β-hydroxybutyryl-HIST1H4A (K91) Antibody

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

Histone lysine β-hydroxybutyrylation (Kbhb) is a post-translational modification identified on histone proteins that functions as an epigenetic regulatory mark. This modification directly connects metabolism to gene expression by utilizing β-hydroxybutyrate, a ketone body that increases during fasting, diabetic ketoacidosis, and other metabolic states. The significance of Kbhb lies in its role as a mechanism through which metabolic states can influence transcriptional regulation. In total, researchers have identified 44 histone Kbhb sites, comparable to the number of known histone acetylation sites, making it a widespread histone modification . As an epigenetic mark, Kbhb is enriched in active gene promoters, and increased H3K9bhb levels during starvation are associated with upregulated genes in starvation-responsive metabolic pathways .

How is histone β-hydroxybutyrylation regulated at the cellular level?

Histone β-hydroxybutyrylation is primarily regulated by cellular β-hydroxybutyrate concentrations. The process likely involves the conversion of β-hydroxybutyrate to β-hydroxybutyryl-CoA, which serves as the cofactor for lysine β-hydroxybutyrylation . This conversion may be catalyzed by short-chain-Coenzyme A synthetase, similar to how acetate and crotonate are converted to their corresponding CoA derivatives. Metabolic labeling experiments using isotopically labeled sodium β-hydroxybutyrate ([2,4-13C2]) have confirmed that exogenous β-hydroxybutyrate can be incorporated into histone proteins as Kbhb modifications . Additionally, removal of Kbhb marks appears to be mediated by histone deacetylases, as treatment with broad class I and II histone deacetylase inhibitors (such as trichostatin A and sodium butyrate) causes Kbhb accumulation in cultured cells .

What are the recommended protocols for using β-hydroxybutyryl-HIST1H4A (K91) antibody in ChIP assays?

When performing Chromatin Immunoprecipitation (ChIP) assays with β-hydroxybutyryl-HIST1H4A (K91) antibody, researchers should follow these methodological steps for optimal results:

• Cross-link chromatin by adding formaldehyde (1% final concentration) directly to the culture medium and incubate for 10 minutes at room temperature.

• Quench cross-linking by adding glycine (125 mM final concentration).

• Harvest cells and prepare nuclear extracts by lysing cells in appropriate buffers containing protease inhibitors.

• Sonicate chromatin to generate fragments of approximately 200-500 bp.

• Pre-clear chromatin with protein G magnetic beads.

• Incubate pre-cleared chromatin with β-hydroxybutyryl-HIST1H4A (K91) antibody (typically 5 μg antibody per 0.5 mg of protein) overnight at 4°C .

• Add protein G magnetic beads and incubate for 2-4 hours.

• Perform sequential washes with increasing salt concentrations to remove non-specific binding.

• Elute bound chromatin, reverse cross-links, and purify DNA for downstream analysis.

• Validate immunoprecipitation efficiency by Western blot, using a small aliquot of the immunoprecipitated material .

The antibody concentration may need optimization based on batch variations and experimental conditions, with initial testing at 1-5 μg per immunoprecipitation reaction.

How should Western blot protocols be optimized for detecting β-hydroxybutyrylated histone H4?

For optimal Western blot detection of β-hydroxybutyrylated histone H4, particularly at K91:

• Sample preparation: Extract histones using acid extraction methods to ensure enrichment of histone proteins.

  • Treat cells with 0.2N HCl for 4 hours at 4°C

  • Collect supernatant containing histones after centrifugation

  • Neutralize with 1M Tris-HCl (pH 8.0)

  • Concentrate histones using TCA precipitation if necessary

• Gel electrophoresis: Use 15-18% SDS-PAGE gels to achieve proper separation of histone proteins (14-20 kDa).

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at 30V overnight at 4°C to ensure complete transfer of small histone proteins.

• Blocking: Block membranes with 5% BSA in TBST for 1 hour at room temperature to reduce background.

• Antibody incubation: Incubate with primary β-hydroxybutyryl-HIST1H4A (K91) antibody at 1 μg/mL in 5% BSA-TBST overnight at 4°C .

• Detection: Use HRP-conjugated secondary antibodies at 1:5000 dilution and develop using enhanced chemiluminescence (ECL) reagents .

• Controls: Include an unmodified histone H4 control antibody to normalize for total H4 levels. The expected molecular weight for histone H4 is approximately 14 kDa .

• Validation: Confirm antibody specificity by pre-treating samples with β-hydroxybutyrate at various concentrations (0-6 mM) to demonstrate dose-dependent increases in signal intensity .

What methods are available for quantifying global changes in histone β-hydroxybutyrylation levels?

Several complementary techniques can be employed to quantify global changes in histone β-hydroxybutyrylation levels:

Researchers should note that the quantification of Kbhb shows a dose-dependent relationship with β-hydroxybutyrate concentration. Western blot analysis reveals 2.27, 4.75, and 6.62-fold increases in Kbhb in cells treated with 2, 4, and 6 mM β-hydroxybutyrate compared to untreated controls .

How do β-hydroxybutyrate levels correlate with histone β-hydroxybutyrylation in different physiological states?

Research has established clear correlations between β-hydroxybutyrate (BHB) levels and histone β-hydroxybutyrylation across various physiological states:

In streptozotocin-induced diabetic mice, blood β-hydroxybutyrate levels increase approximately 10-fold compared to healthy controls, which corresponds with dramatically elevated histone Kbhb levels in liver tissues . Similarly, during prolonged fasting (48 hours), mouse livers show significantly increased histone Kbhb levels, particularly at specific sites like H3K9bhb, while histone acetylation remains relatively unchanged . In cultured cells, treatment with increasing concentrations of β-hydroxybutyrate (2-6 mM) leads to proportional increases in histone Kbhb, with approximately 2.56-fold, 3.91-fold, and 5.22-fold increases in H3K9bhb at 2 mM, 4 mM, and 6 mM BHB, respectively .

What is the relationship between histone β-hydroxybutyrylation and transcriptional regulation?

Histone β-hydroxybutyrylation functions as an epigenetic mark that directly influences transcriptional regulation. ChIP-seq and RNA-seq analyses have demonstrated that histone Kbhb is enriched in active gene promoters . Specifically, increased H3K9bhb levels during starvation are associated with upregulated genes involved in starvation-responsive metabolic pathways . This indicates that Kbhb serves as a mechanism for coupling metabolic state to gene expression programs.

The transcriptional effects of Kbhb appear distinct from those of histone acetylation. Despite similarities in the chemical nature of these modifications (both involve acylation of lysine residues), the genomic distribution and transcriptional consequences differ. For instance, during metabolic stress conditions like fasting or diabetic ketoacidosis, histone Kbhb levels increase dramatically while histone acetylation shows minimal changes . This suggests that Kbhb represents a specific adaptive response to altered metabolic states, potentially activating genes required for adaptation to ketosis or nutrient deprivation.

The relationship between histone deacetylases (HDACs) and Kbhb adds another layer of complexity. Treatment with HDAC inhibitors like sodium butyrate causes accumulation of Kbhb marks , suggesting that these enzymes may also play a role in removing Kbhb modifications. This indicates potential crosstalk between histone acetylation and β-hydroxybutyrylation pathways in regulating gene expression.

What are the differences between histone β-hydroxybutyrylation and histone acetylation mechanisms?

While histone β-hydroxybutyrylation and acetylation both involve acylation of lysine residues, they differ in several key aspects:

Experimentally, treating cells with increasing concentrations of β-hydroxybutyrate leads to dose-dependent increases in histone Kbhb levels, while histone acetylation remains relatively unchanged . For instance, treating fibroblasts with 2-6 mM β-hydroxybutyrate results in significant increases in H3K9bhb (2.56 to 5.22-fold) but minimal changes in H3K9ac . This differential response highlights the specific relationship between β-hydroxybutyrate metabolism and histone β-hydroxybutyrylation.

How can researchers distinguish between different isomers of lysine hydroxybutyrylation in experimental settings?

Distinguishing between different isomers of lysine hydroxybutyrylation presents a significant analytical challenge requiring specialized techniques:

• Synthetic peptide standards: Researchers should synthesize peptide standards containing different hydroxybutyrylation isomers, including:

  • K bhbQLATK (β-hydroxybutyrylation)

  • K bhibQLATK (β-hydroxyisobutyrylation)

  • K 2hbQLATK (2-hydroxybutyrylation)

  • K 2hibQLATK (2-hydroxyisobutyrylation)

  • K 4hbQLATK (4-hydroxybutyrylation)

• HPLC co-elution analysis: Compare HPLC retention times of synthetic peptides with in vivo-derived peptides. Authentic β-hydroxybutyrylated peptides co-elute with synthetic β-hydroxybutyrylated standards but show distinct retention times compared to other isomers .

• High-resolution mass spectrometry: All hydroxybutyrylation isomers yield the same +86.0368 Da mass shift, but they can be distinguished by their fragmentation patterns in MS/MS analysis. Compare MS/MS fragmentation patterns of synthetic standards with in vivo-derived peptides .

• Isotopic labeling: Use isotopically labeled β-hydroxybutyrate ([2,4-13C2]) for metabolic labeling to specifically track β-hydroxybutyrylation. This approach distinguishes β-hydroxybutyrylation from other isomers by producing a characteristic +2 Da mass shift in the modified peptides .

• Antibody specificity testing: Validate antibody specificity using dot blot assays with synthetic peptides containing different hydroxybutyrylation isomers, ensuring recognition of only the β-hydroxybutyryl isomer .

These combined approaches provide rigorous confirmation of β-hydroxybutyrylation versus other potential isomers, which is particularly important when characterizing new modification sites or investigating novel biological contexts.

What are the current challenges in quantitative analysis of site-specific β-hydroxybutyrylation?

Researchers face several significant challenges when attempting quantitative analysis of site-specific β-hydroxybutyrylation:

• Antibody specificity limitations: Most available antibodies recognize β-hydroxybutyrylation at specific lysine residues (e.g., H3K9bhb) or serve as pan-Kbhb antibodies. Developing site-specific antibodies for all 44+ identified Kbhb sites remains challenging, especially for sites with similar flanking sequences.

• Low abundance of modifications: β-hydroxybutyrylation can be a relatively low-abundance modification under physiological conditions, making detection and quantification difficult without enrichment strategies.

• Cross-reactivity concerns: Potential cross-reactivity between antibodies recognizing different acyl modifications (acetylation, butyrylation, crotonylation, β-hydroxybutyrylation) must be rigorously controlled for accurate quantification.

• Dynamic range limitations: The dramatic increases in Kbhb levels under pathological conditions (up to 10-fold in diabetic mice or >50-fold with 20 mM BHB treatment ) create challenges for quantification methods with limited dynamic ranges.

• Heterogeneity of modification patterns: Different cell types and tissues may exhibit distinct patterns of Kbhb, complicating cross-study comparisons and standardization.

• Technical variability in sample preparation: Histones are prone to degradation and artificial modifications during extraction, potentially affecting quantitative measurements.

• Mass spectrometry challenges: The +86.0368 Da mass shift of β-hydroxybutyrylation can be difficult to distinguish from certain combinations of other modifications, requiring high-resolution MS and careful data analysis.

Methodological solutions include developing improved enrichment techniques, utilizing stable isotope labeling for quantitative MS, careful validation of antibody specificity, and the development of multiplexed assays capable of simultaneously measuring multiple histone modifications.

How can researchers investigate the potential enzymatic writers and erasers of histone β-hydroxybutyrylation?

Investigating the enzymatic machinery responsible for writing and erasing histone β-hydroxybutyrylation requires a multifaceted approach:

• Candidate enzyme screening:

  • For writers: Test known histone acetyltransferases (HATs) for β-hydroxybutyrylation activity in vitro using recombinant enzymes, histone substrates, and β-hydroxybutyryl-CoA

  • For erasers: Evaluate histone deacetylases (HDACs) and sirtuins for dehydroxybutyrylation activity

• Inhibitor profiling:

  • Treat cells with specific inhibitors of candidate enzymes (e.g., class-specific HDAC inhibitors)

  • Measure changes in global and site-specific Kbhb levels by immunoblotting and mass spectrometry

  • Current evidence indicates that treatment with broad class I and II HDAC inhibitors (TSA and sodium butyrate) causes Kbhb accumulation, suggesting HDACs may function as erasers

• Genetic manipulation approaches:

  • Perform systematic knockdown or knockout of candidate enzymes

  • Use CRISPR-Cas9 screening to identify genes affecting Kbhb levels

  • Overexpress candidate enzymes and measure effects on Kbhb

• Biochemical purification:

  • Use synthetic β-hydroxybutyrylated peptides as substrates or baits

  • Perform affinity purification to identify interacting proteins

  • Apply activity-based protein profiling methods

• Metabolic regulation studies:

  • Investigate the relationship between β-hydroxybutyryl-CoA levels and Kbhb

  • Manipulate β-hydroxybutyrate metabolism enzymes (e.g., BDH1, OXCT1)

  • Current evidence shows that β-hydroxybutyrate can be converted to β-hydroxybutyryl-CoA in cells, which likely serves as the cofactor for enzymatic β-hydroxybutyrylation reactions

• Site-specificity determination:

  • Compare enzymatic preferences for different histone lysine residues

  • Investigate sequence context requirements using peptide arrays

• Structural biology approaches:

  • Obtain crystal structures of candidate enzymes with β-hydroxybutyryl-CoA or β-hydroxybutyrylated substrates

  • Perform molecular docking and dynamics simulations to understand recognition mechanisms

This comprehensive approach will help elucidate the enzymatic regulation of histone β-hydroxybutyrylation, providing insights into how this modification is dynamically controlled in response to metabolic changes.

What are the potential applications of β-hydroxybutyryl-HIST1H4A (K91) antibody in diabetes and metabolic disorder research?

The β-hydroxybutyryl-HIST1H4A (K91) antibody holds significant potential for advancing diabetes and metabolic disorder research through several applications:

• Biomarker development:

  • Monitoring histone Kbhb levels as an epigenetic biomarker of ketosis severity in diabetes

  • Assessing the efficacy of anti-diabetic interventions by tracking changes in histone β-hydroxybutyrylation patterns

• Mechanistic studies:

  • Investigating how diabetic ketoacidosis affects gene expression through Kbhb-mediated epigenetic regulation

  • Identifying gene targets specifically regulated by H4K91bhb in diabetic models

  • Research has already demonstrated dramatically elevated histone Kbhb levels in streptozotocin-induced diabetic mice, with a 10-fold increase in blood β-hydroxybutyrate corresponding to significantly increased liver histone Kbhb

• Therapeutic target identification:

  • Screening for compounds that modulate histone β-hydroxybutyrylation

  • Evaluating the epigenetic effects of existing diabetes medications

• Nutritional intervention assessment:

  • Studying how ketogenic diets influence gene expression through histone β-hydroxybutyrylation

  • Developing personalized nutrition approaches based on Kbhb profiles

• Comparative studies across metabolic conditions:

  • Comparing Kbhb patterns between type 1 and type 2 diabetes

  • Analyzing differences between physiological ketosis (fasting) and pathological ketoacidosis

• Tissue-specific effects:

  • Investigating how different tissues respond to elevated β-hydroxybutyrate through changes in H4K91bhb

  • Examining the interplay between insulin signaling and histone β-hydroxybutyrylation

• Developmental programming:

  • Studying how maternal ketosis affects offspring epigenetic programming through histone β-hydroxybutyrylation

The ability to specifically detect and quantify β-hydroxybutyrylation at H4K91 provides a powerful tool for understanding the epigenetic dimensions of metabolic disorders, potentially leading to novel diagnostic and therapeutic approaches.

How can researchers integrate β-hydroxybutyrylation data with other multi-omics approaches?

Integrating β-hydroxybutyrylation data with other multi-omics approaches requires sophisticated analytical strategies:

• Integrated experimental design:

  • Collect matched samples for parallel analysis across multiple platforms

  • Include appropriate temporal sampling to capture dynamic changes

  • Incorporate physiological measurements (e.g., blood β-hydroxybutyrate levels)

• Combined epigenomic analyses:

  • Perform ChIP-seq for Kbhb and other histone modifications simultaneously

  • Integrate with DNA methylation data (WGBS or RRBS)

  • Compare with chromatin accessibility (ATAC-seq) profiles

  • Current research has already established that histone Kbhb marks are enriched in active gene promoters

• Transcriptomic integration:

  • Correlate Kbhb ChIP-seq data with RNA-seq to identify relationships between β-hydroxybutyrylation and gene expression

  • Analyze alternative splicing events potentially regulated by Kbhb

• Metabolomic connections:

  • Measure cellular β-hydroxybutyrate, β-hydroxybutyryl-CoA, and related metabolites

  • Develop metabolic flux models incorporating acyl-CoA utilization for histone modifications

  • Studies have shown dose-dependent increases in isotopic bhb-CoA in cells treated with isotopic sodium β-hydroxybutyrate

• Proteomic approaches:

  • Identify non-histone proteins modified by β-hydroxybutyrylation

  • Study protein complexes associated with β-hydroxybutyrylated histones

• Computational integration frameworks:

  • Develop machine learning models to predict β-hydroxybutyrylation sites based on sequence and structural features

  • Create network models linking metabolic pathways to epigenetic modifications

  • Apply dimension reduction techniques to visualize multi-omics data

• Data visualization tools:

  • Develop genome browsers with integrated views of β-hydroxybutyrylation, gene expression, and other epigenetic marks

  • Create metabolic pathway maps highlighting connections to histone modifications

• Statistical approaches:

  • Apply multivariate analysis methods to identify coordinated changes across omics layers

  • Develop causal inference methods to determine directional relationships

This integrated multi-omics approach will provide comprehensive insights into how β-hydroxybutyrate metabolism influences gene expression through histone modifications, advancing our understanding of metabolic regulation at the epigenetic level.

What is the current understanding of species-specific differences in histone β-hydroxybutyrylation patterns?

Current research reveals both conservation and divergence in histone β-hydroxybutyrylation patterns across species:

The presence of histone Kbhb has been detected across diverse eukaryotic species, including human cells, mouse tissues, bovine cells, Drosophila S2 cells, and yeast S. cerevisiae , indicating evolutionary conservation of this epigenetic modification mechanism. This conservation suggests fundamental importance in cellular metabolism and gene regulation.

Human dermal fibroblasts (hDF) and bovine fibroblasts show similar dose-dependent responses to β-hydroxybutyrate treatment. When treated with 2 mM BHB, hDF cells show a ~2.21-fold increase in Kbhb levels relative to untreated controls, while bovine fibroblasts show a ~2.56-fold increase . At 6 mM BHB, human cells exhibit a ~5.97-fold increase in Kbhb, compared to a ~5.22-fold increase in bovine cells . This suggests conservation of the underlying metabolic and enzymatic mechanisms across mammals.

Despite these similarities, some species-specific differences are likely to exist in:

• Site preferences: Different species may show variations in which specific lysine residues are predominantly modified by β-hydroxybutyrylation.

• Metabolic context: The physiological conditions triggering increased β-hydroxybutyrylation may vary between species based on metabolic adaptations.

• Enzymatic machinery: The specific enzymes responsible for writing and erasing Kbhb modifications may differ across species.

• Regulatory outcomes: The genes and pathways regulated by Kbhb might show species-specific patterns reflecting different metabolic priorities.

The evolutionary conservation of histone β-hydroxybutyrylation across diverse species highlights its fundamental importance in linking metabolism to gene regulation, while potential species-specific differences provide insights into metabolic adaptations unique to each organism.