β-hydroxybutyryl-HIST1H2BC (K11) Antibody

Basic Research Questions

• What is β-hydroxybutyryl-HIST1H2BC (K11) Antibody and what are its primary research applications?

  β-hydroxybutyryl-HIST1H2BC (K11) Antibody is a specialized immunological tool that recognizes histone H2B proteins containing a β-hydroxybutyryl modification at the lysine 11 position. This antibody enables detection of a specific histone post-translational modification involved in metabolic regulation of gene expression.

  Primary applications include:

  • Western blotting (WB) at dilutions of 1:100-1:1000

  • Enzyme-linked immunosorbent assay (ELISA) at dilutions of 1:2000-1:10000

  • Immunocytochemistry (ICC) at dilutions of 1:20-1:200

  The antibody is typically developed in rabbits using synthetic peptides corresponding to human histone H2B with β-hydroxybutyrylation at lysine 11 . Validated for human samples, some products also demonstrate reactivity with rat samples .

• How does histone lysine β-hydroxybutyrylation (Kbhb) differ from other histone modifications such as acetylation?

  While both β-hydroxybutyrylation and acetylation occur on lysine residues, they represent distinct modifications with different metabolic origins and functional implications:

  • Chemical structure: β-hydroxybutyrylation incorporates a larger β-hydroxybutyryl group compared to acetylation's acetyl group

  • Metabolic regulation: β-hydroxybutyrylation is specifically linked to elevated β-hydroxybutyrate levels during fasting or ketogenic conditions, whereas acetylation is regulated by glucose metabolism and acetyl-CoA levels

  • Gene activation patterns: Studies using site-specific antibodies show that H3K9bhb, H3K18bhb, and H4K8bhb are induced by β-hydroxybutyrate treatment in a dose-dependent manner, with patterns distinct from their acetylated counterparts (H3K9ac, H3K18ac, H4K8ac)

  • Genomic distribution: ChIP-seq analysis reveals that histone Kbhb marks are enriched at promoters of starvation-responsive metabolic genes, indicating a specialized role in metabolic adaptation

  These differences highlight the unique function of β-hydroxybutyrylation in connecting cellular metabolism to epigenetic regulation.

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

  Histone β-hydroxybutyrylation represents a direct molecular link between ketone body metabolism and epigenetic regulation:

  • β-hydroxybutyrate levels increase during fasting, caloric restriction, or diabetic ketoacidosis

  • Cellular β-hydroxybutyrate can be converted to β-hydroxybutyryl-CoA, which serves as the cofactor for enzymatic addition of β-hydroxybutyryl groups to histone lysine residues

  • Metabolic labeling experiments using isotopically labeled sodium β-hydroxybutyrate ([2,4-13C2]) demonstrate direct incorporation of the β-hydroxybutyryl group onto histones

  • Treatment of human HEK293 cells with sodium β-hydroxybutyrate induces histone Kbhb in a dose-dependent manner, while histone acetylation remains largely unchanged

  • In mouse models, prolonged fasting or streptozotocin-induced diabetic ketoacidosis dramatically increases histone Kbhb levels, particularly in liver tissues

  This metabolic sensing mechanism allows cells to adjust gene expression in response to nutritional status through direct modification of chromatin structure.

• How should researchers validate the specificity of β-hydroxybutyryl-HIST1H2BC (K11) Antibody?

  Comprehensive validation requires multiple approaches:

  • Peptide competition assays: Pre-incubate antibody with β-hydroxybutyrylated and non-modified peptides to verify specific binding to the modified epitope

  • Western blot validation: Compare samples with enhanced β-hydroxybutyrylation (cells treated with sodium β-hydroxybutyrate) against untreated controls - multiple cell lines (HeLa, HEK-293, A549, K562) treated with 50mM sodium 3-hydroxybutyrate for 72 hours show significantly increased signal compared to untreated controls

  • Cross-reactivity testing: Ensure the antibody does not recognize other histone modifications, particularly acetylation at the same position (K11ac)

  • Multiple antibody comparison: When possible, compare results from different antibody clones or sources

  • Mass spectrometry confirmation: Verify modification sites detected by the antibody using mass spectrometry-based approaches

  Manufacturers typically validate antibodies using specific cell treatment protocols; researchers should review these data before experimental design.

Advanced Research Questions

• What methodological approaches can researchers use to experimentally manipulate histone β-hydroxybutyrylation levels?

  Several strategies exist for modulating histone β-hydroxybutyrylation in experimental systems:

  • Direct metabolite supplementation:

    • Treating cells with sodium β-hydroxybutyrate (10-50mM) increases histone Kbhb in a dose-dependent manner

    • Standard protocol involves 50mM sodium 3-hydroxybutyrate treatment for 48-72 hours

  • Metabolic tracing:

    • Using isotopically labeled sodium β-hydroxybutyrate ([2,4-13C2]) confirms direct incorporation into histone proteins

    • Mass spectrometry analysis can detect isotope-labeled peptides with a +2Da mass shift

  • Enzymatic modulation:

    • Targeting HDACs (particularly HDAC1-3) or sirtuins (SIRT1-3) that exhibit de-β-hydroxybutyrylation activity

    • HDAC inhibitors may increase global β-hydroxybutyrylation levels

  • Physiological induction:

    • In animal models, fasting protocols (16-24 hours) naturally elevate β-hydroxybutyrate levels

    • Streptozotocin-induced diabetic ketoacidosis models produce dramatic increases in histone Kbhb

  Verification of β-hydroxybutyrylation changes should employ both antibody-based methods (Western blotting) and, when possible, mass spectrometry analysis.

• How does the genomic distribution of H2BK11bhb compare to other histone modifications, and what does this reveal about its function?

  Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with β-hydroxybutyryl-specific antibodies reveals distinct genomic distribution patterns:

  • Promoter enrichment: H2BK11bhb is predominantly found at gene promoters, similar to active histone marks like H3K4me3 and H3K9ac

  • Correlation with gene activity: Genes marked by H2BK11bhb show higher expression levels, indicating association with transcriptional activation

  • Metabolic pathway specificity: H2BK11bhb shows particular enrichment at genes involved in starvation-responsive metabolic pathways compared to general activating marks like acetylation

  • Dynamic regulation: During fasting or ketosis, H2BK11bhb marks increase at specific metabolic genes, preceding changes in gene expression

  These patterns suggest that H2BK11bhb functions as a metabolic sensor that specifically activates genes required for adaptation to ketogenic conditions, representing a direct link between cellular metabolism and genome regulation.

• What is known about the enzymatic regulation of histone β-hydroxybutyrylation and de-β-hydroxybutyrylation?

  The enzymatic machinery controlling β-hydroxybutyrylation includes:

  • "Writers" (enzymes adding the modification):

    • Specific β-hydroxybutyryltransferases remain to be definitively identified

    • The process requires β-hydroxybutyryl-CoA as the donor molecule

    • Metabolic labeling confirms cellular conversion of β-hydroxybutyrate to β-hydroxybutyryl-CoA

  • "Erasers" (enzymes removing the modification):

    • HDAC1-3 demonstrate significant de-β-hydroxybutyrylation activity against core histones in vitro

    • SIRT1-3 also exhibit de-β-hydroxybutyrylation activity in biochemical assays

    • In cellular contexts, primarily HDAC1 and HDAC2 function as histone Kbhb deacetylases

    • Different HDACs may show selectivity for different chiral forms of β-hydroxybutyrylation

  • "Readers" (proteins recognizing the modification):

    • Specific reader proteins for β-hydroxybutyrylation remain to be fully characterized

    • Likely candidates include bromodomain-containing proteins that recognize various acylated lysine residues

  The enzymatic regulation of histone β-hydroxybutyrylation represents an important area for further research, particularly regarding tissue-specific and context-dependent regulatory mechanisms.

• How can researchers integrate β-hydroxybutyryl-HIST1H2BC (K11) Antibody with other omics approaches to understand metabolic regulation of gene expression?

  Multi-omics integration strategies provide comprehensive insights into β-hydroxybutyrylation function:

  Approach	Methodology	Research Insight
  ChIP-seq + RNA-seq	Map genomic distribution of H2BK11bhb and correlate with transcriptome	Identify direct regulatory targets of β-hydroxybutyrylation
  Metabolomics + ChIP-seq	Measure cellular β-hydroxybutyrate levels alongside histone modifications	Establish dose-response relationships between metabolite levels and epigenetic changes
  ATAC-seq + ChIP-seq	Assess chromatin accessibility in relation to H2BK11bhb	Understand how β-hydroxybutyrylation affects chromatin structure
  Proteomics + ChIP-seq	Identify proteins interacting with β-hydroxybutyrylated histones	Discover potential "reader" proteins that recognize this modification

  Computational integration should include:

  • Network analysis to identify metabolic pathways enriched for H2BK11bhb-marked genes

  • Temporal modeling to capture dynamic relationships between metabolite fluctuations and epigenetic changes

  • Comparative analysis with other histone modifications to distinguish β-hydroxybutyrylation-specific effects

  This integrated approach can reveal how β-hydroxybutyrylation connects metabolic signals to specific gene regulatory programs.

• What considerations are important when using β-hydroxybutyryl-HIST1H2BC (K11) Antibody in ChIP-seq experiments?

  Successful ChIP-seq with β-hydroxybutyryl-HIST1H2BC (K11) Antibody requires careful optimization:

  • Chromatin preparation:

    • Optimal fragmentation to 200-500bp fragments

    • Fixation conditions may need adjustment compared to standard ChIP protocols

  • Antibody validation:

    • Confirm ChIP-grade quality through pilot experiments

    • Determine optimal antibody concentration (typically higher than for Western blotting)

    • Verify enrichment at expected target genes before sequencing

  • Controls:

    • Include input chromatin control

    • Use IgG control to assess non-specific binding

    • Consider parallel ChIP with H2B antibody to normalize for histone occupancy

    • Include positive control targeting abundant histone mark (e.g., H3K4me3)

  • Experimental design:

    • Compare samples with elevated β-hydroxybutyrate levels to control samples

    • Include biological replicates to ensure reproducibility

    • Consider time-course experiments to capture dynamic changes

  • Bioinformatic analysis:

    • Analyze genomic distribution of H2BK11bhb marks

    • Correlate with gene expression data (RNA-seq)

    • Perform motif analysis to identify enriched transcription factor binding sites

    • Compare with other histone modification datasets

  Careful attention to these considerations will maximize the quality and interpretability of ChIP-seq data with β-hydroxybutyryl-HIST1H2BC (K11) Antibody.

• How does the chiral structure of β-hydroxybutyrate affect histone modification and antibody recognition?

  β-hydroxybutyrate exists in D(-) and L(+) enantiomers, adding complexity to histone β-hydroxybutyrylation research:

  • Physiological relevance: D-β-hydroxybutyrate is the predominant form produced during ketosis in humans

  • Enzymatic selectivity: Research suggests different deacetylases (HDACs) may exhibit preferences for removing specific chiral forms of Kbhb

  • Antibody specificity: Commercial antibodies may recognize D-β-hydroxybutyrylation, L-β-hydroxybutyrylation, or both forms

  • Functional differences: The biological significance of different chiral forms remains to be fully elucidated

  Researchers should consider:

  • Verifying which enantiomer(s) their antibody recognizes

  • Using defined stereoisomers in control experiments

  • Investigating potential differences in genomic distribution and functional effects between D- and L-β-hydroxybutyrylation

  • Exploring whether enzymes regulating this modification exhibit stereoselectivity

  Understanding the stereochemical aspects of histone β-hydroxybutyrylation may reveal additional layers of regulatory complexity in metabolic epigenetics.

• What technical challenges exist in detecting low-abundance histone β-hydroxybutyrylation in different experimental systems?

  Researchers face several technical hurdles when studying histone β-hydroxybutyrylation:

  • Antibody specificity:

    • Ensuring antibodies distinguish between β-hydroxybutyrylation and similar modifications (e.g., acetylation) at the same residue

    • Validating performance across different experimental conditions and sample types

  • Signal enhancement strategies:

    • Pre-enrichment of histones before analysis

    • Signal amplification methods for immunodetection

    • Treatment with HDAC inhibitors to prevent removal of the modification

    • Using metabolic treatment (sodium β-hydroxybutyrate) to increase modification levels

  • Mass spectrometry considerations:

    • Developing protocols for efficient enrichment of β-hydroxybutyrylated peptides

    • Optimizing fragmentation methods to distinguish between different acyl modifications

    • Implementing quantitative approaches to compare modification levels across samples

  • Physiological relevance:

    • Determining tissue-specific patterns of β-hydroxybutyrylation

    • Establishing physiologically relevant concentrations of β-hydroxybutyrate that induce histone modifications

    • Accounting for differences between in vitro and in vivo systems

  These challenges necessitate careful experimental design and validation when studying histone β-hydroxybutyrylation, especially in systems with naturally low levels of this modification.

• How does histone H2B β-hydroxybutyrylation interact with the broader histone code and chromatin regulation?

  Histone H2B β-hydroxybutyrylation functions within a complex network of histone modifications:

  • Modification crosstalk: Research suggests potential interactions between β-hydroxybutyrylation and other histone marks, where one modification may influence the presence or function of others

  • Nucleosome structure impact: As a core component of nucleosomes, modified H2B can affect DNA accessibility by altering chromatin compaction

  • Transcriptional machinery interaction: H2BK11bhb likely influences recruitment of transcription factors and RNA polymerase to regulate gene expression

  • Chromatin reader recruitment: The modification may be recognized by specific "reader" proteins that mediate downstream effects

  • Integration with cellular signaling: β-hydroxybutyrylation represents a direct mechanism linking metabolic signaling to chromatin regulation

  Research indicates that histone H2B plays central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . β-hydroxybutyrylation at K11 likely contributes to these functions specifically in the context of metabolic adaptation.

  Understanding how H2BK11bhb integrates with the broader histone code will require comprehensive studies combining genetic, biochemical, and structural approaches with genome-wide analyses of modification patterns.