β-hydroxybutyryl-HIST1H3A (K9) Antibody

What is β-hydroxybutyryl-HIST1H3A (K9) Antibody and what applications has it been validated for?

β-hydroxybutyryl-HIST1H3A (K9) Antibody is a polyclonal antibody that specifically recognizes the β-hydroxybutyryl modification at lysine 9 of histone H3.1 (HIST1H3A). According to product information, this antibody has been validated for multiple applications including:

• Enzyme immunoassay (EIA)

• Immunoassay

• Chromatin immunoprecipitation (ChIP)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunocytochemistry (ICC)

• Immunoprecipitation (IP)

• Western blot (WB)

These applications make it a versatile tool for investigating histone β-hydroxybutyrylation in various experimental contexts.

How does histone lysine β-hydroxybutyrylation (Kbhb) differ from other histone modifications?

Histone lysine β-hydroxybutyrylation (Kbhb) represents a novel class of histone post-translational modification that differs from more well-characterized modifications in several key aspects:

Unlike acetylation, which is predominantly associated with gene activation, Kbhb appears to be a mechanism by which ketone bodies specifically regulate cellular physiology through changes in gene expression .

What experimental methods can be used to detect and quantify histone β-hydroxybutyrylation?

Multiple methodologies can be employed to study histone β-hydroxybutyrylation:

• Antibody-based methods:

  • Western blotting using site-specific antibodies (e.g., anti-H3K9bhb)

  • Immunocytochemistry for cellular localization

  • ChIP assays to identify genomic locations

  • ELISA for quantification

• Mass spectrometry-based methods:

  • HPLC/MS/MS analysis of trypsin-digested histones

  • Isotopic labeling using 13C-labeled β-hydroxybutyrate to track incorporation

  • MALDI-TOF MS for identifying modified peptides

• Enrichment strategies:

  • Functionalized gold nanoparticle probes for enrichment of endogenous binders

  • Crosslinking approaches using UV irradiation (365 nm)

When designing experiments, it's important to include appropriate controls to distinguish β-hydroxybutyrylation from other acylation modifications.

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

Research demonstrates a direct relationship between cellular β-hydroxybutyrate levels and histone β-hydroxybutyrylation:

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

• Treatment of cells with sodium β-hydroxybutyrate induces histone Kbhb levels in a dose-dependent manner

• Histone Kbhb levels increase under physiological conditions that elevate β-hydroxybutyrate, including:

  • Starvation (increasing from 0.1 mM to 2-3.8 mM)

  • Intense exercise

  • Diabetic ketoacidosis

This relationship establishes histone β-hydroxybutyrylation as a mechanism through which metabolic state can directly influence gene expression patterns through epigenetic regulation.

What are the regulatory enzymes ("writers" and "erasers") for histone lysine β-hydroxybutyrylation?

Based on current research, the following enzymes regulate histone lysine β-hydroxybutyrylation:

Writers:

• p300 has been identified as a writer enzyme that can catalyze histone Kbhb

Erasers:
Several histone deacetylases (HDACs) demonstrate de-β-hydroxybutyrylation activity:

• HDAC1, HDAC2, and HDAC3 (Class I HDACs)

• SIRT1 and SIRT2 (Class III HDACs)

In vitro screening of 18 recombinant HDACs (HDAC1-11 and SIRT1-7) showed that these five enzymes exhibited notable de-Kbhb activity toward core histones. This was confirmed using Kbhb-containing histone peptides as substrates followed by HPLC assay.

Cellular validation showed:

• Simultaneous knockdown of HDAC1 and HDAC2 increased levels of Kbhb in both HEK293 and HeLa cells

• Treatment with MS275 (a selective HDAC1/2/3 inhibitor) increased multiple Kbhb site signals in a dose-dependent manner

This regulatory system allows dynamic control of histone β-hydroxybutyrylation levels in response to changing cellular conditions.

How does ENL protein function as a "reader" of histone β-hydroxybutyrylation to modulate gene expression?

ENL (Eleven-nineteen leukemia protein) functions as a "reader" of histone β-hydroxybutyrylation marks. Research using histone peptide probes with and without β-hydroxybutyrylation modification has identified ENL as a protein that specifically recognizes and binds to β-hydroxybutyrylated histones.

Experimental approaches to study this interaction include:

• Using functionalized probes (H3K9bhb and unmodified H3K9) incubated with nuclear extracts

• UV crosslinking at 365 nm to capture binding proteins

• Washing and trypsin digestion followed by HPLC-MS/MS analysis to identify binding partners

The identification of ENL as a reader of histone β-hydroxybutyrylation provides a mechanistic link between this metabolic-responsive histone modification and subsequent changes in gene expression. ENL is known to be involved in transcriptional elongation, suggesting that β-hydroxybutyrylation may influence this process.

What is the functional significance of H3K9bhb compared to other histone modifications at the same residue (H3K9ac, H3K9me3)?

H3K9 is a critical residue that can undergo multiple modifications with distinct functional outcomes:

Intriguingly, research has shown that β-hydroxybutyrate treatment increases H3K9bhb levels while having minimal effect on H3K9ac levels, suggesting specific regulatory mechanisms for these distinct modifications at the same residue .

In the context of class switch recombination (CSR) in B cells, both H3 acetyl K9 and H3 trimethyl K9 have been found to correlate with recombining pairs of donor and recipient switch regions. This is surprising since H3 trimethyl K9 is typically associated with silent genes and heterochromatin .

These findings suggest complex interplay between different modifications at the same residue, potentially allowing for nuanced regulation of gene expression in response to various cellular signals.

How can isotopic labeling approaches be applied to study the dynamics of β-hydroxybutyrylation?

Isotopic labeling provides powerful tools for studying β-hydroxybutyrylation dynamics:

Methodology:

• Treat cells with isotopically labeled β-hydroxybutyrate (e.g., sodium [13C2]-β-hydroxybutyrate)

• Extract histones and perform trypsin digestion

• Analyze by HPLC/MS/MS to detect peptides modified by the isotopic β-hydroxybutyryl group

• Identify specific Kbhb sites through mass shift detection (+2 Da for 13C2-labeled samples)

• Compare fragmentation patterns with unlabeled controls

This approach has successfully demonstrated that:

• Sodium β-hydroxybutyrate can be converted into β-hydroxybutyryl-CoA in cells

• β-hydroxybutyryl-CoA serves as the cofactor for enzymatic lysine β-hydroxybutyrylation

• The process occurs in a dose-dependent manner

Additional applications of isotopic labeling include:

• Pulse-chase experiments to determine turnover rates of Kbhb marks

• Comparative analysis of site-specific Kbhb dynamics under different physiological conditions

• Validation of writer and eraser enzyme activities in cellular contexts

What methodological approaches are recommended for validating the specificity of β-hydroxybutyryl-HIST1H3A (K9) antibodies?

Validating antibody specificity is critical for reliable research. For β-hydroxybutyryl-HIST1H3A (K9) antibodies, consider these approaches:

• Peptide competition assays:

  • Pre-incubate antibody with excess synthetic β-hydroxybutyrylated and unmodified peptides

  • Specificity is demonstrated if only the β-hydroxybutyrylated peptide blocks signal

• Multiple antibody validation:

  • Use antibodies from different commercial sources (confirmed in studies using antibodies from different suppliers)

  • Compare results using antibodies raised against different epitopes containing the same modification

• Genetic/biochemical manipulation:

  • Modulate β-hydroxybutyrate levels (e.g., sodium β-hydroxybutyrate treatment)

  • Manipulate writer (p300) or eraser (HDAC1/2) enzyme levels

  • Confirm antibody signal changes accordingly

• Control modifications:

  • Test cross-reactivity with similar acylations (acetylation, butyrylation)

  • Include acetylated and unmodified histone controls in assays

• Mass spectrometry correlation:

  • Confirm antibody-detected sites match MS-identified β-hydroxybutyrylation sites

These validation steps ensure that experimental results accurately reflect the presence and dynamics of histone β-hydroxybutyrylation.

How can ChIP-seq be optimized for β-hydroxybutyryl-HIST1H3A (K9) antibodies?

Optimizing ChIP-seq for β-hydroxybutyryl-HIST1H3A (K9) antibodies requires special considerations:

• Cross-linking optimization:

  • Standard formaldehyde cross-linking (1% for 10 minutes) works for most histone modifications

  • Consider dual cross-linking approaches (DSG followed by formaldehyde) for improved capture

• Sonication parameters:

  • Target fragment size of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody validation for ChIP:

  • Perform preliminary ChIP-qPCR at known targets before proceeding to sequencing

  • Use at least 2-5 μg of antibody per immunoprecipitation for polyclonal antibodies

• Controls:

  • Include input DNA control

  • Use IgG negative control

  • Consider ChIP for H3K9ac as a comparative modification

  • Include β-hydroxybutyrate treatment conditions to demonstrate specificity

• Data analysis considerations:

  • Compare β-hydroxybutyrylation patterns with transcriptome data

  • Analyze co-occurrence with other histone marks (H3K4me3, H3K27ac) at active genes

  • Look for enrichment in metabolic gene pathways given the connection to β-hydroxybutyrate metabolism

This optimized approach will enable researchers to accurately map the genome-wide distribution of H3K9bhb and correlate it with gene expression patterns under various metabolic conditions.