β-hydroxybutyryl-HIST1H3A (K79) Antibody

What is β-hydroxybutyryl-HIST1H3A (K79) and how was it discovered?

β-hydroxybutyryl-HIST1H3A (K79) is a post-translational modification of histone H3 at lysine 79, where a β-hydroxybutyryl group is added to the amino acid residue. It was discovered as part of a broader investigation into histone acylations using mass spectrometry approaches (HPLC/MS/MS). Researchers identified a total of 44-46 histone Kbhb sites that are dramatically induced during states of elevated β-hydroxybutyrate levels, such as starvation, intermittent fasting, and exercise .

The discovery process involved several methodological steps:

• Initial identification through high-resolution mass spectrometry

• Validation using isotopically labeled β-hydroxybutyrate

• Development of site-specific antibodies

• Confirmation via metabolic labeling experiments

This modification represents a significant link between cellular metabolism and epigenetic regulation of gene expression, offering a new avenue to study chromatin regulation and diverse functions of β-hydroxybutyrate in pathophysiological states .

How does β-hydroxybutyryl-HIST1H3A (K79) differ from other histone modifications?

β-hydroxybutyryl-HIST1H3A (K79) differs from other histone modifications in several key aspects:

• Chemical structure: The β-hydroxybutyryl group contains a hydroxyl group (not present in acetylation) and has a longer carbon chain than acetyl groups, affecting its biochemical properties and recognition by reader proteins .

• Metabolic origin: Unlike many histone modifications, β-hydroxybutyrylation is directly derived from the metabolite β-hydroxybutyrate, a ketone body produced during fasting or ketogenic diets, creating a direct link between cellular metabolism and gene regulation .

• Induction conditions: β-hydroxybutyryl-HIST1H3A (K79) is dramatically induced (10-40 fold) during starvation or elevated β-hydroxybutyrate conditions, whereas most other modifications show more modest changes in response to cellular stimuli .

• Genomic distribution: While sharing some features with histone acetylation (association with active promoters), β-hydroxybutyrylation has distinct enrichment patterns at genes involved in starvation response and lipid metabolism .

• Reader proteins: ENL has been identified as a specific reader of histone β-hydroxybutyrylation, representing a unique interaction mechanism compared to readers of other modifications .

These distinctive features allow β-hydroxybutyryl-HIST1H3A (K79) to serve as a specialized epigenetic mark coupling metabolic state to transcriptional adaptation .

What are the known writers, readers, and erasers of histone β-hydroxybutyrylation at K79?

Research has identified several key molecular regulators of histone β-hydroxybutyrylation at K79:

Writers:
The acetyltransferase p300 has been identified as a primary writer of histone Kbhb marks, catalyzing the addition of β-hydroxybutyryl groups to lysine residues . In vitro studies demonstrate that p300 can utilize β-hydroxybutyryl-CoA as a cofactor to catalyze this reaction, though with lower efficiency compared to acetyl-CoA .

Readers:
ENL (MLLT1) has been established as a novel reader of histone β-hydroxybutyrylation, specifically recognizing this modification to modulate gene expression patterns . Biochemical studies and CUT&Tag analysis suggest that ENL favorably binds to H3K9bhb, co-localizes with it on promoter regions, and modulates expression of genes like MYC that drive cell proliferation .

Erasers:
Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) as well as sirtuins (SIRT1, SIRT2, and SIRT3) have demonstrated capacity to remove β-hydroxybutyryl groups from histones . In vitro screening revealed that HDAC1 to HDAC3 and SIRT1 and SIRT2 exhibit notable de-Kbhb activity toward core histones, while simultaneous knockdown of HDAC1 and HDAC2 increased levels of Kbhb in both HEK293 and HeLa cells .

These enzymes form part of the dynamic regulation of this histone mark, allowing for responsive changes to metabolic state .

What are the challenges in distinguishing β-hydroxybutyryl-HIST1H3A (K79) from other acyl modifications in experimental settings?

Distinguishing β-hydroxybutyryl-HIST1H3A (K79) from other acyl modifications presents several experimental challenges:

• Antibody cross-reactivity: Antibodies raised against β-hydroxybutyryl-K79 may cross-react with chemically similar modifications like acetylation, butyrylation, or crotonylation. Rigorous validation using peptide competition assays and modified versus unmodified protein controls is essential .

• Mass spectrometry resolution: Standard MS approaches may struggle to distinguish between isobaric or near-isobaric acyl modifications. High-resolution MS/MS with electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation methods are often necessary for definitive identification .

• Site-specific detection: When multiple acyl modifications can occur at the same residue (K79), determining the proportion of each modification requires specialized quantitative proteomics approaches, including stable isotope labeling and targeted analysis methods .

• Enzymatic regulators overlap: The writers and erasers of β-hydroxybutyrylation often overlap with those of other acyl modifications (p300 and HDACs), complicating functional studies through enzyme modulation .

• Non-enzymatic modification: β-hydroxybutyrylation can occur non-enzymatically at high β-hydroxybutyrate concentrations, making it difficult to study through traditional enzyme inhibition approaches .

Researchers address these challenges through complementary techniques including chemical derivatization strategies, synthetic peptide standards, and careful experimental design with appropriate controls. Peptide competition assays and modified versus unmodified protein controls are essential for validating antibody specificity .

How does cellular metabolism influence histone β-hydroxybutyrylation at K79?

Histone β-hydroxybutyrylation at K79 represents a direct link between metabolism and gene regulation. This intricate connection functions through several mechanisms:

Through these pathways, βOHB serves not just as a metabolic fuel but as a signaling molecule that directly influences gene expression through histone modification, providing a mechanism for cells to respond to metabolic challenges .

How do dietary interventions affect β-hydroxybutyryl-HIST1H3A (K79) levels?

Dietary interventions significantly impact β-hydroxybutyryl-HIST1H3A (K79) levels through their effects on β-hydroxybutyrate (βOHB) production:

• Fasting protocols: Intermittent fasting (16-24 hours) and prolonged fasting (>24 hours) induce hepatic βOHB production, leading to 10-40 fold increases in histone β-hydroxybutyrylation at K79 and other sites . During fasting periods, the liver shifts from glycolysis to fatty acid oxidation, producing ketone bodies including βOHB.

• Ketogenic diets: High-fat, low-carbohydrate ketogenic diets (typically 70-80% fat, 10-20% protein, 5-10% carbohydrate) elevate circulating βOHB levels to 1-5mM (compared to <0.5mM in fed states), significantly increasing histone β-hydroxybutyrylation . The restricted carbohydrate intake forces the body to utilize fat as the primary energy source, leading to increased hepatic ketogenesis.

• Caloric restriction: Long-term caloric restriction (typically 20-40% reduction from baseline) moderately increases βOHB levels and subsequent histone modifications, though less dramatically than fasting or ketogenic diets .

• Exercise regimens: High-intensity or endurance exercise can transiently increase βOHB levels, particularly when performed in a fasted state, affecting histone β-hydroxybutyrylation patterns .

Methodologically, these interventions are studied using:

• Controlled dietary protocols in both animal models and human subjects

• Western blotting with specific antibodies for semi-quantitative assessment

• Mass spectrometry for precise quantification of β-hydroxybutyryl-HIST1H3A (K79) levels

• ChIP-seq approaches to map genome-wide changes in this modification in response to dietary interventions

These findings suggest potential therapeutic applications through dietary interventions that modulate βOHB levels to influence epigenetic regulation .

How can I establish a ChIP-seq protocol for β-hydroxybutyryl-HIST1H3A (K79) in my research?

Establishing a robust ChIP-seq protocol for β-hydroxybutyryl-HIST1H3A (K79) requires several methodological considerations:

• Antibody selection and validation:

  • Use a highly specific antibody validated for ChIP applications

  • Validate the antibody using peptide competition assays and immunoblotting to ensure specificity for β-hydroxybutyryl-K79 versus other histone modifications

  • Perform western blot analysis to verify single band detection at the expected molecular weight (~17 kDa)

• Sample preparation and crosslinking:

  • Treat cells with β-hydroxybutyrate (10mM sodium β-hydroxybutyrate for 24 hours) or subject to fasting conditions to increase the prevalence of this modification

  • Perform standard formaldehyde crosslinking (1% for 10 minutes at room temperature), though optimization may be necessary for specific cell types

  • Quench crosslinking with glycine (final concentration 0.125M)

• Chromatin fragmentation:

  • Sonicate to produce fragments of 200-500bp

  • Verify fragmentation using gel electrophoresis before proceeding

  • Optimal sonication parameters typically include 10-15 cycles of 30 seconds on/30 seconds off at medium power

• Immunoprecipitation:

  • Use 2-5μg of validated antibody per ChIP reaction

  • Include appropriate controls (IgG control, input samples)

  • Incubate overnight at 4°C with rotation

• Washing and elution:

  • Perform stringent washes to remove non-specific binding

  • Elute DNA-protein complexes from beads

  • Reverse crosslinking by heating at 65°C overnight

• Library preparation and sequencing:

  • Follow standard ChIP-seq library preparation protocols

  • Ensure sufficient sequencing depth (20-30 million reads) for histone modifications

  • Use single-end 50bp or paired-end 2x75bp sequencing

• Bioinformatic analysis:

  • Use established pipelines (MACS2 for peak calling, deepTools for visualization)

  • Identify enrichment patterns and correlate with gene expression data

  • Analyze enrichment at promoter regions and gene bodies

• Validation:

  • Confirm key findings using ChIP-qPCR at selected genomic loci

  • Calculate percent input and fold enrichment using the comparative Ct method

For optimal results, consider integrating ChIP-seq data with other genomic approaches such as RNA-seq, ATAC-seq, or CUT&Tag to comprehensively analyze the relationship between β-hydroxybutyryl-HIST1H3A (K79) and gene expression .

What are the primary techniques for detecting and quantifying β-hydroxybutyryl-HIST1H3A (K79) in research samples?

Several methodological approaches are used to detect and quantify β-hydroxybutyryl-HIST1H3A (K79):

• Antibody-based methods:

  • Western blotting: Using specific antibodies targeting β-hydroxybutyryl-HIST1H3A (K79) with expected band size of ~17-18 kDa. Requires careful validation with positive and negative controls .

  • Immunofluorescence/ICC: For visualizing cellular localization patterns, typically showing nuclear staining with proper optimization of fixation and permeability conditions .

  • ChIP assays: For examining genomic distribution as described in the previous section .

  • ELISA: For quantitative assessment of modification levels in purified histone preparations .

• Mass spectrometry approaches:

  • HPLC-MS/MS: Gold standard for identification and quantification of histone modifications with high sensitivity and specificity.

  • Sample preparation protocols: Include histone extraction, chemical derivatization of unmodified lysines (typically using propionic anhydride), enzymatic digestion with trypsin, followed by LC-MS/MS analysis.

  • Quantification strategies: Label-free quantification, stable isotope labeling (SILAC), or chemical labeling methods (TMT/iTRAQ) .

  • Data analysis: Use specialized software like EpiProfile for histone PTM quantification .

• ChIP-Seq and related techniques:

  • Traditional ChIP-Seq: Combines chromatin immunoprecipitation with next-generation sequencing to map genomic distribution.

  • CUT&Tag: Provides higher resolution and lower background compared to traditional ChIP-seq, with in situ antibody targeting and tagmentation .

  • ATAC-seq integration: Can be combined with ChIP-seq to correlate histone modifications with chromatin accessibility .

• Chemical proteomics approaches:

  • Multivalent photoaffinity probes: Developed to capture binders of histone marks, enabling identification of reader proteins like ENL .

  • Peptide arrays: For testing antibody specificity against various modified peptides, essential for distinguishing between similar modifications .

• Surface enhanced Raman scattering (SERS):

  • A newer biological sensing method using gold nanoparticles as substrate

  • Combines Raman scattering signals for high-sensitivity detection without antibody labeling .

Each technique has specific advantages and limitations, and combining multiple approaches provides the most comprehensive characterization of β-hydroxybutyryl-HIST1H3A (K79) in research samples .

What are the implications of aberrant β-hydroxybutyryl-HIST1H3A (K79) modification in disease states?

Aberrant β-hydroxybutyryl-HIST1H3A (K79) modification has been implicated in several disease processes:

• Cancer biology:

  • Altered histone β-hydroxybutyrylation patterns, including at H3K79, have been observed in hepatocellular carcinoma and other cancers, potentially contributing to oncogenic gene expression programs .

  • Disruption of the interaction between H3K9bhb and ENL via structure-based mutation led to suppressed expression of genes such as MYC that drive cell proliferation, suggesting therapeutic potential .

  • P53, a pivotal tumor suppressor, undergoes modification through β-hydroxybutyrylation at lysines 120, 319, and 370, hampering p53 acetylation and leading to cessation of cellular proliferation and reduction in programmed cell death .

• Metabolic disorders:

  • Given the strong connection between β-hydroxybutyrylation and metabolism, dysregulation of this modification may contribute to metabolic syndrome, diabetes, and obesity through altered transcription of metabolic genes .

  • In diabetes models, histone Kbhb marks are dramatically induced in response to elevated β-hydroxybutyrate levels during diabetic ketoacidosis, altering gene expression patterns .

• Cardiovascular diseases:

  • Research suggests roles for histone β-hydroxybutyrylation in cardiac function and pathology, with potential implications for heart failure and cardiac remodeling .

  • High serum BHB levels were found to correlate with better collateral circulation in patients with myocardial infarction, suggesting a cardioprotective role .

  • BHB promotes angiogenesis post-myocardial infarction through H3K9bhb modification and chromatin opening, enhancing transcription of proangiogenic genes like carnitine palmitoyltransferase 1a (CPT1A) .

• Neuropsychiatric conditions:

  • The ketone body β-hydroxybutyrate and subsequent histone modifications may have neuroprotective effects, with relevance to epilepsy, neurodegenerative disorders, and cognitive function .

  • β-hydroxybutyrylation can provide neuroprotection by mitigating toxic damage to neurons and preventing degenerative changes in dopaminergic neurons among patients with Alzheimer's disease and Parkinson's disease .

• Kidney diseases:

  • In kidney research, significant β-hydroxybutyrylation was observed with the most highly upregulated gene being 3-hydroxy-3-methyglutaryl CoA Synthase 2 (HMGCS2), affecting lipid catabolism .

  • Histone β-hydroxybutyrylation-mediated chromatin compaction of promoter regions contributed to lower transcription and translation of immune function genes like protein tyrosine phosphatase receptor type C (Ptprc) and Lymphocyte cytosolic protein 1 (Lcp1) .

Researchers use both in vitro cell culture models and in vivo animal models to study the causative relationships between aberrant β-hydroxybutyrylation and disease pathogenesis . Integrative approaches combining RNA-seq, ChIP-seq, and ATAC-seq are increasingly employed to understand the molecular mechanisms underlying these disease associations .

Which genes are primarily regulated by β-hydroxybutyryl-HIST1H3A (K79) modification?

β-hydroxybutyryl-HIST1H3A (K79) predominantly regulates genes involved in metabolic adaptation and stress response. ChIP-seq and gene expression studies have identified several gene categories under the influence of this modification:

• Lipid metabolism genes:

  • Particularly those involved in fatty acid oxidation and lipid catabolism

  • Key examples include HMGCS2 (HMG-CoA synthase 2), which is essential for ketogenesis and was upregulated ~15-fold in BHB treated groups .

  • Other targets include Acaa1b, involved in β-oxidation of fatty acids .

• Amino acid catabolism genes:

  • Those supporting gluconeogenesis during starvation conditions

  • Enable the cell to utilize amino acids as carbon sources when glucose is limited .

• Mitochondrial function genes:

  • Including those involved in oxidative phosphorylation and the TCA cycle

  • These genes enhance cellular energy production capacity during metabolic stress .

• FOXO target genes:

  • The FOXO transcription factors, which respond to nutritional stress, have targets that are enriched for β-hydroxybutyryl-HIST1H3A (K79) marks

  • FOXO factors integrate various cellular signals to regulate metabolism, stress resistance, and cell survival .

• Immune function genes:

  • Recent evidence suggests roles in regulating certain aspects of immune cell function and inflammatory responses

  • Downregulation of immune function genes like Ptprc (protein tyrosine phosphatase receptor type C) and Lcp1 (Lymphocyte cytosolic protein 1) was observed through histone β-hydroxybutyrylation-mediated chromatin compaction .

• Angiogenesis-related genes:

  • In cardiovascular research, BHB-mediated H3K9bhb modification promotes transcription of proangiogenic genes like carnitine palmitoyltransferase 1a (CPT1A) .

Genome-wide studies using ChIP-seq have shown that β-hydroxybutyryl-HIST1H3A (K79) is primarily enriched at active gene promoters and is associated with increased transcriptional activity of these metabolically relevant genes during states of elevated β-hydroxybutyrate . Integration of ChIP-seq data with RNA-seq analysis demonstrates strong correlation between H3K9bhb enrichment and upregulation of gene expression, particularly for starvation-responsive metabolic pathways .

The genomic targeting of this modification appears to be highly specific, creating a direct link between metabolic state and gene expression patterns that enable cellular adaptation to nutritional stress .