamics of β-hydroxybutyrylation during metabolic transitions?

  To effectively study β-hydroxybutyrylation dynamics during metabolic transitions, researchers should consider a multi-faceted experimental approach:

  • Metabolic Labeling: Utilize isotopically labeled β-hydroxybutyrate (e.g., [2,4-13C2]-β-hydroxybutyrate) to track incorporation into histone marks. Studies have shown that isotopic β-hydroxybutyrate can be converted to β-hydroxybutyryl-CoA intracellularly and subsequently incorporated into histones .

  • Time-Course Analysis: Design experiments with multiple time points during metabolic shifts (e.g., fed to fasted transition) to capture the temporal dynamics of modification changes.

  • Comparative Profiling: Simultaneously profile multiple histone modifications (β-hydroxybutyrylation, acetylation, methylation) to understand their interrelationships. Data shows that during fasting, Kbhb levels at major histone sites were elevated by 4-40 fold while most acetylation sites showed less than 2-fold changes .

  • Tissue-Specific Analysis: Different tissues respond distinctly to metabolic changes. For example, both liver and kidney show induction of histone Kbhb during fasting, but with tissue-specific patterns .

  • Integration with Metabolomics: Correlate histone modification changes with measurements of cellular metabolites, particularly β-hydroxybutyrate levels and β-hydroxybutyryl-CoA.

  • Gene Expression Correlation: Perform RNA-seq in parallel with ChIP-seq to correlate changes in histone marks with alterations in gene expression patterns .

• What methodological considerations are critical for ChIP-seq experiments using β-hydroxybutyryl-HIST1H3A (K4) antibody?

  For successful ChIP-seq experiments with β-hydroxybutyryl-HIST1H3A (K4) antibody, researchers should consider:

  • Antibody Validation: Verify antibody specificity using dot blot assays with competing modified peptides, as was done for pan-Kbhb antibodies .

  • Chromatin Preparation: Optimize fixation conditions to preserve β-hydroxybutyrylation marks while ensuring efficient chromatin fragmentation.

  • Input Controls: Include appropriate input controls and IgG controls to account for background binding.

  • Spike-in Normalization: Consider using spike-in controls (e.g., Drosophila chromatin) for accurate normalization between different metabolic conditions.

  • Sequential ChIP: For investigating co-occurrence with other modifications, perform sequential ChIP experiments.

  • Bioinformatic Analysis: Develop specific peak-calling and annotation strategies that account for the unique distribution patterns of β-hydroxybutyrylation, which may differ from better-studied modifications like H3K4me3 or H3K27ac.

  • Integration with Metabolic Data: Correlate ChIP-seq findings with measurements of β-hydroxybutyrate levels to establish connections between metabolism and epigenetic patterning.

• How does β-hydroxybutyrylation interact with writer, reader, and eraser enzymes in the epigenetic landscape?

  The enzymatic regulation of β-hydroxybutyrylation is an emerging area of research:

  • Writers: While specific β-hydroxybutyryl transferases have not been definitively identified, evidence suggests that p300 and other histone acetyltransferases may catalyze this reaction using β-hydroxybutyryl-CoA as a cofactor, similar to how they use acetyl-CoA for acetylation .

  • Readers: Proteins containing bromodomains and YEATS domains may recognize β-hydroxybutyrylated lysines. Research is ongoing to identify specific reader proteins that preferentially bind to β-hydroxybutyrylated histones versus other acylation marks.

  • Erasers: Class I and II histone deacetylases (HDACs) may remove β-hydroxybutyryl groups, though with potentially different kinetics than for acetyl groups. Sirtuin family members might also play a role in removing this modification.

  • Crosstalk with Other Modifications: β-hydroxybutyrylation may interact with other histone modifications in complex ways. For example, the presence of H3K4bhb may influence the deposition or removal of methylation or acetylation at nearby residues, creating a dynamic regulatory network.

  • Metabolic Regulation: The concentration of β-hydroxybutyryl-CoA, which is directly influenced by β-hydroxybutyrate levels, appears to be a key determinant in the abundance of histone β-hydroxybutyrylation, establishing a direct link between cellular metabolism and epigenetic regulation .

Methodological Considerations

• What sample preparation methods best preserve β-hydroxybutyrylation marks for accurate analysis?

  Preservation of β-hydroxybutyrylation marks requires careful sample handling:

  • Rapid Fixation: Quick fixation of samples with crosslinking agents (typically formaldehyde) helps preserve the native state of modifications.

  • Cold Processing: Maintain samples at 4°C during processing to minimize enzymatic activity that might remove modifications.

  • Protease Inhibitors: Include comprehensive protease inhibitor cocktails in all buffers.

  • HDAC Inhibitors: Add HDAC inhibitors (e.g., sodium butyrate, trichostatin A) to prevent removal of β-hydroxybutyrylation marks during processing.

  • Deacetylase Inhibitors: Include specific sirtuin inhibitors like nicotinamide, as sirtuins may remove β-hydroxybutyryl groups.

  • Storage Conditions: Store samples at -80°C and avoid repeated freeze-thaw cycles, as recommended for antibodies against these modifications .

  • Buffer Composition: For antibody storage, use preservation buffers such as 0.03% Proclin 300 in 50% Glycerol, 0.01M PBS, pH 7.4 to maintain antibody activity .

• How can researchers validate the specificity of β-hydroxybutyryl-HIST1H3A (K4) antibody in experimental systems?

  Rigorous validation of antibody specificity is essential for reliable results:

  • Peptide Competition Assays: Perform dot blot assays with both modified (β-hydroxybutyrylated) and unmodified peptides to confirm specific recognition .

  • Modified Peptide Arrays: Test antibody against peptide arrays containing various histone modifications to assess cross-reactivity with similar modifications (e.g., acetylation, butyrylation, crotonylation).

  • Western Blot Analysis: Conduct western blots with recombinant histones bearing defined modifications.

  • Mass Spectrometry Correlation: Validate antibody-based findings with mass spectrometry analysis of histone modifications.

  • Knockout/Knockdown Controls: Use genetic approaches to modulate enzymes involved in β-hydroxybutyrylation pathways as validation controls.

  • Metabolic Manipulation: Treat cells with varying concentrations of β-hydroxybutyrate and verify dose-dependent increases in signal, as demonstrated in previous studies .

  • Isotopic Labeling: Employ isotopically labeled β-hydroxybutyrate to confirm antibody detection of newly incorporated modifications .

• What quantification methodologies are most effective for measuring changes in β-hydroxybutyrylation levels?

  Accurate quantification of β-hydroxybutyrylation requires appropriate techniques:

  Method	Application	Advantages	Limitations
  Western Blot	Semi-quantitative analysis of global levels	Simple, widely accessible	Limited resolution, semi-quantitative
  Mass Spectrometry	Precise identification and quantification of modified sites	High accuracy, site-specific	Expensive, requires specialized equipment
  ChIP-qPCR	Quantification at specific genomic loci	Targeted analysis, relatively simple	Limited to pre-selected regions
  ChIP-seq	Genome-wide profiling	Comprehensive coverage	Complex analysis, expensive
  ELISA	Quantitative detection of modifications	High-throughput, quantitative	Limited to global levels
  Imaging-based quantification	Spatial distribution analysis	Provides cellular context	Limited quantitative precision

  For most accurate results, researchers should:

  • Use multiple complementary approaches

  • Include appropriate internal controls

  • Employ isotopically labeled standards for mass spectrometry

  • Utilize spike-in controls for normalization between samples

  • Develop calibration curves with recombinant modified histones

  • Consider the dynamic range of each method relative to expected biological changes

Troubleshooting and Data Interpretation

• How can researchers address potential cross-reactivity with other acylation marks?

  To minimize and address cross-reactivity concerns:

  • Antibody Selection: Choose antibodies that have been rigorously validated for specificity against β-hydroxybutyrylation versus other acylation marks .

  • Competitive Binding Assays: Perform assays with differentially modified peptides to determine the degree of cross-reactivity.

  • Dilution Optimization: Titrate antibody concentrations to maximize specific signal while minimizing non-specific binding. For β-hydroxybutyryl-HIST1H3A (K4) antibody, recommended dilutions are: WB (1:100-1:1000), ICC (1:200-1:500), and IF (1:50-1:200) .

  • Blocking Optimization: Test different blocking reagents to reduce background signal.

  • Secondary Validation: Confirm findings using alternative detection methods, such as mass spectrometry.

  • Metabolic Manipulation: Verify that signals increase under conditions known to elevate β-hydroxybutyrylation (e.g., fasting, ketogenic diet, or direct β-hydroxybutyrate treatment) .

  • Mutational Analysis: Use histone mutants (e.g., K-to-R substitutions) at specific residues to confirm site specificity.

• What experimental controls are essential when studying β-hydroxybutyrylation patterns?

  Proper experimental controls are critical for reliable interpretation:

  • Negative Controls:

    • IgG control antibodies of the same isotype and host species as the β-hydroxybutyryl antibody

    • Samples with enzymatically removed modifications

    • Histone mutants (K-to-R) that cannot be modified

  • Positive Controls:

    • Samples treated with high concentrations of β-hydroxybutyrate (e.g., 10 mM)

    • Samples from fasted animals with verified elevated β-hydroxybutyrate levels

    • Synthetic peptides with defined β-hydroxybutyrylation

  • Specificity Controls:

    • Peptide competition assays

    • Detection with multiple antibodies against the same modification

  • Technical Controls:

    • Input controls for ChIP experiments

    • Loading controls for western blots (total histone H3)

    • Isotopically labeled internal standards for mass spectrometry

  • Biological Controls:

    • Comparison between fed and fasted states

    • Normal vs. diabetic ketoacidosis models

    • Time-course analysis to capture dynamic changes

• How should researchers interpret differences in β-hydroxybutyrylation patterns across experimental conditions?

  Interpretation of β-hydroxybutyrylation data requires careful consideration:

  • Correlation with Metabolic Status: Changes in β-hydroxybutyrylation should be interpreted in the context of β-hydroxybutyrate levels. Studies show that during fasting, when serum β-hydroxybutyrate increases 7-fold, histone Kbhb marks increase 4-40 fold .

  • Comparison with Other Modifications: Analyze how β-hydroxybutyrylation patterns compare with other modifications like acetylation and methylation. Evidence suggests that different modifications may mark distinct sets of genes, even when occurring at the same residue .

  • Genomic Distribution Analysis: Consider the distribution of modifications across genomic features (promoters, enhancers, gene bodies). β-hydroxybutyrylation has been shown to be enriched at promoters of active genes .

  • Integration with Transcriptomic Data: Correlate modification changes with gene expression data to identify functional associations. β-hydroxybutyrylation has been linked to upregulation of starvation-responsive genes .

  • Tissue-Specific Patterns: Recognize that modification patterns may vary significantly between tissues. Both liver and kidney show induction of Kbhb during fasting, but potentially with different patterns .

  • Temporal Dynamics: Consider the time-dependent nature of modifications, as some changes may be transient while others are sustained.

  • Statistical Significance: Apply appropriate statistical tests and multiple testing corrections when comparing modification patterns between conditions.

Advanced Research Insights

• How do β-hydroxybutyrylation marks integrate with the broader network of histone modifications?

  β-hydroxybutyrylation exists within a complex network of histone modifications:

  • Combinatorial Effects: Research suggests that β-hydroxybutyrylation may function in combination with other modifications to create specific "histone codes" that direct gene expression in response to metabolic states .

  • Modification Interplay: The presence of β-hydroxybutyrylation may enhance or inhibit the deposition of other marks. For example, lysine residues that can be both acetylated and β-hydroxybutyrylated (such as H3K4, H3K9, H3K18, H4K8) may show differential regulatory patterns under different metabolic conditions .

  • Shared Enzymatic Machinery: Some enzymes may recognize or modify multiple acylation types with different efficiencies, creating competitive or cooperative relationships between modifications.

  • Metabolic Sensing: While many histone modifications are regulated by signaling pathways, β-hydroxybutyrylation appears to be uniquely positioned as a direct sensor of cellular metabolic state through β-hydroxybutyrate levels .

  • Evolutionary Conservation: The conservation of histone Kbhb across species from yeast to humans suggests fundamental importance in chromatin regulation .

  • Modification Breadth: With 44 identified histone Kbhb sites, the breadth of this modification is comparable to well-established marks like acetylation, indicating its significant role in chromatin regulation .

• What are the implications of β-hydroxybutyrylation for understanding metabolic disease mechanisms?

  β-hydroxybutyrylation provides a direct link between metabolism and gene regulation with important disease implications:

  • Diabetic Ketoacidosis: Studies show that histone Kbhb marks are dramatically induced in livers from mice with streptozotocin-induced diabetic ketoacidosis, suggesting this modification may mediate some of the transcriptional changes in this condition .

  • Fasting Response: The significant induction of histone Kbhb during fasting (4-40 fold increases) compared to minimal changes in acetylation suggests a specialized role in coordinating gene expression during nutrient deprivation .

  • Ketogenic Diet Effects: β-hydroxybutyrylation may mediate some of the transcriptional changes and potential therapeutic benefits associated with ketogenic diets.

  • Metabolic Reprogramming: Cancer cells and other disease states involving metabolic reprogramming may show altered patterns of β-hydroxybutyrylation that contribute to pathological gene expression.

  • Therapeutic Targeting: Understanding the specific genes and pathways regulated by β-hydroxybutyrylation could reveal new therapeutic targets for metabolic disorders.

  • Biomarker Potential: Patterns of histone β-hydroxybutyrylation could serve as biomarkers for metabolic status and disease progression.

• What emerging technologies might advance the study of β-hydroxybutyryl-HIST1H3A (K4) and related modifications?

  Several cutting-edge technologies hold promise for advancing our understanding of β-hydroxybutyrylation:

  • Single-Cell Epigenomics: Technologies that allow mapping of histone modifications at single-cell resolution will reveal cell-type-specific patterns of β-hydroxybutyrylation.

  • Live-Cell Imaging: Development of specific probes to visualize β-hydroxybutyrylation dynamics in living cells could provide unprecedented insights into real-time regulation.

  • CRISPR-Based Epigenome Editing: Targeted modulation of β-hydroxybutyrylation at specific genomic loci will help establish causative relationships with gene expression.

  • Multi-Omics Integration: Combining epigenomic, transcriptomic, proteomic, and metabolomic data will provide systems-level understanding of β-hydroxybutyrylation function.

  • Structural Biology: Structural studies of reader proteins in complex with β-hydroxybutyrylated histones will reveal molecular mechanisms of recognition.

  • Spatial Epigenomics: Technologies that preserve spatial information while mapping modifications will illuminate the nuclear organization of β-hydroxybutyrylated chromatin.

  • Computational Modeling: Machine learning approaches may help predict the functional consequences of β-hydroxybutyrylation patterns and their interplay with other modifications.