β-hydroxybutyryl-HIST1H4A (K5) Antibody

What is β-hydroxybutyrylation of histone H4 and why is it important in epigenetic research?

β-hydroxybutyrylation (bhb) is a post-translational modification (PTM) that occurs on lysine residues of histone proteins, including histone H4 at lysine 5 (K5). Histones are core components of nucleosomes that wrap and compact DNA into chromatin, thereby playing central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability .

The histone code, which includes β-hydroxybutyrylation alongside other modifications like acetylation, methylation, and phosphorylation, regulates DNA accessibility to cellular machinery. β-hydroxybutyrylation specifically has been implicated in metabolic regulation pathways, particularly during starvation conditions or diabetic ketoacidosis . The modification appears to activate transcription of specific genes associated with starvation-responsive metabolic pathways, making it an important target for researchers studying metabolic disorders and cellular energy homeostasis .

To study this modification effectively, researchers employ antibodies specific to β-hydroxybutyrylated histones, such as the β-hydroxybutyryl-HIST1H4A (K5) antibody, which recognizes this specific modification at the lysine 5 position of histone H4.

How does β-hydroxybutyrylation of histone H4K5 differ from other histone acylations?

β-hydroxybutyrylation (bhb) differs from other acylations such as acetylation or butyrylation primarily in its chemical structure, metabolic origins, and potentially in its functional consequences.

From a structural perspective, β-hydroxybutyrylation contains a hydroxyl group on the beta carbon of the butyrate chain, distinguishing it from simple butyrylation . This structural difference stems from its metabolic precursor, β-hydroxybutyrate (BHB), a ketone body produced during fasting, prolonged exercise, or in diabetic ketoacidosis .

Functionally, comparative studies of histone acylations have revealed distinct patterns:

Methodologically, when studying these modifications, researchers must be cautious about antibody cross-reactivity. As demonstrated in recent studies, antibodies for specific acylations (like H3K9bhb) can sometimes recognize alternative histone modifications, potentially undermining experimental reliability .

What are the recommended experimental conditions for detecting β-hydroxybutyryl-HIST1H4A (K5) using Western blotting?

For optimal detection of β-hydroxybutyryl-HIST1H4A (K5) using Western blotting, researchers should follow these methodological guidelines:

Sample preparation:

• Treat cells with β-hydroxybutyrate (BHB) to enhance the signal (typically 50mM for 24-72 hours is effective)

• Harvest cells and prepare whole cell lysates using standard protocols

• Lyse cells in buffer containing protease inhibitors to prevent degradation of histones

Western blotting protocol:

• Resolve proteins using SDS-PAGE (15-18% gels are recommended for histones due to their low molecular weight)

• Transfer to nitrocellulose membranes using a wet or semi-dry transfer system

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

• Incubate with anti-β-hydroxybutyryl-HIST1H4A (K5) antibody at recommended dilutions (1:100-1:1000)

• Use appropriate secondary antibodies (typically HRP-conjugated)

• Develop using chemiluminescent substrate and image with a compatible system

Controls and validation:

• Include positive controls (BHB-treated samples) and negative controls (untreated samples)

• Consider using multiple cell lines to verify consistency (HEK293T, A549, K562, and HepG2 have been successfully used)

• The expected molecular weight for histone H4 is approximately 11-12 kDa

When interpreting results, researchers should be aware that the intensity of Western blot signals may not always directly correlate with the abundance of the specific modification, as demonstrated in studies with other β-hydroxybutyrylated histones .

How can researchers distinguish between enzymatic and non-enzymatic β-hydroxybutyrylation of histones?

Distinguishing between enzymatic and non-enzymatic β-hydroxybutyrylation requires careful experimental design and multiple complementary approaches:

In vitro enzymatic assays:

• Perform in vitro reactions with purified histones, β-hydroxybutyryl-CoA, and candidate histone acetyltransferases (HATs)

• Include appropriate controls without enzymes to assess non-enzymatic modification rates

• Quantify modifications using high-resolution mass spectrometry to accurately measure acylation levels

Correlation analysis:

• Measure intracellular levels of acyl-CoA donors using metabolomic approaches

• Simultaneously quantify histone acyl-PTMs using proteomic methods

• Calculate correlation coefficients between acyl-CoA abundance and corresponding histone modifications

Dose-dependent studies:

• Supplement in nucleo reactions with varying concentrations of β-hydroxybutyryl-CoA

• Observe the dose-dependent increase in histone β-hydroxybutyrylation

• Compare the rate of increase between samples with and without potential enzymatic catalysts

Research has shown that while histone acetyltransferases can catalyze acylations on histones, they are generally less efficient with larger acyl-CoAs compared to acetyl-CoA . Importantly, acyl-CoAs can also directly acylate histones through non-enzymatic mechanisms, particularly at higher concentrations . High correlation (R² > 0.99) between acyl-CoA abundance and corresponding acyl-PTMs suggests that metabolite concentration is a key determinant of modification levels .

For β-hydroxybutyrylation specifically, researchers should consider physiological contexts where BHB levels increase dramatically (starvation, ketogenic diets, diabetic ketoacidosis) as these conditions may favor non-enzymatic modification mechanisms.

What are the key considerations when assessing antibody specificity for β-hydroxybutyryl-HIST1H4A (K5)?

Ensuring antibody specificity for β-hydroxybutyryl-HIST1H4A (K5) is critical for reliable research outcomes. Recent studies highlighting specificity issues with other β-hydroxybutyrylation antibodies (e.g., H3K9bhb) demonstrate the importance of rigorous validation approaches :

Comprehensive cross-reactivity testing:

• Test antibody reactivity against structurally similar modifications (acetylation, butyrylation)

• Treat cells with different metabolites (BHB, butyrate) and HDAC inhibitors (TSA)

• Compare Western blot signals between treated and untreated samples

• Look for unexpected signals that might indicate cross-reactivity

Mass spectrometry validation:

• Perform immunoprecipitation with the antibody of interest

• Analyze enriched proteins by mass spectrometry

• Quantify the percentage of peptides containing the target modification versus other modifications

• For reliable antibodies, target modifications should be significantly enriched in IP samples from appropriately treated cells

Peptide competition assays:

• Pre-incubate antibodies with synthetic peptides containing β-hydroxybutyryl-K5 modifications

• Include control peptides with other modifications (acetylation, butyrylation)

• Observe whether specific peptides block antibody binding in Western blot or ChIP experiments

Examples from existing research with H3K9bhb antibody demonstrate potential pitfalls:

• H3K9bhb antibodies showed unexpected signals with butyrate or TSA treatment

• Mass spectrometry revealed only 1.74% Kbhb-containing peptides in butyrate-treated samples despite strong Western blot signals

• This suggests recognition of alternative modifications that undermine experimental reliability

Researchers should verify whether their specific β-hydroxybutyryl-HIST1H4A (K5) antibody has been validated using these rigorous approaches, particularly if it will be used for chromatin immunoprecipitation (ChIP) experiments to assess gene expression regulation.

How can researchers effectively differentiate between β-hydroxybutyrylation at different lysine residues on histone H4?

Differentiating between β-hydroxybutyrylation at different lysine residues on histone H4 requires sophisticated methodological approaches:

Site-specific antibody selection and validation:

• Use antibodies specifically raised against distinct β-hydroxybutyrylated lysine residues (e.g., H4K5bhb, H4K8bhb, H4K91bhb)

• Validate each antibody's specificity using synthetic peptides containing single modifications

• Test cross-reactivity between antibodies targeting different lysine residues

• Confirm specificity through mass spectrometry analysis of immunoprecipitated material

Mass spectrometry-based approaches:

• Employ bottom-up proteomics with tryptic digestion to generate histone peptides

• Use parallel reaction monitoring (PRM) for targeted quantification of specific modified peptides

• Develop specific transitions for each β-hydroxybutyrylated lysine residue

• Calculate modification stoichiometry (percentage of each lysine that is modified)

The following analytical workflow is recommended:

• Extract histones using acid extraction methods

• Perform propionylation of unmodified lysines (to prevent trypsin cleavage)

• Digest with trypsin

• Analyze by LC-MS/MS with high mass accuracy

• Process data using specialized software for histone PTM quantification

When analyzing multiple modifications, researchers should be aware of potential functional differences between lysine residues. For example, while H4K5 is located in the N-terminal tail accessible for modification , H4K91 is located in the histone fold domain at the histone-histone interface , potentially resulting in different functional consequences when modified.

What approaches should be used to investigate the metabolic conditions that influence β-hydroxybutyryl-HIST1H4A (K5) levels?

Investigating the metabolic conditions influencing β-hydroxybutyryl-HIST1H4A (K5) levels requires integrating metabolomic and epigenomic approaches:

Cellular metabolic manipulation:

• Induce ketogenic states through glucose deprivation, high-fat/low-carbohydrate media, or direct BHB supplementation (typically 5-50mM)

• Model pathological conditions like diabetic ketoacidosis using appropriate cell or animal models

• Time-course experiments to track the dynamic relationship between metabolite levels and histone modifications

Integrated analytical approaches:

• Quantify intracellular β-hydroxybutyryl-CoA levels using LC-MS/MS metabolomics

• Simultaneously measure histone β-hydroxybutyrylation using proteomic methods

• Correlate metabolite concentrations with modification levels

Sample preparation for metabolomic analysis:

• Harvest cells treated under various conditions (control, BHB, butyrate, etc.)

• Extract metabolites using appropriate protocols (e.g., methanol/methyl butyl ether extraction)

• Include internal standards for accurate quantification

From previous research, we know that starvation and diabetic ketoacidosis significantly increase β-hydroxybutyrylation on histones . Researchers should consider the following experimental design:

When analyzing results, researchers should be mindful that changes in modification levels may result from altered acyl-CoA concentrations, enzymatic activity changes, or both .

What are the best experimental designs to elucidate the transcriptional effects of β-hydroxybutyryl-HIST1H4A (K5)?

To elucidate the transcriptional effects of β-hydroxybutyryl-HIST1H4A (K5), researchers should employ multifaceted experimental designs that connect this specific modification to gene expression outcomes:

Chromatin immunoprecipitation approaches:

• Perform ChIP-seq using validated β-hydroxybutyryl-HIST1H4A (K5) antibodies

• Include appropriate controls to account for potential antibody cross-reactivity

• Map genome-wide distribution of H4K5bhb under various metabolic conditions

• Integrate with RNA-seq data to correlate modification patterns with gene expression changes

• Conduct comparative analysis with other histone acylations (acetylation, butyrylation) to identify unique targets

Functional genomics strategies:

• Utilize CRISPR-based epigenome editing to site-specifically modify H4K5 residues

• Generate histone mutants (K5R, K5Q) to mimic unmodified or permanently modified states

• Express engineered reader proteins that specifically recognize β-hydroxybutyrylated H4K5

• Perform reporter assays with promoters identified from ChIP-seq studies

Mechanistic studies:

• Identify proteins that specifically recognize β-hydroxybutyrylated H4K5 using affinity purification with modified peptides

• Characterize enzymes responsible for adding (writers) or removing (erasers) this modification

• Investigate the crosstalk between H4K5bhb and other histone modifications

Based on research on related modifications, β-hydroxybutyrylation likely activates transcription of specific gene sets . Researchers should focus on:

• Metabolic pathway genes, particularly those involved in adaptation to ketogenic states

• Genes regulated during fasting or caloric restriction

• Comparison with genes affected by other acylations to identify modification-specific effects

When interpreting results, consider the physiological context where β-hydroxybutyrylation increases naturally (fasting, ketogenic diet) and how this might relate to adaptive gene expression programs.