β-hydroxybutyryl-HIST1H3A (K18) Antibody

What is histone lysine β-hydroxybutyrylation (Kbhb) and what is its significance?

Histone lysine β-hydroxybutyrylation (Kbhb) is a post-translational modification (PTM) of histone proteins where a β-hydroxybutyryl group is added to specific lysine residues. This modification represents a critical link between cellular metabolism and gene expression regulation. Kbhb is considered an evolutionarily conserved PTM present across diverse eukaryotic species, including yeast (S. cerevisiae), Drosophila, mouse, and human cells . The modification is particularly significant as a mechanism through which the ketone body β-hydroxybutyrate (BHB), produced during fasting, starvation, or ketogenic diets, can directly influence the epigenome and transcriptional regulation.

How does β-hydroxybutyrylation differ from acetylation on histones?

β-hydroxybutyrylation differs from acetylation in several key aspects:

• Metabolic origin: Kbhb is directly linked to β-hydroxybutyrate metabolism, while acetylation is connected to acetyl-CoA levels

• Regulatory response: Treatment of cells with sodium β-hydroxybutyrate significantly increases histone Kbhb levels in a dose-dependent manner, whereas histone acetylation shows minimal changes under the same conditions

• Gene association: Kbhb is associated with genes upregulated in starvation-responsive metabolic pathways, potentially creating a distinct regulatory signature from acetylation

• Molecular structure: The β-hydroxybutyryl group contains a hydroxyl group not present in acetyl modifications, potentially allowing for different interactions with chromatin reader proteins

Importantly, experimental evidence shows that treating cells with β-hydroxybutyrate dramatically increases Kbhb levels (up to 10-fold at high concentrations) while causing only marginal changes in histone acetylation .

What biological processes are associated with H3K18bhb?

H3K18bhb has been associated with several important biological processes:

• Metabolic regulation: H3K18bhb levels increase in response to elevated cellular β-hydroxybutyrate, linking this modification to metabolic states like fasting and ketosis

• Transcriptional activation: Similar to other histone acylations, H3K18bhb is generally associated with active gene transcription

• Starvation response: In liver cells from fasted or diabetic mice, increased H3K18bhb correlates with upregulation of starvation-responsive genes

• Cellular development: BHB treatment and subsequent increase in Kbhb modifications influences genes involved in mitochondrial metabolism and development pathways

How specific are commercially available histone Kbhb antibodies?

The specificity of commercially available histone Kbhb antibodies varies considerably based on the target site and antibody production methods. Research has revealed significant cross-reactivity issues with some Kbhb antibodies:

• Site-specific concerns: Studies have demonstrated that H3K9bhb antibodies recognize additional modifications beyond β-hydroxybutyrylation, likely including acetylation, which undermines their reliability for ChIP experiments

• Validation necessity: Mass spectrometry analysis of immunoprecipitated histones using H3K9bhb antibodies from butyrate-treated cells showed minimal Kbhb-containing peptides (only 1.74%), suggesting significant non-specific binding

• Contrasting reliability: The pan-Kbhb antibody and some site-specific antibodies (like H4K8bhb) demonstrated good specificity, showing strong signals specifically in BHB-treated cells but not with butyrate or TSA treatment

These findings underscore the critical importance of rigorous validation of any Kbhb antibody before use in research applications.

What methods should be used to validate β-hydroxybutyryl histone antibodies?

Comprehensive validation of β-hydroxybutyryl histone antibodies should include multiple approaches:

• Dot blot assays and competition experiments with synthesized peptides containing known modifications

• Western blot analysis comparing cells treated with:

  • β-hydroxybutyrate (should increase signal)

  • Structurally similar compounds like butyrate

  • HDAC inhibitors like Trichostatin A (TSA)

• Immunoprecipitation followed by mass spectrometry to confirm:

  • Enrichment of Kbhb-modified peptides in BHB-treated samples

  • Identification of the specific modified residues

• Dose-dependency tests with increasing BHB concentrations to confirm proportional increases in antibody signal

A properly validated antibody should show robust specificity for β-hydroxybutyrylated histones and minimal cross-reactivity with other modifications.

How can mass spectrometry confirm the specificity of H3K18bhb antibody detection?

Mass spectrometry serves as the gold standard for confirming H3K18bhb antibody specificity by:

• Direct identification of modification: MS/MS can unambiguously identify β-hydroxybutyrylated lysine residues based on their characteristic mass shift and fragmentation patterns

• Isotopic labeling validation: Using isotopically labeled β-hydroxybutyrate (e.g., [13C2]-β-hydroxybutyrate) in cell culture creates a distinctive mass shift that can differentiate genuine Kbhb modifications from other PTMs

• Quantitative assessment: MS can determine the percentage of immunoprecipitated peptides that genuinely contain the Kbhb modification (as demonstrated in the H3K9bhb antibody evaluation where only 13.99% of peptides from BHB-treated samples contained Kbhb)

The mass spectrometry data should reveal the expected mass shift associated with β-hydroxybutyrylation specifically at K18 of histone H3 and show enrichment of this modification in the immunoprecipitated fraction.

What are the optimal protocols for Western blot analysis of H3K18bhb?

For optimal Western blot analysis of H3K18bhb, researchers should follow these methodological guidelines:

• Sample preparation:

  • Extract histones using acid extraction methods or commercial histone extraction kits

  • For whole cell lysates, use PBS-T (PBS/1% Triton X-100) supplemented with protease and phosphatase inhibitors

  • Centrifuge at 10,000 rpm for 5 minutes and collect supernatant

  • Quantify protein using DC Protein Assay Kit or similar

• SDS-PAGE and transfer:

  • Mix samples with 4x Laemmli buffer/10% beta-mercaptoethanol and boil at 95°C for 5 minutes

  • Resolve equivalent amounts of protein by SDS-PAGE

  • Transfer to nitrocellulose membrane using a semi-dry transfer system

• Immunoblotting:

  • Stain with Ponceau S to confirm equal loading

  • Block with 5% milk in TBS-T for 30-60 minutes

  • Incubate with H3K18bhb primary antibody (validated for specificity)

  • Apply HRP-conjugated secondary antibodies

  • Develop using chemiluminescent substrate (SuperSignal West Pico PLUS or Femto Maximum Sensitivity Substrate)

• Controls:

  • Include BHB-treated positive control samples (treatment with 5-10 mM sodium β-hydroxybutyrate for 24 hours)

  • Include total H3 antibody detection as loading control

  • Consider including H3K18ac detection to distinguish between these modifications

How can ChIP-seq be optimized for H3K18bhb detection?

For optimal ChIP-seq results with H3K18bhb antibodies, researchers should:

• Promote target modification:

  • Treat cells with β-hydroxybutyrate (5-10 mM) for 24 hours prior to ChIP to increase Kbhb levels

  • Consider using starvation conditions or ketogenic models if studying physiological contexts

• Cross-linking and chromatin preparation:

  • Fix cells with 1% formaldehyde for 10 minutes at room temperature

  • Extract and sonicate chromatin to fragments of 200-500 bp

  • Reserve a portion of chromatin as input control

• Immunoprecipitation:

  • Use extensively validated H3K18bhb antibodies with confirmed specificity

  • Include appropriate negative controls (IgG, non-modified histone)

  • Perform parallel ChIP with H3K18ac antibody to compare distribution patterns

• Sequencing and analysis:

  • Perform spike-in normalization to account for global changes in modification levels

  • Compare H3K18bhb peaks with transcriptome data to correlate with gene expression

  • Analyze enrichment at promoters, enhancers, and gene bodies separately

• Validation:

  • Confirm key findings with ChIP-qPCR targeting specific genomic regions

  • Correlate ChIP-seq results with RNA-seq data from similarly treated cells

What controls are essential for immunofluorescence detection of H3K18bhb?

When performing immunofluorescence for H3K18bhb detection, include these essential controls:

• Treatment controls:

  • Untreated negative control cells

  • Cells treated with varying BHB concentrations (2 mM, 6 mM, and 20 mM) to demonstrate dose-dependent increases in signal intensity

  • Cells treated with butyrate or HDAC inhibitors as specificity controls

• Antibody controls:

  • Primary antibody omission control

  • Secondary antibody-only control

  • Pre-absorption of antibody with H3K18bhb synthetic peptide to demonstrate specificity

  • Parallel staining with other histone modification antibodies (H3K18ac) for comparison

• Technical considerations:

  • Adjust laser power to avoid saturation at higher BHB concentrations

  • Use quantitative analysis methods to measure fluorescence intensity

  • Include DAPI or other nuclear counterstain for proper nuclear localization

Research with human dermal fibroblasts has shown that 2 mM BHB increases Kbhb levels by ~2.21-fold, 6 mM by ~5.97-fold, and 20 mM by over 10-fold compared to untreated controls . These dramatic differences provide excellent positive controls for validating immunofluorescence protocols.

How does cellular β-hydroxybutyrate concentration affect H3K18bhb levels?

Cellular β-hydroxybutyrate concentration directly regulates H3K18bhb levels through multiple mechanisms:

• Dose-dependent response: Treatment of cells with increasing concentrations of sodium β-hydroxybutyrate leads to proportional increases in H3K18bhb levels

• Metabolic conversion pathway:

  • Sodium β-hydroxybutyrate is converted to β-hydroxybutyryl-CoA in cells

  • β-hydroxybutyryl-CoA serves as the cofactor for enzymatic lysine β-hydroxybutyrylation

  • Isotopic labeling experiments with [13C2]-β-hydroxybutyrate confirm this metabolic conversion pathway

• Physiological relevance: H3K18bhb levels increase significantly in livers from:

  • Fasted mice (where ketone bodies naturally increase)

  • Streptozotocin-induced diabetic mice (a model of type 1 diabetes with elevated ketone production)

This relationship creates a direct link between metabolic state and epigenetic regulation, allowing cells to adapt gene expression in response to nutritional status.

Table 1: Dose-dependent increase in Kbhb levels in human dermal fibroblasts treated with varying concentrations of β-hydroxybutyrate for 24h .

What enzymes are involved in the regulation of histone β-hydroxybutyrylation?

The complete enzymatic machinery regulating histone β-hydroxybutyrylation is still being elucidated, but several key components have been identified:

• Writers (enzymes that add the modification):

  • While specific histone β-hydroxybutyryltransferases have not been definitively identified, evidence suggests p300/CBP and other histone acetyltransferases may catalyze this reaction

  • The cofactor for this reaction is β-hydroxybutyryl-CoA, which increases in concentration when cellular β-hydroxybutyrate levels rise

• Erasers (enzymes that remove the modification):

  • Class I histone deacetylases (HDACs) likely remove β-hydroxybutyryl groups

  • Sirtuin family members (particularly SIRT3) may also act as de-β-hydroxybutyrylases

• Readers (proteins that recognize the modification):

  • Bromodomain-containing proteins that typically recognize acetylated lysines may also bind to β-hydroxybutyrylated histones

  • The specific reader proteins for H3K18bhb remain to be fully characterized

The balance between these enzymatic activities determines the dynamic regulation of H3K18bhb levels in response to metabolic changes.

How does H3K18bhb compare to other histone β-hydroxybutyrylation sites?

H3K18bhb exists among a diverse landscape of histone β-hydroxybutyrylation sites:

• Widespread modification: Research has identified at least 44 distinct histone Kbhb sites across human and mouse samples, indicating extensive modification of histones by β-hydroxybutyrylation

• Comparative dynamics:

  • H3K18bhb, H3K9bhb, H4K8bhb, and H3K4bhb all show induction in a β-hydroxybutyrate dose-dependent manner

  • Different sites may show varying sensitivity to BHB concentration changes

  • Tissue-specific patterns of Kbhb sites have been observed, with some modifications more prevalent in certain cell types

• Functional significance: Kbhb marks occur on lysine residues known to be important for chromatin structure and function when acetylated or methylated, including H3K4, H3K9, H3K56, H4K8, and H4K12

• Cell-type specificity: H3K9bhb is strongly induced in cumulus cells after BHB treatment, but only faintly detected in oocytes, suggesting cell-type specific regulation of different Kbhb sites

How can researchers address antibody cross-reactivity issues?

To address antibody cross-reactivity issues with histone Kbhb antibodies:

• Comprehensive validation:

  • Always validate antibodies using multiple techniques (Western blot, dot blot, immunoprecipitation followed by mass spectrometry)

  • Test antibody specificity against cells treated with BHB, butyrate, and HDAC inhibitors like TSA

  • Consider testing the antibody against synthetic peptides containing different modifications

• Mass spectrometry confirmation:

  • Perform IP-MS analysis to determine what percentage of immunoprecipitated peptides contain the actual Kbhb modification

  • Investigate whether other modifications are being recognized by the antibody

• Alternative approaches:

  • Use isotopically labeled BHB ([13C2]-β-hydroxybutyrate) and mass spectrometry to directly detect and quantify specific Kbhb sites

  • Consider developing new, more specific antibodies if existing ones show cross-reactivity

  • Employ pan-Kbhb antibodies for initial screening and validation

• Data interpretation caution:

  • Review published datasets using H3K9bhb and similar antibodies with caution

  • Consider potential detection of H3K9ac or other PTMs in these datasets

  • Clearly report antibody validation results in publications

What factors affect the detection sensitivity of H3K18bhb in experimental systems?

Several factors significantly impact H3K18bhb detection sensitivity:

• Basal modification levels:

  • H3K18bhb may have low prevalence in cells, even after treatment with high BHB concentrations

  • Starting with metabolically active cells or tissues (e.g., liver) may improve detection

• Treatment conditions:

  • BHB concentration and treatment duration significantly affect Kbhb levels

  • Physiological concentrations (2-6 mM) show moderate increases

  • Higher concentrations (20 mM) show dramatic increases but may not be physiologically relevant

• Technical considerations:

  • Sample preparation method affects histone extraction efficiency

  • Antibody quality and specificity are crucial determinants

  • Detection method sensitivity (chemiluminescence vs. fluorescence)

  • For microscopy, laser power settings need careful adjustment to avoid saturation at higher BHB concentrations

• Biological variables:

  • Cell type impacts the degree of Kbhb induction (e.g., cumulus cells vs. oocytes)

  • Metabolic state of the cells affects baseline levels

  • Species differences may exist in Kbhb distribution patterns

How can discrepancies between different detection methods for H3K18bhb be reconciled?

When facing discrepancies between different H3K18bhb detection methods:

• Understand method-specific limitations:

  • Western blots provide a population average but may miss cell-to-cell variability

  • Immunofluorescence shows cellular heterogeneity but can be challenging to quantify precisely

  • ChIP measures genomic distribution but not absolute abundance

  • Mass spectrometry offers direct identification but may have sensitivity limitations for low-abundance modifications

• Reconciliation strategies:

  • Use isotopically labeled BHB to generate definitive mass spectrometry reference data

  • Perform parallel analyses with multiple methods on the same samples

  • Include appropriate positive controls (BHB-treated cells) and negative controls in all experiments

  • Quantify results when possible and compare fold-changes rather than absolute values

• Technical standardization:

  • Use consistent sample preparation protocols across detection methods

  • Apply the same antibody concentration and lot for all experiments

  • Standardize BHB treatment conditions (concentration, duration, cell density)

  • Validate key findings using orthogonal approaches

• Data interpretation:

  • Consider that different methods may measure different aspects of the same modification

  • Account for potential cross-reactivity in antibody-based methods

  • When discrepancies persist, rely on mass spectrometry as the definitive approach