β-hydroxybutyryl-HIST1H2BC (K120) Antibody

What is histone lysine β-hydroxybutyrylation and why is it significant?

Histone lysine β-hydroxybutyrylation (Kbhb) is a post-translational modification where a β-hydroxybutyryl group is added to lysine residues on histone proteins. This modification has emerged as an important epigenetic mark with implications for cellular metabolism and gene regulation. It represents a critical link between metabolic state and gene expression, particularly in response to metabolic stress conditions. Research has identified 44 histone Kbhb sites, comparable to the number of known histone acetylation sites, indicating it is a widespread histone mark . This modification is especially significant because it connects nutritional status with chromatin modification and subsequent gene expression regulation.

How does β-hydroxybutyrylation differ from other histone modifications?

β-hydroxybutyrylation is distinct from other acylation modifications like acetylation, butyrylation, and propionylation in both structure and function. The modification involves the addition of a β-hydroxybutyryl group, which contains a hydroxyl group that is not present in other acylations. Unlike acetylation, which primarily responds to acetyl-CoA levels, β-hydroxybutyrylation specifically responds to β-hydroxybutyrate (BHB) levels, which are elevated during fasting, caloric restriction, and diabetic ketoacidosis . This makes it a unique metabolic sensor. Structurally, synthetic peptides containing other modification isomers (such as 2-hydroxyisobutyrylation, 2-hydroxybutyrylation, etc.) show HPLC retention times distinct from β-hydroxybutyrylated peptides, confirming the structural uniqueness of this modification .

What is the evolutionary conservation status of histone Kbhb?

Histone Kbhb appears to be highly conserved across diverse eukaryotic species. Using pan anti-Kbhb antibodies, researchers have detected histone Kbhb in yeast (S. cerevisiae), Drosophila S2 cells, mouse embryonic fibroblast (MEF) cells, and human HEK293 cells . This evolutionary conservation suggests that β-hydroxybutyrylation plays a fundamental role in eukaryotic gene regulation that has been maintained throughout evolution, underscoring its biological importance.

What are the key specifications of commercially available β-hydroxybutyryl-HIST1H2BC (K20) antibodies?

Commercial β-hydroxybutyryl-HIST1H2BC (K20) antibodies are typically polyclonal antibodies raised in rabbits. They specifically target the β-hydroxybutyrylation of histone H2B at lysine 20. These antibodies are validated for various applications including ELISA (recommended dilution 1:2000-1:10000), Western blotting (WB, recommended dilution 1:100-1:1000), and immunocytochemistry (ICC, recommended dilution 1:20-1:200) . The immunogen used is generally a peptide sequence around the β-hydroxybutyryl-Lys (20) site derived from Human Histone H2B type 1-C/E/F/G/I. These antibodies are typically supplied in liquid form in a storage buffer containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% Glycerol, 0.01M PBS, pH 7.4) .

How can researchers validate the specificity of β-hydroxybutyryl-HIST1H2BC antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For β-hydroxybutyryl-HIST1H2BC antibodies, researchers should employ multiple complementary approaches:

• Dot blot assays and competition experiments to confirm specificity against β-hydroxybutyrylation versus other modifications

• Mass spectrometry validation by comparing synthetic peptides with β-hydroxybutyrylation at specific lysine residues with in vivo-derived peptides

• HPLC retention time comparison between synthetic peptides and in vivo-derived peptides

• Fragmentation pattern analysis by high-resolution MS/MS

• Testing antibody reactivity against related modifications (acetylation, butyrylation, propionylation)

• Dose-response experiments using sodium β-hydroxybutyrate treatment to confirm increased signal with increased modification

When using site-specific antibodies like anti-H3K9bhb, researchers should be aware that at very high BHB concentrations, the antibody may recognize other β-hydroxybutyrylated sites due to structural similarities between different histone acylations .

What is the recommended storage and handling procedure for these antibodies?

For optimal performance and longevity of β-hydroxybutyryl-HIST1H2BC antibodies, they should be stored according to manufacturer recommendations, typically at -20°C in a storage buffer containing 50% glycerol and 0.01M PBS at pH 7.4 with preservatives like 0.03% Proclin 300 . For working solutions, aliquoting is recommended to avoid repeated freeze-thaw cycles. When handling the antibody, researchers should use proper laboratory techniques to prevent contamination and degradation. Diluted working solutions should be prepared fresh and used within recommended time frames to ensure consistent results across experiments.

What methods can be used to study histone β-hydroxybutyrylation in cell culture models?

Several methodological approaches can be employed to study histone β-hydroxybutyrylation in cell culture:

• Sodium β-hydroxybutyrate treatment: Cells can be treated with various concentrations of sodium β-hydroxybutyrate (typically 1-10 mM) to induce histone Kbhb in a dose-dependent manner .

• Isotopic labeling experiments: Treating cells with isotopically labeled β-hydroxybutyrate (e.g., [2,4-13C2]) allows tracking of β-hydroxybutyrylation through mass spectrometry .

• Western blotting: Using pan-Kbhb antibodies or site-specific antibodies (e.g., H3K9bhb, H3K18bhb, H4K8bhb) to detect changes in β-hydroxybutyrylation levels .

• Immunocytochemistry: Visualizing the cellular localization of β-hydroxybutyrylated histones .

• ChIP-seq: Chromatin immunoprecipitation followed by sequencing to identify genomic regions associated with β-hydroxybutyrylated histones .

• RNA-seq: Transcriptomic analysis to correlate changes in gene expression with alterations in histone β-hydroxybutyrylation .

These methods can be combined to provide comprehensive insights into the roles and regulation of histone β-hydroxybutyrylation in cellular processes.

How can histone β-hydroxybutyrylation be studied in animal models?

To study histone β-hydroxybutyrylation in animal models, researchers can employ these methodological approaches:

• Fasting models: Prolonged fasting (typically 24-48 hours) induces elevated β-hydroxybutyrate levels and subsequent increases in histone Kbhb, particularly in liver tissue .

• Diabetic models: Streptozotocin (STZ)-induced Type 1 Diabetes Mellitus models show dramatically elevated blood β-hydroxybutyrate levels (approximately 10-fold) and corresponding increases in histone Kbhb levels .

• Tissue analysis: Histones can be extracted from tissues (especially liver) for Western blot analysis using pan-Kbhb or site-specific antibodies .

• MS/MS analysis: Histone extraction followed by trypsin digestion and HPLC/MS/MS analysis to identify and quantify specific β-hydroxybutyrylation sites .

• ChIP-seq and RNA-seq: These techniques can be applied to tissue samples to correlate β-hydroxybutyrylation patterns with gene expression changes in physiological or pathological conditions .

When designing these experiments, researchers should consider appropriate controls and account for potential confounding factors such as age, sex, and genetic background of the animals.

What is the relationship between β-hydroxybutyrate metabolism and histone β-hydroxybutyrylation?

The relationship between β-hydroxybutyrate metabolism and histone β-hydroxybutyrylation represents a direct link between cellular metabolism and epigenetic regulation:

• Metabolic conversion: Sodium β-hydroxybutyrate can be converted into β-hydroxybutyryl-CoA in cells, which serves as the cofactor for lysine β-hydroxybutyrylation .

• Dose-dependent relationship: Treating cells with increasing concentrations of β-hydroxybutyrate leads to a dose-dependent increase in histone Kbhb levels, while histone acetylation remains relatively unchanged .

• Physiological relevance: During fasting or in diabetic ketoacidosis, blood β-hydroxybutyrate levels increase significantly, leading to elevated histone Kbhb levels in tissues, particularly the liver .

• Enzyme mediation: While the specific enzymes responsible for adding and removing β-hydroxybutyryl groups remain under investigation, evidence suggests that β-hydroxybutyryl-CoA can tag histones through nucleophilic attack mechanisms .

• Transcriptional impact: Increased histone Kbhb levels are associated with altered gene expression profiles, particularly in starvation-responsive metabolic pathways .

This relationship provides a mechanism by which cells can adapt their gene expression profiles in response to changing metabolic conditions, particularly during nutritional stress.

How does histone β-hydroxybutyrylation influence transcriptional regulation?

Histone β-hydroxybutyrylation plays a significant role in transcriptional regulation through several mechanisms:

• Promoter enrichment: ChIP-seq analyses demonstrate that histone Kbhb is predominantly enriched in active gene promoters .

• Starvation-responsive gene regulation: Increased H3K9bhb levels during starvation are associated with upregulation of genes involved in starvation-responsive metabolic pathways .

• Differential gene expression: RNA-seq analysis of BHB-treated cells reveals significant transcriptional changes. In bovine cumulus cells treated with BHB, approximately 2.38% of detected genes (345 out of 14,459) showed differential expression, with 166 genes upregulated and 179 downregulated .

• Distinct transcriptional signature: Principal Component Analysis of RNA-seq data from BHB-treated cells shows distinct clustering from control samples, with 83% of variance accumulated in the first two principal components .

This transcriptional influence appears to be part of a cellular adaptation mechanism that responds to metabolic stress by adjusting gene expression profiles to meet changing energy demands.

What is the interplay between histone β-hydroxybutyrylation and other epigenetic modifications?

Understanding the interplay between histone β-hydroxybutyrylation and other epigenetic modifications is an evolving area of research:

• Co-occurrence with acetylation: β-hydroxybutyrylation can occur on many of the same lysine residues that are targets for acetylation (e.g., H4K8, H4K12, H3K9), suggesting potential competition between these modifications .

• Differential regulation: While histone Kbhb levels are dramatically induced by elevated β-hydroxybutyrate, histone acetylation levels generally remain stable, indicating separate regulatory mechanisms .

• Shared and distinct targets: The 44 identified histone Kbhb sites include lysine residues (H4K8, H4K12, H3K4, H3K9, H3K56) whose acetylation and methylation are important for chromatin structure and function .

• Metabolic influence: While acetylation is primarily influenced by acetyl-CoA levels, β-hydroxybutyrylation responds specifically to β-hydroxybutyrate levels, providing a distinct metabolic sensing mechanism .

• Reader proteins: Further identification and characterization of Kbhb readers or effector proteins are needed to fully decipher the "Kbhb code" and its relationship with other histone modifications .

This complex interplay suggests a sophisticated epigenetic regulatory system that integrates various metabolic signals to fine-tune gene expression.

What are the current limitations in β-hydroxybutyrylation research and how might they be addressed?

Several key limitations currently exist in β-hydroxybutyrylation research:

• Antibody specificity issues: At high BHB concentrations, antibodies may recognize β-hydroxybutyrylation at sites beyond their intended targets due to structural similarities between different histone acylations . This can be addressed through more rigorous antibody validation, development of higher-specificity antibodies, and complementary non-antibody-based detection methods.

• Incomplete understanding of enzymatic regulation: The specific enzymes responsible for adding and removing β-hydroxybutyryl groups (writers and erasers) remain incompletely characterized. Targeted enzymatic screens and proteomic approaches could help identify these regulatory proteins.

• Limited knowledge of reader proteins: The proteins that specifically recognize and bind to β-hydroxybutyrylated histones to mediate downstream effects are not fully identified . Affinity purification using β-hydroxybutyrylated peptides followed by mass spectrometry could help identify these reader proteins.

• Tissue-specific effects: Most studies have focused on liver tissue or cell lines, with limited understanding of tissue-specific effects. Expanding research to diverse tissue types would provide a more comprehensive understanding of β-hydroxybutyrylation biology.

• Functional significance: While correlations between β-hydroxybutyrylation and gene expression have been established, the causal relationships and functional significance require further investigation. CRISPR-based approaches to specifically modulate β-hydroxybutyrylation at targeted genomic loci could help address this limitation.

Addressing these limitations will require interdisciplinary approaches combining biochemistry, molecular biology, genetics, and computational biology.

What are common technical challenges when working with β-hydroxybutyryl-HIST1H2BC antibodies and how can they be overcome?

Researchers frequently encounter several technical challenges when working with β-hydroxybutyryl-HIST1H2BC antibodies:

• Cross-reactivity issues: Anti-Kbhb antibodies may recognize other β-hydroxybutyrylated sites or even structurally similar modifications at high BHB concentrations . This can be mitigated by:

  • Using lower antibody concentrations

  • Including appropriate blocking peptides in control experiments

  • Validating results with multiple antibodies or complementary techniques

• Background signal: High background can obscure specific signals. To reduce background:

  • Optimize blocking conditions (concentration, time, temperature)

  • Increase washing steps

  • Use freshly prepared buffers

  • Consider alternative detection systems

• Variability between antibody lots: Lot-to-lot variations can affect consistency. Researchers should:

  • Test each new lot against previous lots

  • Maintain detailed records of antibody performance

  • Consider pooling antibodies from multiple lots for long-term studies

• Sample preparation issues: Improper histone extraction can affect detection. For optimal results:

  • Use standardized histone extraction protocols

  • Minimize protein degradation by adding protease inhibitors

  • Consider acid extraction methods for histone purification

• Interference from neighboring modifications: Adjacent histone modifications can affect antibody binding . Researchers should:

  • Use peptide competition assays to assess specificity

  • Consider mass spectrometry validation for critical experiments

How should data from β-hydroxybutyrylation studies be analyzed and interpreted?

Proper analysis and interpretation of β-hydroxybutyrylation data require careful consideration of several factors:

• Western blot quantification:

  • Normalize Kbhb signals to total histone loading controls

  • Use appropriate statistical methods for comparing treatment groups

  • Consider relative changes rather than absolute values when comparing different sites or antibodies

• ChIP-seq data analysis:

  • Use appropriate peak-calling algorithms suitable for histone modifications

  • Compare Kbhb enrichment with other histone marks to contextualize findings

  • Correlate Kbhb peaks with gene expression data to identify functional associations

• RNA-seq data interpretation:

  • Apply robust statistical frameworks (e.g., DESeq2) for differential expression analysis

  • Use appropriate cutoffs (e.g., adjusted p-value <0.1) for identifying significant changes

  • Perform pathway and gene ontology analyses to identify biological processes affected

• Mass spectrometry data:

  • Validate identified sites through comparison with synthetic standards

  • Confirm modification identity through fragmentation patterns

  • Quantify relative abundances of different modification sites

• Physiological context:

  • Interpret changes in β-hydroxybutyrylation in relation to metabolic parameters (e.g., blood glucose, β-hydroxybutyrate levels)

  • Consider temporal dynamics and tissue-specific effects

  • Relate findings to known metabolic pathways and physiological states

How can researchers distinguish between biological effects of β-hydroxybutyrylation and technical artifacts?

Distinguishing genuine biological effects from technical artifacts requires systematic experimental design and controls:

• Dose-response relationships: True biological effects typically show consistent dose-response relationships with BHB treatment . Erratic responses may indicate technical artifacts.

• Multiple detection methods: Confirm key findings using complementary techniques (e.g., antibody-based detection, mass spectrometry, isotopic labeling) .

• Temporal dynamics: Biological processes often show characteristic temporal patterns. Time-course experiments can help distinguish biological effects from random fluctuations.

• Biological replicates: Use sufficient biological replicates (minimum 3-5) to establish statistical significance and reproducibility .

• Genetic manipulation: CRISPR-based approaches to manipulate BHB metabolism or specific Kbhb sites can help establish causality.

• Physiological correlation: Correlate changes in Kbhb with physiological parameters (e.g., BHB levels, fasting duration, diabetic status) to support biological relevance.

• Controls for antibody specificity: Include peptide competition assays and isotype controls to confirm antibody specificity .

• Reproducibility across models: Effects that reproduce across different cell types, animal models, or experimental conditions are more likely to represent genuine biological phenomena.

What are promising areas for future research on histone β-hydroxybutyrylation?

Several promising research directions could significantly advance our understanding of histone β-hydroxybutyrylation:

• Identification of enzymatic regulators: Discovering the specific enzymes (writers, erasers) that regulate β-hydroxybutyrylation would enable targeted manipulation of this modification.

• Reader protein characterization: Identifying and characterizing the proteins that specifically recognize β-hydroxybutyrylated histones will help decipher the downstream functional consequences .

• Tissue-specific effects: Exploring β-hydroxybutyrylation patterns across different tissues beyond the liver would provide insights into tissue-specific regulatory mechanisms.

• Disease relevance: Investigating alterations in histone β-hydroxybutyrylation in various disease states beyond diabetes, particularly metabolic disorders and cancer.

• Therapeutic targeting: Developing compounds that can specifically modulate histone β-hydroxybutyrylation could have therapeutic potential for metabolic disorders.

• Interplay with the gut microbiome: Exploring how gut microbiota-derived short-chain fatty acids affect histone β-hydroxybutyrylation patterns.

• Transgenerational effects: Investigating whether altered β-hydroxybutyrylation patterns can be transmitted across generations through epigenetic inheritance mechanisms.

• Non-histone targets: Examining β-hydroxybutyrylation of non-histone proteins and its functional consequences.

How might advances in technology improve the study of histone β-hydroxybutyrylation?

Technological advances hold significant promise for advancing β-hydroxybutyrylation research:

• Single-cell epigenomics: Single-cell ChIP-seq and related technologies could reveal cell-to-cell heterogeneity in β-hydroxybutyrylation patterns within tissues.

• CRISPR-based epigenome editing: Targeted modulation of β-hydroxybutyrylation at specific genomic loci using CRISPR-based approaches would help establish causal relationships with gene expression.

• Advanced mass spectrometry: Improvements in sensitivity and throughput of mass spectrometry would enable more comprehensive profiling of β-hydroxybutyrylation sites and their dynamics.

• Live-cell imaging: Development of specific probes for visualizing β-hydroxybutyrylation in living cells would provide insights into real-time dynamics.

• Computational modeling: Advanced algorithms for integrating multi-omics data could help predict functional consequences of β-hydroxybutyrylation patterns.

• Protein structure analysis: Cryo-EM and other structural biology approaches could reveal how β-hydroxybutyrylation affects chromatin structure and protein-protein interactions.

• Improved antibodies: Development of highly specific monoclonal antibodies against various β-hydroxybutyrylated sites would enhance detection specificity and reliability .

• Metabolic tracing: Advanced metabolic tracing techniques could provide insights into the flux of β-hydroxybutyrate into histone modification pathways.

What are the potential implications of histone β-hydroxybutyrylation research for human health and disease?

Research on histone β-hydroxybutyrylation has several important implications for human health and disease:

• Metabolic disorders: Understanding how β-hydroxybutyrylation links metabolism to gene expression could provide new insights into diabetes, obesity, and metabolic syndrome pathophysiology .

• Ketogenic diets: Elucidating the epigenetic effects of elevated β-hydroxybutyrate levels could help explain the molecular mechanisms behind the therapeutic effects of ketogenic diets in epilepsy and other neurological conditions.

• Fasting and caloric restriction: Understanding how fasting-induced β-hydroxybutyrylation affects gene expression could clarify the health benefits associated with intermittent fasting and caloric restriction .

• Biomarker development: Changes in specific β-hydroxybutyrylation patterns could serve as biomarkers for metabolic stress or disease states.

• Drug development: Targeting the enzymatic machinery regulating β-hydroxybutyrylation could lead to novel therapeutics for metabolic disorders.

• Personalized nutrition: Individual variations in β-hydroxybutyrylation responses could inform personalized dietary recommendations.

• Cancer metabolism: Given the altered metabolism in cancer cells, understanding β-hydroxybutyrylation could provide insights into cancer epigenetics and potential therapeutic targets.

• Aging-related processes: Metabolic changes during aging might affect β-hydroxybutyrylation patterns, potentially contributing to age-related gene expression changes.

How does histone β-hydroxybutyrylation compare with acetylation in terms of tissue distribution and regulation?

The tissue distribution and regulation of histone β-hydroxybutyrylation show distinct patterns compared to acetylation:

While acetylation is relatively stable across various metabolic conditions, β-hydroxybutyrylation shows dramatic fluctuations in response to metabolic states that alter β-hydroxybutyrate levels, such as fasting and diabetic ketoacidosis . This suggests that β-hydroxybutyrylation serves as a more dynamic sensor of specific metabolic states compared to acetylation.

What is known about the differentiation between β-hydroxybutyrylation and other acyl modifications in mass spectrometry analysis?

Mass spectrometry can effectively differentiate β-hydroxybutyrylation from other structurally similar acyl modifications:

• Mass difference: β-hydroxybutyrylation adds a mass of +86.0368 Da to modified lysine residues, which is distinct from other acyl modifications .

• HPLC retention time: Synthetic peptides containing β-hydroxybutyrylation show distinct HPLC retention times compared to peptides with other modification isomers such as 2-hydroxyisobutyrylation (2hib), 2-hydroxybutyrylation (2hb), or 4-hydroxybutyrylation (4hb) .

• Fragmentation patterns: High-resolution MS/MS fragmentation patterns of β-hydroxybutyrylated peptides are distinctive and can be compared with synthetic standards for confirmation .

• Isotopic labeling: Using isotopically labeled β-hydroxybutyrate (e.g., [2,4-13C2]) in cell culture experiments results in a characteristic mass shift (+2 Da) in β-hydroxybutyrylated peptides, confirming the direct incorporation of β-hydroxybutyrate into the modification .

• Co-elution experiments: Co-elution of synthetic β-hydroxybutyrylated peptides with in vivo-derived peptides on HPLC provides further confirmation of modification identity .