β-hydroxybutyryl-HIST1H3A (K4) Antibody

What is histone lysine β-hydroxybutyrylation (Kbhb) and how does it relate to HIST1H3A (K4)?

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 was identified as a new type of histone mark that is dramatically induced in response to elevated β-hydroxybutyrate levels. HIST1H3A (K4) refers specifically to the lysine at position 4 on histone H3.1, which is one of the 44 identified histone Kbhb sites. This modification is particularly important as H3K4 modifications are known to play critical roles in transcription regulation and chromatin function .

How is β-hydroxybutyrylation different from other histone modifications like acetylation or methylation?

β-hydroxybutyrylation (Kbhb) differs from acetylation or methylation in several ways:

• Chemical structure: Kbhb involves the addition of a β-hydroxybutyryl group (causing a +86.0368 Da mass shift), which contains a hydroxyl group not present in acetylation.

• Metabolic connection: Kbhb is directly linked to β-hydroxybutyrate metabolism, particularly elevated during fasting or diabetic ketoacidosis, while acetylation relates to acetyl-CoA levels.

• Regulatory mechanisms: Kbhb is regulated by cellular β-hydroxybutyrate concentrations, while acetylation and methylation have different regulatory pathways.

• Functional outcomes: While H3K4 methylation is associated with active transcription and H3K27 methylation with gene silencing, H3K4bhb appears to mark active gene promoters specifically during metabolic stress conditions .

What are the common applications for β-hydroxybutyryl-HIST1H3A (K4) antibodies in research?

Common research applications include:

• Chromatin Immunoprecipitation (ChIP): To identify genomic regions associated with H3K4bhb modification

• Western Blotting (WB): To detect and quantify H3K4bhb levels in various experimental conditions

• Immunocytochemistry (ICC): To visualize the nuclear localization and distribution of H3K4bhb

• ELISA: For quantitative measurement of H3K4bhb in histone samples

These applications enable researchers to study how metabolic changes affect gene expression through histone modifications .

How can one validate the specificity of β-hydroxybutyryl-HIST1H3A (K4) antibodies for ChIP experiments?

Validating antibody specificity for ChIP experiments requires a multi-faceted approach:

• Mass spectrometry validation: Compare the enrichment profiles of immunoprecipitated samples with synthetic peptides containing the K4bhb modification using high-resolution MS/MS.

• Competition assays: Pre-incubate the antibody with synthesized peptides containing K4bhb versus other modifications (K4ac, K4me3) to demonstrate specific binding inhibition.

• Quantitative specificity assessment: Use quantitative mass spectrometry to characterize the fold enrichment over background for the intended target and measure bias against other modifications.

• Cross-reactivity testing: Test against other hydroxybutyryl isomers (2-hydroxybutyryl, 4-hydroxybutyryl) to ensure specificity for β-hydroxybutyrylation .

How do metabolic conditions affect histone H3K4bhb levels, and what are the optimal experimental designs to study these changes?

Metabolic conditions significantly impact H3K4bhb levels, with implications for experimental design:

• Starvation/fasting models: In mice, prolonged fasting (typically 24-48 hours) induces elevated liver β-hydroxybutyrate levels and corresponding increases in H3K4bhb. Time-course experiments are essential to capture dynamic changes.

• Diabetic models: Streptozotocin-induced diabetic ketoacidosis in mice provides a model for studying pathological elevation of β-hydroxybutyrate and resultant H3K4bhb increases.

• Cell culture models: Treating cells with sodium β-hydroxybutyrate (5-20 mM range) induces H3K4bhb in a dose-dependent manner.

• Isotopic labeling: Using isotopically labeled β-hydroxybutyrate ([2,4-13C2]-β-hydroxybutyrate) allows tracking the direct incorporation into histone marks.

For optimal experimental design, researchers should include time-course measurements, dose-response assessments, and parallel measurements of cellular β-hydroxybutyrate levels alongside H3K4bhb quantification .

What is the relationship between SIRT3 and H3K4bhb, and how can this interaction be effectively studied?

SIRT3 displays class-selective histone de-β-hydroxybutyrylase activities with specific preference for certain sites including H3K4bhb. To study this relationship effectively:

• In vitro deacylation assays: Using purified SIRT3 and synthetic H3K4bhb peptides to measure direct enzymatic activity.

• Structural studies: X-ray crystallography has revealed that SIRT3 contains a hydrogen bond-lined hydrophobic pocket that preferentially recognizes the S-form of Kbhb.

• Site-specificity analysis: SIRT3 shows hierarchical activity toward different Kbhb sites, preferring H3K4, K9, K18, K23, K27, and H4K16, but not H4K5, K8, or K12.

• Sequence motif recognition: β-backbone-mediated interactions around Kbhb, rather than side chain interactions, dominate sequence motif recognition by SIRT3.

For comprehensive study designs, researchers should compare SIRT3 knockout/knockdown models with wild-type under various metabolic conditions to assess the physiological relevance of this enzymatic relationship .

What are the optimal conditions for ChIP-seq experiments using β-hydroxybutyryl-HIST1H3A (K4) antibodies?

For optimal ChIP-seq with β-hydroxybutyryl-HIST1H3A (K4) antibodies:

• Crosslinking: Standard 1% formaldehyde for 10 minutes at room temperature is typically sufficient.

• Sonication: Adjust to obtain DNA fragments between 200-500 bp.

• Antibody concentration: Use 2-5 μg of antibody per ChIP reaction for 25 μl of protein A/G-agarose beads.

• Incubation conditions: Overnight incubation at 4°C with gentle agitation.

• Washing: Use stringent washing conditions (three times with ChIP buffer) to reduce non-specific binding.

• Controls: Include:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control (non-specific antibody)

  • Pan-H3 antibody (for normalization)

  • Peptide competition control with synthetic K4bhb peptide

For data analysis, normalize to input and pan-H3 signal to account for nucleosome occupancy variations .

How can isotopic labeling be used to confirm the specificity of β-hydroxybutyryl-HIST1H3A (K4) modification?

Isotopic labeling provides powerful confirmation of β-hydroxybutyrylation specificity:

• Stable isotope labeling: Treat cells with isotopically labeled sodium β-hydroxybutyrate ([2,4-13C2]-β-hydroxybutyrate) at 10 mM concentration.

• Mass shift detection: After treatment, extract histones and perform mass spectrometry to detect the expected mass shift from the labeled β-hydroxybutyrate incorporation.

• Cofactor analysis: Detect the dose-dependent increase of isotopic bhb-CoA formation, which is the direct cofactor for lysine β-hydroxybutyrylation.

• Antibody validation: Confirm that the β-hydroxybutyryl-HIST1H3A (K4) antibody recognizes both labeled and unlabeled forms.

• Time-course analysis: Monitor the isotope incorporation rate to understand the dynamics of K4bhb turnover.

This approach provides definitive evidence that cellular β-hydroxybutyrate is directly converted to bhb-CoA and subsequently incorporated into histone marks .

What techniques can be used to quantify changes in global and site-specific H3K4bhb levels?

Multiple complementary techniques can quantify H3K4bhb changes:

• Western blotting: For semi-quantitative assessment of global H3K4bhb levels:

  • Use site-specific antibodies

  • Include loading controls (total H3)

  • Normalize to housekeeping proteins

• Mass spectrometry:

  • Extracted ion chromatograms (XICs) for absolute quantification

  • Multiple reaction monitoring (MRM) for targeted quantification

  • SILAC labeling for comparative studies

• ELISA assays:

  • Direct ELISA with immobilized histones

  • Sandwich ELISA for improved sensitivity

• ChIP-seq with spike-in normalization:

  • Add exogenous chromatin (e.g., Drosophila) as internal control

  • Use for genome-wide quantitative comparisons between conditions

For accurate global measurements, a combination of Western blotting and mass spectrometry is recommended, while site-specific changes are best quantified through mass spectrometry or ChIP-seq approaches .

How can researchers distinguish between true H3K4bhb signals and antibody cross-reactivity with other modifications?

Distinguishing true H3K4bhb signals from cross-reactivity requires rigorous validation:

• Peptide competition assays: Pre-incubate the antibody with:

  • H3K4bhb synthetic peptides (should diminish signal)

  • H3K4ac, H3K4me1/2/3 peptides (should not affect signal if specific)

  • Other hydroxybutyrylated lysine peptides (e.g., H3K9bhb)

• Mass spectrometry validation:

  • Compare retention times of immunoprecipitated peptides with synthetic K4bhb standards

  • Verify fragmentation patterns match true β-hydroxybutyrylation, not isomers

• Quantitative specificity assessment:

  • Calculate enrichment ratios for target vs. non-target modifications

  • Use heat maps to visualize fold enrichment over background

• Orthogonal validation:

  • Metabolic labeling with isotopic β-hydroxybutyrate

  • Enzymatic removal using purified SIRT3 (should reduce signal)

The quantitative assessment method using mass spectrometry is particularly valuable, as it measures both target enrichment and bias against other modifications at the same site .

What are common issues in ChIP experiments with β-hydroxybutyryl-HIST1H3A (K4) antibodies and how can they be resolved?

Common ChIP issues and solutions include:

• Low signal-to-noise ratio:

  • Increase antibody specificity through additional pre-clearing steps

  • Optimize antibody concentration (typically 2-5 μg for H3K4bhb ChIP)

  • Use more stringent washing conditions

• High background:

  • Increase blocking with BSA or non-fat milk

  • Pre-clear lysates with protein A/G beads

  • Include appropriate controls (IgG, input)

• Cross-reactivity:

  • Validate antibody with peptide competition assays

  • Use antibodies tested for minimal cross-reactivity with other modifications

• Variable enrichment:

  • Normalize to input and pan-H3 signals

  • Use spike-in controls for between-sample comparisons

  • Consider cellular β-hydroxybutyrate levels as a confounding factor

• Inconsistent results between replicates:

  • Standardize crosslinking conditions

  • Control cell growth and metabolic state carefully

  • Consider fasting/feeding status of experimental animals

When working with metabolically sensitive modifications like H3K4bhb, careful control of the metabolic state is particularly important for reproducible results .

How should researchers interpret changes in H3K4bhb levels in relation to other histone modifications and gene expression?

Interpreting H3K4bhb changes requires consideration of multiple factors:

• Co-occurrence with other modifications:

  • Analyze correlation between H3K4bhb and other active marks (H3K4me3, H3K27ac)

  • Consider mutual exclusivity with repressive marks (H3K27me3, H3K9me3)

  • Assess sequential ChIP (Re-ChIP) for co-occurrence on the same nucleosomes

• Relationship with gene expression:

  • Integrate RNA-seq data to correlate H3K4bhb enrichment with transcriptional changes

  • Focus on starvation-responsive metabolic pathways, which show strong correlation

  • Consider time-course experiments to establish causality

• Metabolic context:

  • Measure cellular β-hydroxybutyrate levels concurrently

  • Consider ketogenic diet effects or fasting conditions

  • Account for circadian fluctuations in metabolism

• Functional interpretation:

  • H3K4bhb generally marks active gene promoters

  • Enrichment is associated with genes up-regulated in starvation-responsive pathways

  • Consider enzymatic regulation by SIRT3 and other deacylases

A comprehensive interpretation requires integration of epigenomic data with transcriptomics and metabolomics to understand the functional significance of H3K4bhb changes .

How does the enzymatic regulation of H3K4bhb by SIRT3 influence experimental design and interpretation?

The role of SIRT3 as a histone de-β-hydroxybutyrylase has significant implications:

• Experimental considerations:

  • Include SIRT3 inhibitors/activators in experimental designs

  • Compare tissues/cells with different SIRT3 expression levels

  • Consider subcellular localization of SIRT3 (primarily mitochondrial, but with nuclear function)

• Mechanistic insights:

  • SIRT3 shows class-selective activity, preferring H3K4bhb among other sites

  • Contains a hydrogen bond-lined hydrophobic pocket for S-form Kbhb recognition

  • Displays hierarchical deacylation activity based on sequence context

• Physiological relevance:

  • SIRT3 activity increases during caloric restriction

  • May serve as a metabolic sensor linking β-hydroxybutyrate levels to epigenetic regulation

  • Could explain temporal dynamics of H3K4bhb marks

When designing experiments involving H3K4bhb, researchers should consider SIRT3 expression and activity as potential confounding variables, especially in metabolically active tissues like liver and kidney .

What is known about the relationship between ketogenic diets, β-hydroxybutyrate levels, and H3K4bhb in various tissues?

The ketogenic diet-β-hydroxybutyrate-H3K4bhb axis reveals tissue-specific responses:

• Liver:

  • Fasting and ketogenic diets dramatically increase hepatic β-hydroxybutyrate and H3K4bhb

  • Marks genes involved in gluconeogenesis and fatty acid oxidation

  • Shows rapid response to metabolic changes

• Brain:

  • Ketogenic diets are used therapeutically for neurological conditions

  • β-hydroxybutyrate serves as an alternative energy source

  • H3K4bhb changes may mediate neuroprotective effects

• Kidney:

  • Filters and reabsorbs β-hydroxybutyrate

  • May show significant H3K4bhb changes during ketosis

  • Less studied than liver in this context

• Muscle:

  • Utilizes β-hydroxybutyrate during prolonged fasting

  • H3K4bhb may regulate substrate utilization genes

When designing studies examining the relationship between diet and H3K4bhb, researchers should:

• Include tissue-specific controls

• Monitor serum and tissue β-hydroxybutyrate levels

• Consider time-course experiments to capture dynamic responses

• Account for other confounding dietary factors

The liver shows the most robust and well-characterized H3K4bhb response to ketogenic conditions, making it an ideal model tissue for initial studies .