2-hydroxyisobutyryl-HIST1H4A (K59) Antibody

What is 2-hydroxyisobutyryl-HIST1H4A (K59) and its significance in epigenetic research?

2-hydroxyisobutyryl-HIST1H4A (K59) refers to a specific post-translational modification where a 2-hydroxyisobutyryl group is attached to the lysine 59 residue of histone H4. Histone H4 is one of the core components of nucleosomes, which wrap and compact DNA into chromatin structures . This modification is significant in epigenetic research because it represents one of several histone marks that regulate DNA accessibility to cellular machinery involved in transcription, replication, and repair processes . The 2-hydroxyisobutyrylation of lysine residues is a relatively newly identified modification that appears to play distinct roles from other better-studied modifications like acetylation and methylation . Understanding this modification provides insights into the complex "histone code" that regulates chromatin dynamics and gene expression patterns .

How does the antibody specifically recognize the 2-hydroxyisobutyrylation modification?

The 2-hydroxyisobutyryl-HIST1H4A (K59) antibody is designed with high specificity to recognize this particular post-translational modification at lysine 59 of histone H4. The antibody was developed using a synthesized peptide derived from human histone H4 protein (amino acids 52-63) that contains the 2-hydroxyisobutyrylated K59 residue . This immunogen design ensures that the antibody's binding pocket specifically recognizes both the 2-hydroxyisobutyryl chemical group and its position within the surrounding amino acid sequence context of histone H4. The polyclonal nature of this antibody means it contains a mixture of antibodies that recognize multiple epitopes on the target, which can enhance sensitivity while maintaining specificity for the modification . Validation studies have confirmed its specificity in detecting the 2-hydroxyisobutyrylated K59 of histone H4 in human samples, distinguishing it from other modifications that may occur at nearby residues .

What are the validated applications for 2-hydroxyisobutyryl-HIST1H4A (K59) antibody?

The 2-hydroxyisobutyryl-HIST1H4A (K59) antibody has been validated for several research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The antibody has been verified for quantitative detection of the target modification in protein samples .

• ICC (Immunocytochemistry): Validated for visualization of the modification in fixed cells, with successful implementation in HeLa cells .

• IF (Immunofluorescence): The antibody has shown reliable performance in fluorescence-based detection systems, allowing for subcellular localization studies .

Recommended dilutions for these applications vary, with ICC/IF applications typically using dilutions in the range of 1:10-1:100 . The antibody has been particularly tested in human samples, with confirmation of reactivity to human 2-hydroxyisobutyrylated histone H4 . When designing experiments, researchers should consider that optimal dilutions may need to be determined empirically for each specific experimental system and condition .

What is the recommended protocol for immunocytochemistry with this antibody?

For immunocytochemistry applications using the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody, the following protocol has been validated:

Sample Preparation:

• Fix cells in 4% formaldehyde solution .

• Permeabilize cells using 0.2% Triton X-100 .

• Block with 10% normal goat serum for 30 minutes at room temperature .

Antibody Incubation:

• Dilute primary antibody (2-hydroxyisobutyryl-HIST1H4A K59) at 1:20 in 1% BSA solution .

• Incubate with primary antibody overnight at 4°C .

• Detect using a biotinylated secondary antibody against rabbit IgG .

• Visualize using an HRP-conjugated detection system .

Special Considerations:

• Pre-treatment of cells with sodium butyrate (30mM for 4 hours) has been shown to enhance the signal, as it increases histone modification levels .

• The antibody works well on the Leica BondTM system for automated immunostaining .

• For fluorescence detection, appropriate fluorophore-conjugated secondary antibodies can be substituted for the HRP system .

• Recommended dilution range for ICC is 1:10-1:100, though optimal concentration should be determined experimentally for each application .

How can researchers use this antibody to study the dynamics of histone 2-hydroxyisobutyrylation in response to cellular stimuli?

To study the dynamics of histone 2-hydroxyisobutyrylation in response to cellular stimuli, researchers can implement several advanced approaches using the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody:

Time-Course Experiments:
Design experiments that track changes in 2-hydroxyisobutyrylation at K59 following exposure to various stimuli at multiple time points. For example, researchers can treat cells with sodium butyrate (30mM) and collect samples at 0, 2, 4, 8, 12, and 24 hours post-treatment for immunoblotting or immunofluorescence analysis . This approach would reveal the temporal dynamics of this modification in response to histone deacetylase inhibition.

Stimulus-Response Quantification:
Utilize quantitative approaches such as ELISA or automated image analysis of immunofluorescence data to measure changes in 2-hydroxyisobutyrylation levels. The antibody's validation for ELISA applications makes it suitable for generating quantitative data on modification levels across different experimental conditions .

Multi-Omics Integration:
Combine ChIP-seq approaches (if the antibody can be validated for chromatin immunoprecipitation) with transcriptomics to correlate 2-hydroxyisobutyrylation patterns with gene expression changes. This would provide insights into the functional consequences of this modification in response to specific stimuli.

Comparative Analysis with Other Modifications:
Design experiments that simultaneously track multiple histone modifications (e.g., acetylation at H4K5 compared to 2-hydroxyisobutyrylation at H4K59) to understand the relationships between different epigenetic marks during cellular responses . This would require parallel experiments with antibodies targeting different modifications.

By implementing these approaches, researchers can generate comprehensive data on how 2-hydroxyisobutyrylation at H4K59 contributes to epigenetic regulation in response to various cellular conditions and stimuli.

What are the methodological considerations for using this antibody in ChIP-seq experiments?

While the search results don't explicitly mention ChIP-seq validation for the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody, researchers interested in adapting it for this application should consider the following methodological considerations:

Antibody Qualification Testing:

• Perform preliminary ChIP experiments followed by qPCR for known targets to assess enrichment efficacy.

• Test multiple antibody concentrations (starting with 2-5 μg per ChIP reaction) to determine optimal performance.

• Include appropriate negative controls (IgG from the same species) and positive controls (regions known to be enriched for the modification).

Crosslinking Optimization:
Since 2-hydroxyisobutyrylation may affect protein-DNA interactions differently than other modifications, test different crosslinking conditions:

• Standard formaldehyde crosslinking (1% for 10 minutes)

• Dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Varying crosslinking times to identify optimal conditions

Sonication Parameters:
Optimize sonication conditions to generate chromatin fragments of 200-500 bp, which is ideal for high-resolution mapping of histone modifications.

Sequencing Considerations:

• Include input controls for normalization

• Consider sequencing depth of at least 20 million uniquely mapped reads for histone modification profiling

• Implement appropriate peak calling algorithms that account for the typically broad enrichment patterns of histone modifications

Validation Strategies:

• Confirm enrichment at select loci by ChIP-qPCR before proceeding to sequencing

• Consider spike-in controls for quantitative comparisons between samples

• Validate findings using orthogonal methods such as mass spectrometry

Researchers should note that additional optimization beyond standard ChIP-seq protocols may be necessary given the relatively novel nature of this histone modification.

How does sodium butyrate treatment affect HIST1H4A K59 2-hydroxyisobutyrylation?

Sodium butyrate treatment has been shown to significantly impact HIST1H4A K59 2-hydroxyisobutyrylation levels in experimental systems. In immunocytochemistry studies, HeLa cells treated with 30mM sodium butyrate for 4 hours demonstrated enhanced 2-hydroxyisobutyrylation at the K59 position of histone H4, allowing for improved detection with the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody . This effect is likely related to sodium butyrate's known function as a histone deacetylase (HDAC) inhibitor.

The mechanism behind this enhancement appears to involve:

• Metabolic Regulation: Sodium butyrate can serve as a precursor for 2-hydroxyisobutyryl-CoA, potentially increasing the substrate availability for histone 2-hydroxyisobutyryltransferases.

• Enzyme Inhibition: By inhibiting HDACs, sodium butyrate may indirectly influence the activity of enzymes responsible for adding or removing 2-hydroxyisobutyryl groups.

• Chromatin Accessibility: The general relaxation of chromatin structure following HDAC inhibition may make histone residues more accessible to 2-hydroxyisobutyrylation.

Similar effects have been observed with acetylation at different lysine residues on histone H4. For instance, treatment with 10mM sodium butyrate for 24 hours significantly increases acetylation at lysine 5 (K5) of histone H4 in HeLa cells, as detected by western blot . This suggests that sodium butyrate may broadly affect multiple types of acylation modifications on histones, including both acetylation and 2-hydroxyisobutyrylation.

Researchers investigating the biological significance of 2-hydroxyisobutyrylation should consider sodium butyrate treatment as a useful tool for amplifying this modification in experimental systems, particularly when using detection methods like immunocytochemistry or western blotting.

What are the differences between 2-hydroxyisobutyrylation and acetylation of histone H4?

2-hydroxyisobutyrylation and acetylation represent distinct post-translational modifications of histone H4, with important differences in their chemical properties, occurrence sites, and potential biological functions:

Chemical Structure Differences:

• 2-hydroxyisobutyrylation: Involves the addition of a 2-hydroxyisobutyryl group (C₄H₇O₃), which contains a hydroxyl group and a larger carbon chain .

• Acetylation: Involves the addition of an acetyl group (C₂H₃O), which is smaller and lacks the hydroxyl group present in 2-hydroxyisobutyrylation .

Site-Specific Preferences:

• The 2-hydroxyisobutyryl-HIST1H4A antibody targets modification at lysine 59 (K59) .

• Acetylation of histone H4 commonly occurs at several lysine residues, including K5, K8, K12, and K16, with the search results specifically discussing K5 acetylation .

Functional Implications:

• Both modifications reduce the positive charge on histones, potentially relaxing chromatin structure .

• Acetylation at H4K5 is associated with higher levels of gene transcription .

• While specific functional data for 2-hydroxyisobutyrylation at K59 isn't detailed in the search results, this modification likely has distinct regulatory roles given its different chemical properties and location.

Detection Methods:

• Different antibodies are required for specific detection: the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody for 2-hydroxyisobutyrylation and acetyl Histone H4 (Lys5) antibody for acetylation .

• Both modifications can be induced or enhanced by sodium butyrate treatment, though possibly through different mechanisms .

Disease Associations:

• Misregulation of histone acetylation has been linked to developmental and neurodegenerative disorders including Alzheimer's disease, and various cancers .

• The disease associations for 2-hydroxyisobutyrylation misregulation are less established in the literature based on the search results.

Understanding these differences is crucial for researchers investigating the specific roles of these modifications in epigenetic regulation and their potential contributions to normal physiology and disease states.

What are the key optimization parameters for using this antibody in different applications?

When using the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody across different applications, researchers should optimize several key parameters to ensure reliable and reproducible results:

For ELISA Applications:

• Antibody Concentration: Start with manufacturer's recommended dilutions and optimize through titration experiments .

• Antigen Coating: Determine optimal coating concentration of target protein or peptide.

• Blocking Conditions: Test different blocking buffers (BSA, milk proteins) to minimize background.

• Incubation Parameters: Optimize both temperature and time for primary antibody binding.

• Detection System: Select appropriate enzyme-conjugated secondary antibody and substrate.

For Immunocytochemistry/Immunofluorescence:

• Fixation Method: 4% formaldehyde has been validated, but other fixatives may be compared for optimal epitope preservation .

• Permeabilization: 0.2% Triton X-100 is recommended, but concentration may need adjustment for different cell types .

• Blocking Parameters: 10% normal goat serum for 30 minutes at room temperature is suggested, but alternative blocking reagents may be tested .

• Antibody Dilution: Recommended range is 1:10-1:100; perform titration to determine optimal concentration for your specific sample .

• Incubation Conditions: Overnight incubation at 4°C has been validated, but shorter incubations at higher temperatures may be tested .

• Signal Enhancement: Pre-treatment with sodium butyrate (30mM for 4 hours) can increase 2-hydroxyisobutyrylation levels and improve signal detection .

• Detection Method: Choose between enzymatic (HRP) or fluorescent secondary antibodies based on experimental needs .

General Considerations:

• Sample Preparation: Ensure proper preservation of the modification during sample preparation.

• Positive Controls: Include samples known to have high levels of the modification (e.g., sodium butyrate-treated HeLa cells) .

• Negative Controls: Include appropriate controls lacking the primary antibody or using non-specific IgG.

• Batch Consistency: When comparing multiple samples, process them simultaneously under identical conditions.

Each of these parameters may require systematic optimization for individual experimental systems to achieve optimal signal-to-noise ratio and reliable detection of the target modification.

How should researchers troubleshoot weak or non-specific signals when using this antibody?

When encountering weak or non-specific signals with the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody, researchers should implement a systematic troubleshooting approach:

For Weak Signals:

• Increase Antibody Concentration: If using dilutions at the upper end of the recommended range (closer to 1:100), try a higher concentration (closer to 1:10) .

• Enhance Target Modification Levels: Pre-treat cells with sodium butyrate (30mM for 4 hours) to increase 2-hydroxyisobutyrylation levels, which has been validated to improve signal detection .

• Optimize Antigen Retrieval: For fixed tissues or cells, test different antigen retrieval methods to ensure the epitope is accessible.

• Extend Incubation Time: Consider extending primary antibody incubation from overnight to 24-48 hours at 4°C for difficult samples.

• Adjust Detection System: Switch to a more sensitive detection system, such as tyramide signal amplification or a higher-sensitivity fluorophore.

• Reduce Washing Stringency: Decrease salt concentration or washing duration if signal is consistently faint.

For Non-Specific Signals:

• Increase Blocking Stringency: Extend blocking time beyond 30 minutes or increase normal goat serum concentration above 10% .

• Optimize Antibody Dilution: Test more dilute antibody solutions to reduce non-specific binding.

• Add Protein Competitors: Include additional BSA (2-5%) during antibody incubation to reduce non-specific interactions .

• Modify Wash Protocol: Increase washing duration or add detergents (0.05-0.1% Tween-20) to wash buffers.

• Pre-adsorb Antibody: Consider pre-adsorbing the antibody with cell/tissue lysates lacking the target to remove antibodies that might cross-react.

• Validate Specificity: Perform peptide competition assays using the immunizing peptide (human histone H4 protein aa 52-63) to confirm signal specificity .

Additional Considerations:

• Sample Quality: Ensure proper fixation and preservation of the modification during sample preparation.

• Reagent Quality: Check antibody storage conditions (should be stored at -20°C to -80°C) and avoid repeated freeze-thaw cycles .

• Compare Multiple Detection Methods: If possible, validate findings using an orthogonal method (e.g., if ICC results are ambiguous, try ELISA) .

By systematically addressing these factors, researchers can optimize signal quality and ensure reliable detection of 2-hydroxyisobutyrylation at histone H4 K59.

What controls should be included when using this antibody for experimental validation?

For rigorous experimental validation using the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody, researchers should include a comprehensive set of controls:

Essential Positive Controls:

• Sodium Butyrate-Treated Cells: HeLa cells treated with 30mM sodium butyrate for 4 hours serve as a validated positive control with enhanced 2-hydroxyisobutyrylation at H4K59 .

• Purified Modified Peptide: Include a synthetic peptide containing 2-hydroxyisobutyrylated K59 (from human histone H4 residues 52-63) when possible, especially for ELISA validation .

Essential Negative Controls:

• Primary Antibody Omission: Samples processed identically but without the primary antibody to assess secondary antibody specificity and background.

• Isotype Control: Rabbit IgG at the same concentration as the primary antibody to evaluate non-specific binding .

• Unmodified Samples: When possible, include samples known to have low or undetectable levels of the modification (e.g., specific cell types or treatment conditions that reduce 2-hydroxyisobutyrylation).

Specificity Controls:

• Peptide Competition Assay: Pre-incubate the antibody with excess 2-hydroxyisobutyrylated peptide (H4 residues 52-63) to block specific binding sites, which should eliminate true positive signals.

• Cross-Reactivity Assessment: Test the antibody against peptides with similar modifications (e.g., acetylation, butyrylation) at the same or nearby residues to confirm specificity.

• Mutant Cell Lines: If available, use cell lines with mutations affecting the target lysine residue (K59) or enzymes responsible for the modification.

Methodological Controls:

• Dilution Series: Prepare a dilution series of the antibody to determine optimal concentration and assess signal linearity .

• Technical Replicates: Perform at least three technical replicates to assess reproducibility.

• Batch Controls: Include identical samples across different experimental batches to detect and correct for batch effects.

Validation Controls:

• Orthogonal Detection: When possible, validate findings using an alternative method (e.g., mass spectrometry) to confirm the presence and abundance of the modification.

• Biological Context Validation: Demonstrate the biological relevance by showing expected patterns (e.g., nuclear localization, chromatin association).

Implementing this comprehensive set of controls will significantly enhance the reliability and interpretability of experimental results obtained with the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody.

What fixation and permeabilization methods are optimal for preserving this histone modification?

Optimal fixation and permeabilization methods are critical for preserving 2-hydroxyisobutyrylation at H4K59 while maintaining cellular morphology and accessibility for antibody detection. Based on validated protocols, the following methods are recommended:

Validated Fixation Protocol:

• Formaldehyde Fixation: 4% formaldehyde has been successfully used to preserve this modification in HeLa cells . This cross-linking fixative effectively maintains protein-protein and protein-DNA interactions while preserving histone modifications.

• Fixation Duration: Standard fixation times of 10-15 minutes at room temperature are typically sufficient, though this may need optimization for different cell types.

• Temperature Considerations: Room temperature fixation is generally recommended to balance fixation efficiency with epitope preservation.

Validated Permeabilization Protocol:

• Triton X-100: 0.2% Triton X-100 has been demonstrated to provide effective permeabilization for detection of 2-hydroxyisobutyrylated H4K59 .

• Permeabilization Duration: Brief permeabilization (5-10 minutes) at room temperature typically provides sufficient access while minimizing loss of nuclear contents.

Alternative Methods to Consider:

• Methanol Fixation/Permeabilization: For some histone modifications, ice-cold methanol (-20°C for 10 minutes) can serve as both a fixative and permeabilizer, which might be tested as an alternative.

• Paraformaldehyde with Glutaraldehyde: For exceptionally sensitive epitopes, a combination of 4% paraformaldehyde with 0.1-0.5% glutaraldehyde may provide stronger fixation.

• Digitonin Permeabilization: For more gentle permeabilization, 50-100 μg/mL digitonin may be considered as an alternative to Triton X-100.

Considerations for Optimization:

• Over-fixation Risks: Excessive cross-linking can mask epitopes. If signal is weak despite high target abundance, reducing fixation time or concentration may help.

• Under-fixation Risks: Insufficient fixation may result in loss of target proteins during subsequent steps. If nuclear staining appears weak or patchy, increasing fixation time may be beneficial.

• Cell Type Variations: Different cell types may require adjusted protocols; thicker or more robust cells might need increased permeabilization.

• Antibody Accessibility: The location of the H4K59 epitope within chromatin may require more extensive permeabilization than some other targets.

Based on the validated protocol, 4% formaldehyde fixation followed by 0.2% Triton X-100 permeabilization represents a reliable starting point for detecting 2-hydroxyisobutyrylation at H4K59, with specific parameters optimized based on experimental conditions and cell types .

How does 2-hydroxyisobutyrylation at K59 compare with other histone H4 modifications in terms of function and regulation?

While the complete functional characterization of 2-hydroxyisobutyrylation at H4K59 is still emerging, comparative analysis with better-studied histone H4 modifications provides important contextual insights:

Structural and Chemical Comparisons:

Functional Comparisons:

• Chromatin Structure Impact:

  • Acetylation of H4 (particularly at K5) reduces positive charge on histones, relaxing nucleosome structure and generally promoting transcriptional activation .

  • 2-hydroxyisobutyrylation at K59 likely also affects chromatin structure due to its charge-neutralizing effect, though its specific impact may differ due to the larger chemical group and different position within the histone .

• Genomic Distribution:

  • While the search results don't provide specific data on genomic distribution of H4K59 2-hydroxyisobutyrylation, the location of K59 within the histone fold domain (rather than the N-terminal tail where many other modifications occur) suggests potentially distinct functional roles.

• Enzymatic Regulation:

  • Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) .

  • The enzymes specifically responsible for adding and removing 2-hydroxyisobutyryl groups at H4K59 are less well characterized in the search results, though the response to sodium butyrate (an HDAC inhibitor) suggests potential regulatory overlap with acetylation pathways .

Evolutionary Conservation:
The high conservation of histone H4 (P62805) across species suggests that modifications at key residues like K59 may have evolutionarily conserved functions, though the prevalence of 2-hydroxyisobutyrylation across different organisms is not detailed in the search results .

Associated Cellular Processes:

• Acetylation of H4K5 is associated with transcriptional activation and has been linked to various developmental processes and pathological conditions including neurodegenerative disorders and cancer .

• The specific cellular processes associated with 2-hydroxyisobutyrylation at H4K59 are less extensively characterized in the search results, representing an area for future research.

While both modifications can be enhanced by sodium butyrate treatment , the distinct chemical properties and positions of these modifications suggest they likely serve non-redundant functions in chromatin regulation, with 2-hydroxyisobutyrylation potentially representing a modification with unique regulatory capabilities that complement the better-studied acetylation marks.

What cell types or experimental systems show significant 2-hydroxyisobutyrylation patterns relevant to research?

Based on the available search results and broader context, several cell types and experimental systems have demonstrated significant 2-hydroxyisobutyrylation patterns that are valuable for research:

Validated Cell Lines:

• HeLa Cells: Human cervical epithelial carcinoma cells have been specifically validated for studying 2-hydroxyisobutyrylation at H4K59. Treatment with 30mM sodium butyrate for 4 hours significantly enhances the detection of this modification in HeLa cells .

• Cancer Cell Lines: While not explicitly mentioned for 2-hydroxyisobutyrylation, various cancer cell lines have been used to study histone modifications including acetylation of histone H4 , suggesting they may also be suitable for studying 2-hydroxyisobutyrylation.

Experimental Enhancement Systems:

• Sodium Butyrate Treatment: Treatment with sodium butyrate (30mM for 4 hours) has been demonstrated to enhance 2-hydroxyisobutyrylation levels, making it a valuable tool for studying this modification . Similarly, a 24-hour treatment with 10mM sodium butyrate increases acetylation at H4K5 , suggesting that HDAC inhibition broadly affects various acylation marks.

• Other HDAC Inhibitors: While not specifically mentioned in the search results, other HDAC inhibitors (such as trichostatin A, valproic acid, or suberoylanilide hydroxamic acid) might also affect 2-hydroxyisobutyrylation levels and could be explored in experimental systems.

Potential Biological Systems for Investigation:

Based on what is known about histone modifications generally, the following systems may be particularly relevant for 2-hydroxyisobutyrylation research:

• Developing Embryos: Histone modifications play crucial roles in embryonic development and cell differentiation.

• Neural Tissues: Given the association of histone acetylation with neurodegenerative disorders , neural tissues may exhibit important 2-hydroxyisobutyrylation patterns.

• Metabolically Active Tissues: Since 2-hydroxyisobutyryl-CoA is derived from metabolic pathways, tissues with unique metabolic profiles (liver, muscle, adipose) may show distinct patterns.

• Immune Cells: Rapid transcriptional changes in immune cell activation may involve dynamic histone modifications including 2-hydroxyisobutyrylation.

Methodological Considerations for Different Systems:

When working with different cell types or tissues, researchers should consider:

• Fixation Optimization: Different tissues may require modified fixation protocols to preserve the modification while ensuring antibody accessibility.

• Background Levels: Endogenous levels of 2-hydroxyisobutyrylation may vary significantly between cell types, requiring adjustment of detection parameters.

• Functional Validation: The biological significance of the modification should be validated in each system, potentially through correlation with transcriptional activity or chromatin accessibility.

HeLa cells treated with sodium butyrate currently represent the best-validated system for studying 2-hydroxyisobutyrylation at H4K59 based on the available search results .

How might researchers correlate 2-hydroxyisobutyrylation patterns with gene expression or chromatin accessibility data?

To correlate 2-hydroxyisobutyrylation patterns with gene expression or chromatin accessibility, researchers can implement several integrated approaches:

Integrated Multi-Omics Experimental Design:

• ChIP-seq for 2-hydroxyisobutyrylated H4K59:

  • If the antibody can be validated for ChIP applications, perform ChIP-seq to map genomic locations of this modification.

  • Include appropriate controls (input DNA, IgG ChIP) and consider spike-in normalization for quantitative comparisons.

  • Analyze peak distribution relative to genomic features (promoters, enhancers, gene bodies).

• RNA-seq for Gene Expression Correlation:

  • Perform RNA-seq on the same cell populations used for ChIP-seq.

  • Implement differential expression analysis between conditions with varying 2-hydroxyisobutyrylation levels (e.g., with/without sodium butyrate treatment ).

  • Correlate 2-hydroxyisobutyrylation peak intensity with expression levels of nearby genes.

• ATAC-seq or DNase-seq for Chromatin Accessibility:

  • Map open chromatin regions in matching samples.

  • Compare accessibility profiles with 2-hydroxyisobutyrylation patterns to identify relationships between this modification and chromatin state.

Advanced Analytical Approaches:

• Integrated Data Visualization:

  • Use genome browsers to visually inspect alignment of 2-hydroxyisobutyrylation, expression, and accessibility at specific loci.

  • Develop heatmaps clustering genes based on 2-hydroxyisobutyrylation patterns and expression levels.

• Statistical Integration Methods:

  • Implement correlation analyses (Pearson, Spearman) between modification levels and expression.

  • Apply machine learning approaches to identify patterns predictive of gene expression or accessibility.

  • Use dimension reduction techniques (PCA, t-SNE) to identify relationships across multi-omic datasets.

• Functional Enrichment Analysis:

  • Perform GO term and pathway analysis on genes with correlated 2-hydroxyisobutyrylation and expression patterns.

  • Compare with enrichment patterns known for other histone modifications (e.g., H4K5 acetylation ).

Experimental Validation Approaches:

• Targeted Manipulation:

  • Use CRISPR-based approaches to mutate specific lysine residues (K59→R) and assess effects on gene expression.

  • Employ chemical inhibitors or genetic knockdown of enzymes involved in 2-hydroxyisobutyrylation.

  • Modulate 2-hydroxyisobutyrylation using sodium butyrate treatment at varying concentrations and durations .

• Single-Cell Approaches:

  • Implement single-cell RNA-seq combined with CUT&Tag or other single-cell epigenomic methods to capture cell-to-cell variation in modification-expression relationships.

• Time-Course Experiments:

  • Follow changes in 2-hydroxyisobutyrylation and corresponding gene expression over time after stimulation or treatment.

  • Identify temporal relationships (does modification precede or follow expression changes?).

By integrating these approaches, researchers can develop comprehensive insights into the functional implications of 2-hydroxyisobutyrylation at H4K59 and its relationship to gene regulation and chromatin architecture.

What are the known or hypothesized roles of 2-hydroxyisobutyrylation in disease contexts?

While the search results don't provide extensive information on the specific roles of 2-hydroxyisobutyrylation at H4K59 in disease contexts, we can infer potential implications based on the available information and the broader role of histone modifications in pathology:

Potential Cancer Connections:

The validation of the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody in HeLa cells , a cervical cancer cell line, suggests this modification may be relevant in cancer biology. Given that histone modifications broadly affect gene expression patterns, dysregulation of 2-hydroxyisobutyrylation could potentially contribute to:

• Altered Gene Expression Programs: Similar to how acetylation affects transcription , aberrant 2-hydroxyisobutyrylation patterns could lead to inappropriate activation or repression of cancer-related genes.

• Chromatin Structure Abnormalities: As this modification likely affects nucleosome dynamics and chromatin accessibility , its dysregulation could contribute to genomic instability observed in cancer cells.

• Metabolic Reprogramming: Since 2-hydroxyisobutyrylation depends on metabolic precursors, it may represent a link between altered cancer metabolism and epigenetic regulation.

Neurodegenerative Disease Implications:

The search results mention that misregulation of histone acetylation has been linked to neurodegenerative disorders including Alzheimer's disease . By extension, 2-hydroxyisobutyrylation may also play roles in:

• Neuronal Gene Expression: Dysregulation could affect expression of genes critical for neuronal function and survival.

• Protein Aggregation Processes: Altered histone modifications might influence expression of genes involved in protein quality control and aggregation prevention.

• Neuroinflammatory Responses: Modified epigenetic regulation could affect inflammatory pathways implicated in neurodegenerative processes.

Developmental and Metabolic Disorders:

Given the fundamental role of histone modifications in development and cellular metabolism:

• Developmental Abnormalities: Disruption of 2-hydroxyisobutyrylation patterns during critical developmental windows could potentially contribute to congenital disorders.

• Metabolic Syndrome and Diabetes: Alterations in metabolic pathways that generate 2-hydroxyisobutyryl-CoA could link metabolic disorders with epigenetic dysregulation.

Research Directions to Establish Disease Connections:

To establish clearer links between 2-hydroxyisobutyrylation at H4K59 and specific diseases, researchers should consider:

• Comparative Profiling: Compare 2-hydroxyisobutyrylation patterns between normal and diseased tissues using the validated antibody .

• Genetic Association Studies: Investigate correlations between disease risk and genetic variants in enzymes that regulate 2-hydroxyisobutyrylation.

• Therapeutic Intervention Studies: Assess how treatments that affect 2-hydroxyisobutyrylation (e.g., sodium butyrate ) influence disease progression in model systems.

• Integration with Known Disease Mechanisms: Investigate how 2-hydroxyisobutyrylation interfaces with established disease-associated pathways.

While specific disease associations for 2-hydroxyisobutyrylation at H4K59 remain to be fully established, the centrality of histone modifications to gene regulation suggests potentially significant roles in various pathological contexts that warrant further investigation.

What emerging technologies might enhance the study of 2-hydroxyisobutyrylation?

Several emerging technologies hold significant promise for advancing our understanding of 2-hydroxyisobutyrylation at H4K59 and other histone residues:

Advanced Microscopy Techniques:

• Super-Resolution Microscopy: Technologies like STORM, PALM, or STED could enable visualization of 2-hydroxyisobutyrylation patterns at sub-diffraction resolution, potentially revealing spatial organization within the nucleus not detectable with conventional immunofluorescence approaches used currently .

• Live-Cell Modification Tracking: Development of specific probes for real-time tracking of 2-hydroxyisobutyrylation in living cells could provide unprecedented insights into the dynamics of this modification during cellular processes.

Next-Generation Sequencing Approaches:

• CUT&Tag and CUT&RUN: These technologies offer advantages over traditional ChIP-seq for mapping histone modifications with higher sensitivity and lower background, potentially enabling genome-wide mapping of 2-hydroxyisobutyrylation patterns with fewer cells.

• Single-Cell Epigenomics: Techniques for mapping histone modifications in individual cells could reveal cell-to-cell heterogeneity in 2-hydroxyisobutyrylation patterns and correlate them with single-cell transcriptomes.

• Long-Read Sequencing: Technologies like Oxford Nanopore could enable detection of multiple histone modifications simultaneously on the same histone tail, providing insights into the combinatorial patterns involving 2-hydroxyisobutyrylation.

Mass Spectrometry Innovations:

• Top-Down Proteomics: Analyzing intact histone proteins rather than digested peptides would provide a more complete picture of how 2-hydroxyisobutyrylation co-occurs with other modifications.

• Targeted MS Approaches: Development of selective reaction monitoring (SRM) or parallel reaction monitoring (PRM) methods specifically for 2-hydroxyisobutyrylated peptides could improve quantification of this modification.

• Crosslinking Mass Spectrometry: Could reveal protein interactions specifically mediated by or affected by 2-hydroxyisobutyrylation at H4K59.

Genome Editing and Synthetic Biology:

• CRISPR Base Editing: Precise modification of lysine codons to create K59R mutations could enable detailed functional studies of this modification site.

• Engineered "Writers" and "Erasers": Development of tools to selectively add or remove 2-hydroxyisobutyryl groups at specific genomic loci would allow causal studies of this modification's function.

• Synthetic Histone Systems: Engineering systems with modified histones containing non-natural amino acids at position 59 that mimic or prevent 2-hydroxyisobutyrylation could provide mechanistic insights.

Computational and AI Approaches:

• Deep Learning for Pattern Recognition: AI algorithms could identify subtle patterns in 2-hydroxyisobutyrylation distribution across the genome and correlate them with functional genomic elements.

• Integrative Multi-Omics Analysis: Advanced computational frameworks could integrate 2-hydroxyisobutyrylation data with other epigenomic, transcriptomic, and proteomic datasets to develop comprehensive models of how this modification contributes to genome regulation.

These emerging technologies, combined with the continued development and validation of specific antibodies like the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody , will likely drive significant advances in our understanding of this histone modification in the coming years.

What are the most pressing unresolved questions about 2-hydroxyisobutyrylation of histone H4?

Despite the availability of specific antibodies for 2-hydroxyisobutyrylation at H4K59 , numerous fundamental questions about this modification remain unresolved, representing critical areas for future research:

Enzymology and Regulation:

• Writer Enzymes: What are the specific enzymes responsible for adding 2-hydroxyisobutyryl groups to H4K59? Are they distinct from or overlapping with enzymes that catalyze other acylation modifications?

• Eraser Enzymes: Which deacylases remove 2-hydroxyisobutyryl groups from H4K59? Do classical HDACs play a role, explaining the effect of sodium butyrate treatment ?

• Metabolic Regulation: How is the cellular pool of 2-hydroxyisobutyryl-CoA (the likely donor for this modification) regulated? What metabolic pathways contribute most significantly to this pool?

• Signal Integration: What cellular signals trigger changes in 2-hydroxyisobutyrylation patterns? How do these integrate with other histone modification pathways?

Functional Genomics:

• Genomic Distribution: What is the genome-wide distribution of 2-hydroxyisobutyrylation at H4K59? Does it associate with specific genomic features or regulatory elements?

• Transcriptional Impact: Does 2-hydroxyisobutyrylation at H4K59 activate or repress transcription? Is its effect context-dependent?

• Reader Proteins: What nuclear proteins specifically recognize and bind to 2-hydroxyisobutyrylated H4K59? How do these differ from readers of other histone modifications?

• Chromatin Structure Effects: How does this modification affect nucleosome stability, higher-order chromatin structure, and DNA accessibility?

Biological Significance:

• Developmental Roles: How do patterns of 2-hydroxyisobutyrylation change during cellular differentiation and development? Are there critical developmental processes requiring this modification?

• Tissue Specificity: Do different cell types exhibit distinct patterns of 2-hydroxyisobutyrylation? Are there tissue-specific regulatory mechanisms?

• Environmental Responses: How do environmental stimuli (stress, nutrients, toxins) affect 2-hydroxyisobutyrylation patterns? Could this modification mediate environmental influences on gene expression?

• Evolutionary Conservation: How conserved is this modification across species? Does its functional significance vary between organisms?

Disease Relationships:

• Cancer Connections: Are 2-hydroxyisobutyrylation patterns altered in cancer? Could these changes contribute to oncogenesis or tumor progression?

• Neurodegeneration: Given the links between histone acetylation and neurodegenerative disorders , does 2-hydroxyisobutyrylation play a role in neuronal function and dysfunction?

• Metabolic Disorders: How does metabolic disease affect 2-hydroxyisobutyrylation, and vice versa? Is there a bidirectional relationship?

• Therapeutic Targeting: Could modulation of 2-hydroxyisobutyrylation represent a viable therapeutic approach for certain conditions?

Methodological Challenges:

• Antibody Specificity: How can we further validate and improve antibody specificity for this modification given the chemical similarities to other acylations?

• Detection Sensitivity: What are the limits of detection for current methods? How can we improve sensitivity for cells or tissues with low modification levels?

• Temporal Dynamics: How stable is this modification? What is its half-life and turnover rate in different cellular contexts?

Addressing these questions will require interdisciplinary approaches combining biochemistry, genomics, cell biology, and computational analysis, with the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody serving as a key tool in these investigations.

How might 2-hydroxyisobutyrylation connect to metabolic regulation in cells?

The presence of 2-hydroxyisobutyryl groups on histone H4 suggests intriguing connections between cellular metabolism and epigenetic regulation that warrant further investigation:

Metabolic Precursors and Pathways:

• Amino Acid Metabolism: 2-hydroxyisobutyryl-CoA may be derived from the metabolism of branched-chain amino acids, particularly valine, suggesting connections between protein catabolism and histone modification.

• Short-Chain Fatty Acid Metabolism: The enhancement of 2-hydroxyisobutyrylation by sodium butyrate treatment indicates potential links with short-chain fatty acid metabolism, which is particularly relevant in contexts like the gut microbiome's influence on host epigenetics.

• CoA Donor Availability: The cellular availability of 2-hydroxyisobutyryl-CoA likely influences modification rates, creating a direct link between metabolic state and epigenetic marking.

Nutrient Sensing and Adaptation:

• Metabolic State Signaling: 2-hydroxyisobutyrylation could serve as a mechanism for translating cellular metabolic state directly into chromatin modifications that regulate gene expression.

• Fasting/Feeding Cycles: Changes in nutrient availability during fasting/feeding cycles might influence 2-hydroxyisobutyrylation patterns, potentially regulating genes involved in metabolic adaptation.

• Stress Response: Metabolic stress (hypoxia, nutrient deprivation) could alter 2-hydroxyisobutyrylation, providing a mechanism to coordinate gene expression with cellular stress responses.

Cellular Energy Status:

• NAD⁺/NADH Ratio: If the removal of 2-hydroxyisobutyryl groups involves NAD⁺-dependent enzymes (similar to some HDACs), then cellular energy status could directly influence modification dynamics.

• ATP Levels: The addition of 2-hydroxyisobutyryl groups may require ATP, making this modification sensitive to cellular energy availability.

• Mitochondrial Function: Changes in mitochondrial activity, which affects numerous metabolic pathways, could impact 2-hydroxyisobutyrylation through altered precursor availability.

Potential Research Approaches:

• Metabolic Perturbation Studies: Systematically alter metabolic pathways and measure effects on 2-hydroxyisobutyrylation at H4K59 using the available antibody .

• Nutrient Modulation: Assess 2-hydroxyisobutyrylation changes in response to altered nutrient availability (glucose, amino acids, fatty acids) or dietary interventions.

• Metabolic Disease Models: Investigate 2-hydroxyisobutyrylation patterns in models of metabolic disorders (diabetes, obesity) to identify potential dysregulation.

• Isotope Tracing: Use isotopically labeled metabolic precursors to track their incorporation into 2-hydroxyisobutyrylated histones, establishing direct metabolic connections.

• Enzyme Identification: Identify and characterize the enzymes that catalyze the addition and removal of 2-hydroxyisobutyryl groups, exploring their regulation by metabolic factors.

The connection between 2-hydroxyisobutyrylation and metabolism represents an exciting frontier in chromatin biology, potentially explaining how cells adapt their transcriptional programs to changing metabolic conditions. The availability of specific antibodies like the 2-hydroxyisobutyryl-HIST1H4A (K59) antibody provides a valuable tool for investigating these connections.

What interdisciplinary approaches could accelerate understanding of this histone modification?

Advancing our understanding of 2-hydroxyisobutyrylation at H4K59 would benefit significantly from interdisciplinary approaches that combine expertise and methodologies from multiple scientific fields:

Biochemistry and Structural Biology Integration:

• Structural Studies: Crystallography or cryo-EM analysis of nucleosomes containing 2-hydroxyisobutyrylated H4K59 would reveal how this modification affects chromatin architecture.

• Biophysical Characterization: Techniques like hydrogen-deuterium exchange mass spectrometry or NMR could elucidate how this modification alters histone-DNA interactions and nucleosome dynamics.

• In Vitro Reconstitution Systems: Biochemical systems with purified components could identify enzymes responsible for adding and removing 2-hydroxyisobutyryl groups.

Genomics and Computational Biology Collaboration:

• Integrative Analysis Platforms: Develop computational frameworks that integrate 2-hydroxyisobutyrylation data with other epigenomic, transcriptomic, and proteomic datasets.

• Machine Learning Approaches: Apply AI techniques to identify patterns in 2-hydroxyisobutyrylation distribution and predict functional outcomes.

• Evolutionary Bioinformatics: Compare 2-hydroxyisobutyrylation across species to identify conserved patterns suggesting fundamental functional importance.

Metabolomics and Chemical Biology Partnership:

• Metabolite Profiling: Correlate cellular metabolite levels with 2-hydroxyisobutyrylation patterns to establish connections between metabolism and epigenetic regulation.

• Chemical Probes: Develop specific inhibitors or activators of enzymes involved in 2-hydroxyisobutyrylation to enable precise experimental manipulation.

• Click Chemistry Approaches: Implement click-chemistry methods to track newly added 2-hydroxyisobutyryl groups on histones in real-time.

Cell Biology and Disease Modeling Collaboration:

• Organoid Systems: Study 2-hydroxyisobutyrylation in 3D organoid models that better recapitulate tissue environments than traditional cell cultures.

• Patient-Derived Systems: Analyze 2-hydroxyisobutyrylation patterns in patient-derived cells to identify disease-specific alterations.

• Animal Models: Develop animal models with mutations affecting 2-hydroxyisobutyrylation to assess physiological consequences.

Systems Biology Framework:

• Modeling Epigenetic Networks: Develop mathematical models that predict how 2-hydroxyisobutyrylation interacts with other epigenetic modifications in regulatory networks.

• Perturbation Studies: Systematically perturb cellular systems and measure effects on 2-hydroxyisobutyrylation to identify regulatory relationships.

• Multi-scale Analysis: Connect molecular-level 2-hydroxyisobutyrylation changes to cellular, tissue, and organism-level phenotypes.

Practical Implementation Strategies:

• Collaborative Research Consortia: Establish multidisciplinary research teams specifically focused on 2-hydroxyisobutyrylation biology.

• Standardized Methods and Reagent Sharing: Develop standardized protocols for 2-hydroxyisobutyrylation analysis and make key reagents (including validated antibodies like the one described ) widely available.

• Open Data Platforms: Create repositories for 2-hydroxyisobutyrylation datasets that facilitate integration with other types of biological data.

• Interdisciplinary Training Programs: Develop training opportunities that equip researchers with cross-disciplinary skills needed to study complex histone modifications like 2-hydroxyisobutyrylation.

By implementing these interdisciplinary approaches, the scientific community can accelerate understanding of 2-hydroxyisobutyrylation at H4K59, potentially revealing new principles of epigenetic regulation and identifying novel therapeutic targets for diseases involving epigenetic dysregulation.