2-hydroxyisobutyryl-HIST1H3A (K18) Antibody

What is lysine 2-hydroxyisobutyrylation and how does it differ from other histone modifications?

Lysine 2-hydroxyisobutyrylation (Khib) is a post-translational modification derived from 2-hydroxyisobutyrate and 2-hydroxyisobutyryl-CoA. Unlike more common modifications such as acetylation or methylation, Khib has a unique chemical structure and specific genomic distribution patterns. This modification has been shown to be associated with active gene expression, particularly in spermatogenic cells. The modification exhibits varied dynamics among diverse model systems and appears to be mechanistically and functionally distinct from histone lysine acetylation (Kac) and methylation (Kme) . The unique regulatory function of this modification is linked to two cellular metabolites: 2-hydroxyisobutyrate and 2-hydroxyisobutyryl-CoA, which serve as precursors for this PTM .

What is the 2-hydroxyisobutyryl-HIST1H3A (K18) antibody and what epitope does it recognize?

The 2-hydroxyisobutyryl-HIST1H3A (K18) antibody is a polyclonal antibody raised in rabbits that specifically recognizes the 2-hydroxyisobutyryl modification at lysine 18 of histone H3.1. The immunogen used for producing this antibody is a peptide sequence surrounding the site of 2-hydroxyisobutyryl-Lys (18) derived from Human Histone H3.1 . This antibody is purified through antigen affinity methods and is designed to be highly specific for the modified lysine residue, allowing researchers to detect and quantify this specific post-translational modification in experimental settings .

What is the structural basis for enzyme recognition of 2-hydroxyisobutyryl-CoA?

Structural modeling has revealed important insights about enzyme interaction with 2-hydroxyisobutyryl-CoA. For example, studies of Esa1p (a yeast acetyltransferase) bound to acetyl-CoA showed that its catalytic pocket is large enough to accommodate 2-hydroxyisobutyryl-CoA with its larger acyl tail. When researchers modeled an Esa1p/2-hydroxyisobutyryl-CoA structure based on the acetyl-CoA-bound Esa1p structure, they found that 2-hydroxyisobutyryl-CoA binds in a similar manner, with the 2-hydroxyisobutyryl group fitting well into the catalytic pocket . This structural compatibility explains how enzymes like Esa1p and its human homolog Tip60 can catalyze 2-hydroxyisobutyrylation despite their primary evolution as acetyltransferases .

What are the validated applications for 2-hydroxyisobutyryl-HIST1H3A (K18) antibody?

The 2-hydroxyisobutyryl-HIST1H3A (K18) antibody has been validated for multiple experimental applications, including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Chromatin Immunoprecipitation (ChIP)

These diverse applications make the antibody versatile for different research questions, from protein detection and quantification to localization studies and analysis of chromatin interactions . The broad application spectrum highlights the antibody's utility in both biochemical and cell-based assays.

How should researchers design ChIP experiments using 2-hydroxyisobutyryl-HIST1H3A (K18) antibody?

When designing ChIP experiments with the 2-hydroxyisobutyryl-HIST1H3A (K18) antibody, researchers should consider:

• Cross-linking protocol: Standard formaldehyde fixation (1% for 10 minutes) is typically sufficient for histone modifications.

• Sonication conditions: Aim for chromatin fragments of 200-500 bp for optimal resolution.

• Antibody concentration: Start with manufacturer's recommended dilution, typically around 1:200 for immunoprecipitation.

• Controls: Include:

  • Input DNA (pre-immunoprecipitation sample)

  • IgG control (non-specific rabbit IgG)

  • Positive control (antibody against a well-characterized histone mark)

• Validation: Consider parallel ChIP-qPCR of known regions before proceeding to sequencing.

This approach leverages the antibody's specificity for the 2-hydroxyisobutyryl modification at K18 of histone H3 and enables mapping of this modification across the genome to identify associated regulatory elements .

What are the optimal protein extraction methods for detecting 2-hydroxyisobutyryl-HIST1H3A (K18) in Western blots?

For optimal detection of 2-hydroxyisobutyryl-HIST1H3A (K18) in Western blots, researchers should:

• Extract histones using acid extraction:

  • Lyse cells in Triton Extraction Buffer (PBS with 0.5% Triton X-100, 2mM PMSF, 0.02% NaN₃)

  • Resuspend nuclei in 0.2N HCl

  • Incubate overnight at 4°C

  • Collect supernatant containing histones

• Include deacetylase inhibitors in all buffers:

  • Add sodium butyrate (5-30 mM) to preserve butyrl modifications

  • Include trichostatin A (TSA) for class I and II HDAC inhibition

• Gel electrophoresis conditions:

  • Use 15-18% SDS-PAGE for optimal histone separation

  • Load 10-20 μg of acid-extracted histones

• Antibody incubation:

  • Dilute primary antibody at 1:2000 in 1% BSA

  • Incubate overnight at 4°C for optimal binding

This protocol is based on successful detection of similar histone modifications and has been shown to effectively preserve the 2-hydroxyisobutyryl modification during extraction .

How can researchers distinguish between 2-hydroxyisobutyrylation and other acylation marks on histone H3?

Distinguishing between 2-hydroxyisobutyrylation and other acylation marks requires careful experimental design:

• Antibody specificity validation:

  • Perform peptide competition assays with modified and unmodified peptides

  • Use dot blots with various modified peptides to test cross-reactivity

  • Conduct Western blots with samples enriched for different modifications

• Mass spectrometry approaches:

  • Use LC-MS/MS with collision-induced dissociation (CID) or electron transfer dissociation (ETD)

  • Analyze fragment ion patterns for diagnostic ions specific to 2-hydroxyisobutyrylation

  • Employ targeted multiple reaction monitoring for specific modified peptides

• Parallel immunoprecipitations:

  • Compare ChIP-seq profiles of 2-hydroxyisobutyrylation with other marks like butyrylation or acetylation

  • Analyze co-occurrence patterns and unique genomic distributions

Research shows that H4K8hib has unique genomic distribution patterns compared to acetylation and methylation marks, supporting the distinction between these modifications . Mass spectrometry has identified H4K8, H4K12, H4K16, and H4K31 as K2hib substrates of Tip60, providing analytical targets for differentiation .

What are the known "writers" and "erasers" of the 2-hydroxyisobutyryl mark, and how should researchers design experiments to study them?

To study these enzymes:

• For writers (Esa1p/Tip60):

  • Use in vitro assays with purified enzymes and nucleosome core particles

  • Supply 2-hydroxyisobutyryl-CoA as donor

  • Use specific inhibitors like TH1834 for Tip60 to confirm activity

  • Employ genetic approaches (knockdown/overexpression)

• For erasers (HDAC2/HDAC3):

  • Use in vitro screening with core histones as substrates

  • Apply HDAC inhibitors (NaBu, TSA) to confirm specificity

  • Implement genetic approaches (SILAC quantification after HDAC manipulation)

The search results show that overexpression of HDAC2 or HDAC3 reduced global histone K2hib levels by approximately 30%, while double depletion of HDAC2 and HDAC3 significantly increased global K2hib levels .

What cellular signaling pathways influence 2-hydroxyisobutyrylation levels?

2-hydroxyisobutyrylation levels are influenced by multiple cellular pathways:

• Metabolic pathways:

  • 2-hydroxyisobutyrate production is linked to symbiotic gut microbiota metabolism

  • Cellular concentrations of 2-hydroxyisobutyryl-CoA affect modification rates

  • The dynamics of these metabolites are associated with diverse host metabolic phenotypes

• Enzymatic regulation:

  • Tip60 acetyltransferase complex activity is regulated by cellular signaling

  • HDAC2/3 activity responds to various cellular conditions and signaling events

  • The balance between writer and eraser activities determines steady-state levels

• Transcriptional activation:

  • H4K8hib is associated with regions of active gene transcription

  • The modification is dynamically regulated during meiotic and post-meiotic processes

Experimental approaches to study these pathways include metabolite supplementation (e.g., sodium butyrate treatment), enzyme inhibition studies, and genetic manipulation of writers and erasers in combination with transcriptional analysis . Studies have shown that treatment with 30 mM sodium butyrate for 4 hours can significantly alter histone modification patterns in HeLa and Jurkat cell lines .

What are common pitfalls in 2-hydroxyisobutyryl-HIST1H3A (K18) antibody experiments and how can they be addressed?

Common pitfalls and solutions include:

• Low signal intensity:

  • Increase antibody concentration (try 1:100 - 1:500 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Pre-treat samples with HDAC inhibitors (sodium butyrate, TSA) to preserve modifications

  • Use enhanced detection systems for Western blots (e.g., biotin-streptavidin amplification)

• High background or non-specific binding:

  • Increase blocking time and concentration (5-10% normal serum)

  • Include additional washing steps with higher detergent concentration

  • Pre-absorb antibody with unmodified histone peptides

  • Use more stringent antibody dilution buffers (1% BSA with 0.1% Tween-20)

• Inconsistent results between replicates:

  • Standardize cell culture conditions affecting metabolism

  • Control fixation and permeabilization parameters carefully

  • Ensure consistent sample processing times to prevent modification loss

  • Use internal controls (total H3 detection) for normalization

Research protocols show that successful immunostaining involves fixation in 4% formaldehyde, permeabilization with 0.2% Triton X-100, and blocking with 10% normal goat serum for 30 minutes at room temperature .

How should researchers interpret changes in 2-hydroxyisobutyryl-HIST1H3A (K18) levels in different experimental contexts?

Interpreting changes in 2-hydroxyisobutyryl-HIST1H3A (K18) levels requires consideration of:

• Baseline modification levels:

  • Establish normal ranges in your experimental system

  • Compare to other histone modifications (acetylation, methylation) at similar residues

  • Determine cell-type specificity of the modification

• Context-dependent interpretation:

  • Increased levels may indicate:

    • Enhanced gene activation (as Khib is associated with active transcription)

    • Metabolic changes affecting 2-hydroxyisobutyryl-CoA availability

    • Decreased activity of eraser enzymes (HDAC2/HDAC3)

  • Decreased levels may suggest:

    • Reduced transcriptional activity

    • Metabolic shifts away from 2-hydroxyisobutyrate production

    • Increased eraser enzyme activity

• Integrated analysis:

  • Correlate with transcriptomic data to link modifications to gene expression

  • Compare with ChIP-seq data of transcription factors and other histone marks

  • Analyze in context of metabolomic data when possible

Research has shown that 2-hydroxyisobutyrylation has distinct genomic distribution patterns compared to acetylation and functions in regulating gene expression, particularly in spermatogenic cells . SILAC quantification studies have demonstrated that HDAC3 overexpression can decrease Khib levels by approximately 30%, providing benchmarks for expected changes .

What is the current understanding of the 2-hydroxyisobutyryl proteome beyond histones?

The current understanding of the 2-hydroxyisobutyryl proteome reveals:

• Scope and distribution:

  • Global profiling has identified 6,548 Khib sites on 1,725 substrate proteins in mammalian cells

  • The modification extends far beyond histones to various cellular proteins

  • The extensive nature of this modification suggests broad regulatory roles

• Functional protein categories:

  • Khib-modified proteins are involved in diverse cellular processes

  • The modification appears on both nuclear and cytoplasmic proteins

  • Functional clustering reveals enrichment in specific biological pathways

• Substrate specificity patterns:

  • Analysis of flanking sequences around Khib sites reveals potential consensus motifs

  • Structural analysis indicates accessibility factors for modification sites

  • Comparison with other acylation sites shows both overlap and unique targets

This comprehensive modification landscape provides a foundation for understanding the broader biological functions of 2-hydroxyisobutyrylation and opens new avenues for investigating how cellular metabolites influence protein function .

How does the 2-hydroxyisobutyryl modification coordinate with other histone marks in gene regulation?

The coordination between 2-hydroxyisobutyryl modification and other histone marks involves:

Understanding these coordination patterns is crucial for deciphering the histone code and the specific role of 2-hydroxyisobutyrylation within this complex regulatory system . Research has shown that H4K8hib marks are associated with regions of active gene transcription in both meiotic and post-meiotic cells, suggesting a specific role in gene regulation during these developmental processes .

What experimental approaches are emerging for studying the metabolic regulation of 2-hydroxyisobutyrylation?

Emerging experimental approaches for studying metabolic regulation include:

• Metabolite tracing studies:

  • Isotope-labeled 2-hydroxyisobutyrate to track metabolic incorporation

  • Metabolomic profiling combined with histone modification analysis

  • Microbiome manipulation to alter 2-hydroxyisobutyrate production

• Advanced enzyme assays:

  • Development of fluorescent or bioluminescent reporters for real-time transferase activity

  • High-throughput screening for novel writers and erasers

  • Structure-guided design of specific inhibitors for functional studies

• Integrated multi-omics:

  • Combined analysis of metabolomics, proteomics, and transcriptomics data

  • Correlation of gut microbiome profiles with host 2-hydroxyisobutyrylation patterns

  • Mathematical modeling of metabolite-modification relationships

Research has established connections between symbiotic gut microbiota-associated metabolites, including 2-hydroxyisobutyrate, and diverse host metabolic phenotypes, suggesting complex regulation of this modification pathway . The dynamics of these metabolites influence 2-hydroxyisobutyrylation levels, opening new avenues for investigating how microbial communities might influence host epigenetics through this modification pathway .