β-hydroxybutyryl-HIST1H3A (K27) Antibody

What is Histone β-hydroxybutyrylation and Why is it Important for Epigenetic Research?

Histone β-hydroxybutyrylation (Kbhb) is a post-translational modification that occurs on lysine residues of histone proteins, including the K27 position of histone H3.1 (HIST1H3A). This modification is part of the complex "histone code" that regulates DNA accessibility and chromatin structure.

Histones are core components of nucleosomes that wrap and compact DNA into chromatin, playing a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . β-hydroxybutyrylation represents a metabolically-sensitive epigenetic mark that connects cellular metabolism with gene expression regulation.

Research has shown that β-hydroxybutyryl-CoA serves as the cofactor for lysine β-hydroxybutyrylation, and cellular β-hydroxybutyrate concentrations can directly impact histone Kbhb levels . This connection between metabolism and epigenetic regulation makes Kbhb particularly important for understanding how metabolic states influence gene expression patterns.

The significance of this modification extends beyond basic chromatin biology to potential applications in understanding metabolic diseases, fasting responses, and diabetic conditions, as studies have identified elevated histone Kbhb levels in fasted and diabetic mouse livers .

How Does β-hydroxybutyryl-HIST1H3A (K27) Differ from Other Histone Modifications at the Same Residue?

The lysine 27 residue on histone H3 can undergo several different post-translational modifications, each with distinct functional consequences for chromatin regulation:

Comparison of H3K27 Modifications:

H3K27me3 is specifically associated with the downregulation of nearby genes through the formation of heterochromatic regions . In contrast, β-hydroxybutyrylation at K27 is thought to be responsive to cellular metabolic states, particularly elevated β-hydroxybutyrate levels that occur during fasting, ketogenic diets, or diabetic states .

These modifications can be mutually exclusive on the same residue, creating a dynamic regulatory system where metabolic signals can potentially override existing chromatin states through changes in histone modifications.

What are the Recommended Applications for β-hydroxybutyryl-HIST1H3A (K27) Antibody?

The β-hydroxybutyryl-HIST1H3A (K27) antibody has been validated for several experimental applications in epigenetic and chromatin research:

Validated Applications:

• Western Blotting (WB): Recommended dilution range of 1:100-1:1000

• Immunocytochemistry (ICC): Recommended dilution range of 1:20-1:200

• Chromatin Immunoprecipitation (ChIP): For detecting genomic regions enriched for this modification

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of the modification

For Western blotting, the expected band size is approximately 16 KDa, which corresponds to the molecular weight of histone H3 . This technique is particularly useful for tracking global changes in H3K27bhb levels in response to metabolic interventions or disease states.

ChIP experiments combined with sequencing (ChIP-seq) can provide genome-wide mapping of this modification, allowing researchers to identify specific genes and regulatory elements that may be influenced by metabolic changes through this epigenetic mechanism.

How Should Researchers Validate the Specificity of β-hydroxybutyryl-HIST1H3A (K27) Antibody?

Antibody specificity is critical for accurate interpretation of epigenetic modification studies. Recent research has highlighted concerns about non-specific recognition by some histone modification antibodies , making validation essential.

Recommended Validation Protocol:

• Peptide Competition Assay:

  • Pre-incubate antibody with increasing concentrations of synthetic β-hydroxybutyrylated K27 peptides

  • Perform Western blotting on histone extracts

  • Signal reduction indicates specific binding

• Cross-reactivity Testing:

  • Test antibody against peptide arrays containing various histone modifications

  • Include other modifications at K27 (acetylation, methylation) and β-hydroxybutyrylation at other positions

• Mass Spectrometry Validation:

  • Perform immunoprecipitation using the antibody on histones from cells treated with and without sodium β-hydroxybutyrate

  • Analyze enriched peptides by mass spectrometry to confirm specific enrichment of K27bhb

• Metabolic Induction Control:

  • Compare antibody signal in cells treated with sodium β-hydroxybutyrate versus untreated cells

  • A specific increase should be observed in treated cells

• Knockout/Knockdown Controls:

  • Generate cells with mutations at K27 (K27R) to prevent modification

  • Antibody signal should be absent in these cells

Recent studies have shown that some histone modification antibodies can recognize multiple modifications. For example, research on H3K9bhb antibodies revealed recognition of non-specific targets , highlighting the importance of thorough validation before experimental use.

What is the Metabolic Regulation of Histone β-hydroxybutyrylation?

Histone β-hydroxybutyrylation represents a direct link between cellular metabolism and epigenetic regulation, with several key mechanisms:

Metabolic Pathways Influencing β-hydroxybutyrylation:

Research has demonstrated that isotopic sodium β-hydroxybutyrate treatment leads to a dose-dependent increase of isotopic bhb-CoA in cells, confirming the metabolic pathway from exogenous β-hydroxybutyrate to the active cofactor for histone modification .

What Controls Should Be Included When Using β-hydroxybutyryl-HIST1H3A (K27) Antibody in ChIP Experiments?

Chromatin immunoprecipitation (ChIP) with β-hydroxybutyryl-HIST1H3A (K27) antibody requires rigorous controls to ensure reliable and interpretable results:

Essential Controls for ChIP Experiments:

• Input Control:

  • A portion of chromatin sample prior to immunoprecipitation

  • Used for normalization of enrichment calculations

• Negative Controls:

  • IgG control from the same species as the primary antibody (rabbit)

  • No-antibody control to assess non-specific binding

  • Regions known to lack histone H3 or the K27bhb modification

• Positive Controls:

  • Cells treated with sodium β-hydroxybutyrate to increase K27bhb levels

  • Genomic regions known to be enriched for the modification

• Specificity Controls:

  • Pre-block antibody with β-hydroxybutyrylated K27 peptide

  • Compare enrichment patterns with other K27 modifications (K27me3, K27ac)

• Biological Validation:

  • Metabolic manipulations that alter cellular β-hydroxybutyrate levels

  • Genetic manipulations of enzymes involved in β-hydroxybutyrate metabolism

When analyzing ChIP data, researchers should be aware that changes in chromatin accessibility can affect antibody binding independently of actual modification levels. Therefore, combining ChIP with other techniques like mass spectrometry is recommended for conclusive results.

How Does β-hydroxybutyrylation at K27 Relate to Other Histone Modifications?

The interplay between β-hydroxybutyrylation at K27 and other histone modifications creates a complex regulatory network:

Relationships with Other Modifications:

• Competition for the Same Residue:

  • K27 can be modified by acetylation, methylation, or β-hydroxybutyrylation

  • These modifications are mutually exclusive at the single-molecule level

  • Changes in cellular metabolism may shift the balance between these modifications

• Functional Interplay:

  • H3K27me3 is associated with gene repression and heterochromatin

  • H3K27ac is generally associated with active enhancers and promoters

  • H3K27bhb may represent a metabolic override of these regulatory states

• Cross-talk with Other Sites:

  • β-hydroxybutyrylation has been identified at 44 different histone sites

  • Many of these sites, such as H4K8, H4K12, H3K4, H3K9, and H3K56, are also targets for acetylation and methylation

  • This suggests complex regulatory interactions across the histone code

Understanding the temporal dynamics and relative abundance of these modifications requires sophisticated approaches combining ChIP-seq, mass spectrometry, and functional genomics.

What are the Optimal Storage and Handling Conditions for β-hydroxybutyryl-HIST1H3A (K27) Antibody?

Proper storage and handling are essential for maintaining antibody activity and ensuring consistent experimental results:

Storage Recommendations:

• Short-term Storage (≤1 week):

  • Store at +4°C

• Long-term Storage:

  • Aliquot into small volumes to minimize freeze-thaw cycles

  • Store at -20°C or -80°C

  • Avoid repeated freeze and thaw cycles, as each cycle can reduce antibody binding activity by approximately half

• Working Solution Preparation:

  • Dilute in appropriate buffer immediately before use

  • For Western blotting, prepare dilutions in the range of 1:100-1:1000

  • For immunocytochemistry, prepare dilutions in the range of 1:20-1:200

• Quality Control:

  • Monitor antibody performance over time using positive control samples

  • Consider validation with fresh antibody if unexpected results occur after storage

Following these storage guidelines will help maintain antibody specificity and sensitivity for reliable detection of β-hydroxybutyryl-HIST1H3A (K27).

How Can Researchers Experimentally Induce β-hydroxybutyrylation to Study Its Functional Consequences?

Several experimental approaches can be used to modulate histone β-hydroxybutyrylation levels:

Experimental Induction Methods:

• Direct Metabolite Treatment:

  • Treat cells with sodium β-hydroxybutyrate (10 mM is commonly used)

  • This leads to dose-dependent increases in histone Kbhb levels

  • Can be detected using Western blotting with specific antibodies

• Metabolic Manipulation:

  • Fasting conditions (in vivo or in vitro)

  • Ketogenic diet in animal models

  • Streptozotocin (STZ) treatment to induce diabetic conditions

  • These conditions naturally elevate β-hydroxybutyrate levels

• Isotopic Labeling:

  • Treat cells with isotopically labeled sodium β-hydroxybutyrate

  • Track conversion to bhb-CoA and incorporation into histones

  • Analyze by mass spectrometry to confirm site-specific modification

• Enzyme Modulation:

  • Target enzymes involved in β-hydroxybutyrate metabolism

  • Inhibit deacylases that might remove the modification

Research has shown that histone Kbhb levels at specific sites (H3K9bhb, H3K18bhb, H4K8bhb, and H3K4bhb) can all be induced in a β-hydroxybutyrate dose-dependent manner, while corresponding acetylation levels show minimal changes .

What Technical Challenges Exist in β-hydroxybutyryl-HIST1H3A (K27) Antibody Research?

Researchers should be aware of several technical challenges that may impact experimental results:

Common Technical Challenges:

• Antibody Specificity Issues:

  • Recent research has identified non-specific recognition by some histone modification antibodies

  • Cross-reactivity with other modifications may occur

  • Rigorous validation is essential before experimental use

• Detecting Low Abundance Modifications:

  • β-hydroxybutyrylation may exist at low levels in some biological conditions

  • Enrichment techniques may be necessary before detection

  • Signal amplification methods may improve detection sensitivity

• Distinguishing Between Similar Modifications:

  • β-hydroxybutyrylation and butyrylation have similar structures

  • Mass spectrometry can differentiate these modifications but requires specialized equipment

• Quantification Challenges:

  • Semi-quantitative methods like Western blotting have limitations

  • Consider mass spectrometry for absolute quantification

  • Internal standards may improve quantification accuracy

• Functional Analysis Complexity:

  • Determining causal relationships between the modification and gene expression

  • Separating the effects of β-hydroxybutyrylation from other metabolic consequences

  • Need for genome-editing approaches to specifically manipulate K27 modification

Researchers have observed that antibody signal intensity in Western blots may not always accurately reflect modification abundance as measured by mass spectrometry , highlighting the importance of orthogonal validation approaches.