2-hydroxyisobutyryl-HIST1H4A (K8) Antibody

What is 2-hydroxyisobutyryl-HIST1H4A (K8) and why is it significant in epigenetic research?

2-hydroxyisobutyryl-HIST1H4A (K8) refers to the 2-hydroxyisobutyrylation post-translational modification at lysine 8 of histone H4. This modification represents an evolutionarily conserved active mark enriched in active chromatin . The significance of this modification lies in its role as a dynamic epigenetic regulator that responds to metabolic conditions, particularly glucose availability, positioning it at the intersection of cellular metabolism and gene regulation. H4K8hib has been identified as a key chromatin modification that allows access to genetic information by disrupting DNA-histone complexes, making it crucial for understanding how metabolic states influence gene expression patterns .

How does 2-hydroxyisobutyrylation differ from other histone post-translational modifications?

2-hydroxyisobutyrylation is distinct from better-known modifications such as acetylation in both its chemical structure and metabolic regulation. While both modifications occur on lysine residues, 2-hydroxyisobutyrylation involves the addition of a 2-hydroxyisobutyryl group derived from 2-hydroxyisobutyryl-CoA, compared to the acetyl group from acetyl-CoA in acetylation . Functionally, H4K8hib shows unique glucose-dependent regulation not observed with all histone modifications, as demonstrated by its significant reduction during glucose deprivation but minimal response to other stresses including DNA damage, temperature changes, osmotic pressure, or nitrogen starvation . This metabolic sensitivity suggests 2-hydroxyisobutyrylation serves as a specific link between cellular energy metabolism and chromatin structure.

What applications does the 2-hydroxyisobutyryl-HIST1H4A (K8) antibody have in epigenetic research?

The 2-hydroxyisobutyryl-HIST1H4A (K8) antibody serves as a critical tool for investigating the presence and dynamics of H4K8hib in chromatin. Primary applications include:

• Western blot analysis to monitor changes in H4K8hib levels under various physiological conditions

• Immunofluorescence (IF) assays to visualize cellular localization of the modification

• Chromatin immunoprecipitation (ChIP) experiments to identify genomic regions enriched with H4K8hib

• Enzyme-linked immunosorbent assays (ELISA) to quantify H4K8hib levels across different experimental conditions

These applications enable researchers to establish correlations between metabolic states, particularly glucose availability, and epigenetic regulation through H4K8hib modifications, offering insights into how cells adapt gene expression in response to environmental changes.

What are the optimal storage and handling conditions for 2-hydroxyisobutyryl-HIST1H4A (K8) antibodies?

For optimal storage and handling of 2-hydroxyisobutyryl-HIST1H4A (K8) antibodies, researchers should follow these evidence-based protocols:

• Upon receipt, store antibodies at -20°C or -80°C to maintain long-term stability

• Avoid repeated freeze-thaw cycles as they can compromise antibody integrity and performance

• The antibodies are typically supplied in liquid form with a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4

• When working with the antibody, maintain cold chain conditions and use appropriate laboratory safety protocols for handling research-grade antibodies

• For western blot applications, dilute the antibody in the range of 1:100-1:1000, and for immunofluorescence applications, use dilutions of 1:50-1:200

Following these handling protocols ensures maximum antibody performance and reproducibility in experimental applications.

How should researchers design experiments to monitor dynamic changes in H4K8hib levels?

When designing experiments to monitor dynamic changes in H4K8hib levels, researchers should consider the following methodological approach:

This experimental design allows researchers to capture the glucose-dependent dynamics of H4K8hib modification, providing insights into its regulatory mechanisms and potential biological functions.

What dilution and incubation protocols yield optimal results for different applications?

The optimal protocols for various applications of the 2-hydroxyisobutyryl-HIST1H4A (K8) antibody are as follows:

These protocols should be optimized for specific experimental systems, as factors such as cell type, fixation method, and detection system can influence antibody performance . Including appropriate positive and negative controls is essential for validating antibody specificity and experimental outcomes.

How can researchers investigate the enzymes responsible for adding and removing the H4K8hib modification?

Investigating the enzymatic machinery responsible for H4K8hib regulation requires a multi-faceted approach:

For identifying "writers" (enzymes that add the modification):

• Candidate approach: Based on research showing Esa1p (the catalytic subunit of the nucleosome acetyltransferase of H4 - NuA4 complex) functions as an H4K8hib writer in yeast, researchers should examine mammalian homologs like Tip60/KAT5 .

• In vitro enzymatic assays: Use purified recombinant enzymes (like picNuA4 complex) with nucleosome core particles and synthetic 2-hydroxyisobutyryl-CoA to assess catalytic activity, as demonstrated in previous studies .

• Structural modeling: Employ computational approaches to predict binding potential of candidate enzymes with 2-hydroxyisobutyryl-CoA based on crystal structures .

For identifying "erasers" (enzymes that remove the modification):

• Systematic deletion approach: Following the strategy used to identify Rpd3p and Hos3p as H4K8hib erasers in yeast, researchers should create single and combinatorial knockouts of histone deacetylase (HDAC) family members .

• Glucose starvation assays: Subject these knockout models to glucose deprivation and monitor H4K8hib levels using western blot to identify enzymes whose absence prevents H4K8hib reduction .

• Biochemical validation: Perform in vitro deacylation assays using purified enzymes and synthetic H4K8hib peptides to confirm direct enzymatic activity.

This comprehensive approach enables identification of the complete enzymatic machinery regulating H4K8hib dynamics in various model systems.

What are the recommended protocols for using 2-hydroxyisobutyryl-HIST1H4A (K8) antibody in ChIP-seq experiments?

For successful ChIP-seq experiments using the 2-hydroxyisobutyryl-HIST1H4A (K8) antibody, researchers should follow this optimized protocol:

• Crosslinking and chromatin preparation:

  • Fix cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Isolate nuclei and sonicate chromatin to fragments of 200-500 bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads for 1-2 hours at 4°C

  • Incubate pre-cleared chromatin with 2-5 μg of 2-hydroxyisobutyryl-HIST1H4A (K8) antibody overnight at 4°C

  • Include appropriate controls: IgG negative control and total H3 or H4 antibody as positive control

  • Add protein A/G beads and incubate for 2-3 hours at 4°C

  • Perform stringent washing steps to remove non-specific binding

• DNA purification and library preparation:

  • Reverse crosslinks at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using column-based methods

  • Prepare sequencing libraries following platform-specific protocols

  • Include input controls for normalization

• Data analysis considerations:

  • Use peak-calling algorithms optimized for histone modifications (e.g., MACS2)

  • Compare H4K8hib enrichment with other active chromatin marks

  • Correlate with gene expression data to establish functional relationships

  • Consider metabolic state of cells (glucose availability) when interpreting results

This protocol integrates standard ChIP-seq methodology with specific considerations for H4K8hib analysis, ensuring high-quality data that captures the true biological distribution of this modification across the genome.

How can researchers distinguish between 2-hydroxyisobutyrylation and other acylation modifications in their experiments?

Distinguishing between 2-hydroxyisobutyrylation and other acylation modifications requires multiple complementary approaches:

• Antibody specificity validation:

  • Perform dot blot or ELISA assays using synthetic peptides containing various acylation modifications (acetylation, propionylation, butyrylation, crotonylation, 2-hydroxyisobutyrylation) at the same lysine position

  • Test antibody cross-reactivity through competitive binding assays with modified peptides

  • Include western blot controls with samples known to contain specific modifications

• Mass spectrometry-based approaches:

  • Use high-resolution MS/MS to distinguish between modifications based on their distinct molecular weights

  • Implement targeted proteomics approaches focusing on diagnostic fragment ions specific to each modification

  • Apply ETD (electron transfer dissociation) or EThcD fragmentation, which better preserves post-translational modifications

• Metabolic regulation experiments:

  • Exploit the unique glucose-dependent regulation of H4K8hib to distinguish it from other modifications

  • Compare responses to different metabolic perturbations, as each acylation may respond differently based on its corresponding acyl-CoA donor availability

• Enzyme specificity assays:

  • Utilize the specific "writers" and "erasers" of H4K8hib (Esa1p and Rpd3p/Hos3p in yeast) as tools to selectively modulate this modification

  • Compare with enzymes known to specifically regulate other acylations

This multi-faceted approach ensures accurate identification and characterization of 2-hydroxyisobutyrylation distinct from other chemically similar histone modifications.

What are common challenges in detecting 2-hydroxyisobutyryl-HIST1H4A (K8) and how can they be addressed?

Researchers commonly encounter several challenges when detecting H4K8hib, each requiring specific troubleshooting strategies:

• Low signal intensity:

  • Increase antibody concentration within recommended range (1:100-1:500 for WB)

  • Extend primary antibody incubation time to overnight at 4°C

  • Enhance detection sensitivity using signal amplification systems

  • Enrich for histones through acid extraction protocols prior to analysis

• High background:

  • Increase blocking stringency (5% BSA or 5% non-fat dry milk in TBST)

  • Extend blocking time to 2 hours at room temperature

  • Use more stringent washing protocols (5 × 5 min in TBST)

  • Pre-absorb antibody with non-specific proteins

• Specificity concerns:

  • Include peptide competition controls using 2-hydroxyisobutyrylated and non-modified peptides

  • Compare patterns with other known histone marks

  • Use samples from glucose-starved cells as biological negative controls

• Inconsistent results:

  • Standardize cell culture conditions, particularly glucose concentrations

  • Maintain strict harvesting protocols to avoid stress-induced changes

  • Use internal loading controls for normalization

  • Prepare fresh buffers and reagents for each experiment

By implementing these targeted solutions, researchers can overcome technical challenges and obtain reliable, reproducible data on H4K8hib levels and distribution.

How should researchers interpret changes in H4K8hib levels in response to different experimental conditions?

Interpreting changes in H4K8hib levels requires careful consideration of multiple factors:

• Glucose-dependent regulation:

  • Decreased H4K8hib levels following glucose deprivation likely reflect normal physiological response rather than experimental artifacts

  • Restoration of levels upon glucose reintroduction confirms the specificity of this regulation

  • Compare with other histone marks to determine whether the response is modification-specific or represents global histone changes

• Enzymatic regulation interpretation:

  • Persistent H4K8hib levels in cells lacking specific HDACs (e.g., Rpd3p and Hos3p in yeast) during glucose starvation indicate these enzymes' roles as "erasers"

  • Changes in H4K8hib following manipulation of writer enzymes (e.g., Esa1p) confirm their regulatory role

• Correlation with transcriptional activity:

  • As an active mark, increased H4K8hib levels at specific genomic regions typically correlate with enhanced transcriptional activity

  • Integration with RNA-seq data can validate functional outcomes of H4K8hib changes

• Metabolic context consideration:

  • Interpret H4K8hib changes in relation to cellular metabolic state

  • Consider availability of 2-hydroxyisobutyryl-CoA, which may be altered in different metabolic conditions

  • Recognize potential connection to glycolysis pathway activity

This interpretative framework allows researchers to extract meaningful biological insights from observed changes in H4K8hib levels across experimental conditions.

What analytical approaches should be used to correlate H4K8hib patterns with gene expression and cellular metabolism?

To establish meaningful correlations between H4K8hib patterns, gene expression, and metabolism, researchers should implement these analytical approaches:

• Integrated genomics analysis:

  • Combine ChIP-seq data for H4K8hib with RNA-seq to correlate modification enrichment with transcriptional activity

  • Implement peak-gene association algorithms to identify genes potentially regulated by H4K8hib

  • Apply gene set enrichment analysis (GSEA) to identify biological pathways associated with H4K8hib-marked genes

• Metabolic profiling correlation:

  • Measure levels of key metabolites, particularly those in glycolysis and those related to acyl-CoA production

  • Quantify 2-hydroxyisobutyryl-CoA levels under different metabolic conditions

  • Perform correlation analysis between metabolite levels, H4K8hib abundance, and gene expression

• Network analysis approaches:

  • Construct protein-protein interaction networks including identified writers/erasers

  • Build gene regulatory networks integrating transcription factors, H4K8hib distribution, and gene expression

  • Develop metabolic-epigenetic interaction models to visualize relationships

• Comparative analysis across conditions:

  • Use differential binding analysis to identify genomic regions with significant changes in H4K8hib

  • Apply machine learning approaches to identify patterns predictive of gene expression changes

  • Develop visualization tools that integrate metabolic states with epigenetic patterns

This multi-omics analytical framework enables researchers to establish causal relationships between metabolism, H4K8hib modification, and downstream gene regulatory consequences, providing a comprehensive understanding of this epigenetic mechanism.

What are emerging applications for 2-hydroxyisobutyryl-HIST1H4A (K8) antibody in disease research?

Several promising research directions are emerging for applying H4K8hib analysis to disease contexts:

• Cancer metabolism and epigenetics:

  • Investigate H4K8hib patterns in tumors characterized by altered glucose metabolism (Warburg effect)

  • Examine correlations between oncogene-driven metabolic reprogramming and changes in H4K8hib distribution

  • Explore potential for targeting writer/eraser enzymes as therapeutic strategies

• Metabolic disorders:

  • Study H4K8hib alterations in diabetes and obesity models, where glucose metabolism is dysregulated

  • Investigate how insulin signaling impacts H4K8hib patterns

  • Examine potential roles in metabolic memory phenomena

• Neurodegenerative diseases:

  • Explore connections between altered brain energy metabolism in conditions like Alzheimer's and changes in H4K8hib

  • Investigate roles in neuronal gene expression regulation during disease progression

  • Examine potential neuroprotective effects of modulating H4K8hib levels

• Aging research:

  • Study H4K8hib changes during chronological aging, particularly in connection with documented metabolic shifts

  • Investigate connections to longevity pathways and caloric restriction responses

  • Examine potential roles in age-related chromatin reorganization

These emerging applications position H4K8hib antibodies as valuable tools for understanding the epigenetic dimension of metabolic dysregulation in disease, potentially revealing new therapeutic targets and biomarkers.

How can researchers integrate H4K8hib analysis with other omics approaches?

Effective integration of H4K8hib analysis with other omics technologies requires strategic experimental design and computational approaches:

• Multi-omics experimental design:

  • Perform parallel ChIP-seq (H4K8hib), RNA-seq, ATAC-seq, and metabolomics on the same biological samples

  • Include conditions that manipulate glucose availability to capture dynamic relationships

  • Design time-course experiments to establish causality between metabolic changes, H4K8hib modifications, and gene expression

• Computational integration frameworks:

  • Apply multi-omics data integration tools (e.g., MOFA, mixOmics, DIABLO)

  • Develop custom pipelines that account for the unique relationship between metabolism and epigenetics

  • Implement network-based approaches to visualize relationships across different data types

• Single-cell multi-omics approaches:

  • Adapt H4K8hib detection for single-cell epigenomics platforms

  • Integrate with single-cell transcriptomics and metabolomics

  • Analyze cellular heterogeneity in H4K8hib patterns and their relationship to metabolic states

• Causal inference methodologies:

  • Apply directed acyclic graphs to model causal relationships

  • Implement Mendelian randomization-inspired approaches using genetic perturbations

  • Develop mathematical models that capture the dynamic interplay between metabolism and epigenetic regulation

This integrated approach enables researchers to construct comprehensive models of how metabolic signals are translated into epigenetic modifications that ultimately affect gene expression and cellular phenotypes.

What technological advancements are needed to enhance the specificity and sensitivity of H4K8hib detection?

Advancing H4K8hib research requires several technological developments:

• Improved antibody technologies:

  • Development of recombinant antibodies with enhanced specificity

  • Creation of antibody fragments (Fab, scFv) for applications requiring smaller probe size

  • Generation of modification-specific nanobodies for live-cell imaging applications

• Advanced mass spectrometry approaches:

  • Development of targeted, site-specific MS methods for absolute quantification of H4K8hib

  • Implementation of top-down proteomics to analyze intact histone proteoforms

  • Advances in spatial MS techniques to analyze H4K8hib distribution in tissue contexts

• In situ detection methods:

  • Development of proximity ligation assays specific for H4K8hib and associated proteins

  • Adaptation of CUT&RUN or CUT&Tag technologies for improved genomic mapping

  • Creation of FRET-based sensors to monitor H4K8hib dynamics in living cells

• Synthetic biology tools:

  • Engineering of site-specific writer and eraser enzymes for precise manipulation of H4K8hib

  • Development of optogenetic or chemically-inducible systems to control H4K8hib levels

  • Creation of synthetic H4K8hib readers to manipulate downstream effects

These technological advancements would significantly enhance researchers' ability to detect, quantify, and functionally characterize H4K8hib in diverse experimental systems, accelerating discovery in this emerging field of epigenetic regulation.