β-hydroxybutyryl-HIST1H4A (K5) Antibody

What is β-hydroxybutyrylation of Histone H4 and its biological significance?

β-hydroxybutyrylation (Kbhb) is a post-translational modification of histones that connects metabolism to gene expression. Specifically, β-hydroxybutyryl-HIST1H4A (K5) refers to the β-hydroxybutyrylation of the lysine 5 residue on histone H4. This modification represents a metabolism-mediated epigenetic change that enables cellular signaling beyond the historically studied lysine acetylation and methylation mechanisms. The modification plays a crucial role in coupling metabolic states to chromatin structure and gene regulation, allowing cells to adapt transcriptional responses to changing metabolic conditions .

β-hydroxybutyrate serves as the precursor for this modification and can increase from basal levels (0.1 mM or lower) to 2-3.8 mM during physiological states such as starvation, intense exercise, or pathological conditions like diabetic ketoacidosis. This links nutritional status directly to epigenetic regulation, representing a fundamental mechanism by which metabolism influences gene expression patterns .

How does β-hydroxybutyryl-HIST1H4A (K5) modification differ from other histone modifications?

β-hydroxybutyrylation at histone H4K5 is distinct from other histone modifications in several key aspects. Unlike acetylation, which simply transfers an acetyl group, β-hydroxybutyrylation involves the addition of a more complex β-hydroxybutyrate moiety to lysine residues. The regulatory enzymes also differ - while p300 can catalyze both acetylation and β-hydroxybutyrylation, the latter appears to be more specifically regulated by HDAC1 and HDAC2 for removal .

Functionally, β-hydroxybutyrylation is particularly responsive to metabolic states associated with elevated β-hydroxybutyrate levels, such as fasting or ketogenic conditions. This allows for a direct coupling of these specific metabolic states to gene regulation that other modifications may not provide. Evidence suggests that histone Kbhb can directly mediate in vitro transcription, demonstrating its mechanistic role in gene expression regulation .

What are the known regulatory enzymes for β-hydroxybutyrylation?

The β-hydroxybutyrylation pathway is enzymatically regulated through specific writer and eraser proteins:

These findings establish that lysine β-hydroxybutyrylation is an enzymatically regulated process, with p300-dependent histone Kbhb being the major mechanism for nuclear Kbhb .

What are the key technical specifications of commercially available β-hydroxybutyryl-HIST1H4A (K5) antibodies?

Currently available β-hydroxybutyryl-HIST1H4A (K5) antibodies share several important technical characteristics that researchers should consider when selecting reagents:

When selecting an antibody, researchers should consider these specifications in relation to their specific experimental requirements, particularly the applications they intend to use and the species they are studying .

How should researchers validate the specificity of β-hydroxybutyryl-HIST1H4A (K5) antibodies before experimental use?

Validation of antibody specificity is critical for ensuring reliable experimental results. For β-hydroxybutyryl-HIST1H4A (K5) antibodies, a comprehensive validation approach should include:

• Positive and negative controls: Compare cells treated with sodium β-hydroxybutyrate (typically 30-50mM) against untreated cells. Western blot analysis should show increased signal in treated cells and minimal signal in untreated cells, as demonstrated in multiple cell lines (HEK-293, A549, K562, HepG2) .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (containing β-hydroxybutyrylated K5) before applying to samples, which should abolish specific signals.

• Cross-reactivity testing: Validate that the antibody does not cross-react with other histone modifications (acetylation, methylation) at the same or nearby residues by comparing signal patterns with antibodies specific to these other modifications.

• HDAC inhibitor treatment: Cells treated with HDAC inhibitors like sodium butyrate (NaBu), trichostatin A (TSA), or FK228 should show increased β-hydroxybutyrylation signal due to inhibition of the eraser enzymes HDAC1 and HDAC2 .

These validation steps ensure that the observed signals specifically represent β-hydroxybutyrylation at H4K5 rather than other modifications or non-specific binding .

What are the optimal conditions for using β-hydroxybutyryl-HIST1H4A (K5) antibody in Western blot applications?

For optimal Western blot results with β-hydroxybutyryl-HIST1H4A (K5) antibodies, researchers should follow these protocol guidelines:

• Sample preparation:

  • For positive controls, treat cells with 30-50mM sodium β-hydroxybutyrate for 4-72 hours

  • Extract histones using acid extraction or prepare whole cell lysates depending on experimental goals

  • Load 10-20μg of protein per lane

• Antibody dilution and incubation:

  • Use antibody at 1:500-1:5000 dilution (optimize for each lot)

  • Incubate membranes overnight at 4°C or for 2 hours at room temperature

• Detection conditions:

  • Use appropriate secondary antibody (anti-rabbit IgG)

  • Expected band size: approximately 11-14 kDa for histone H4

• Controls to include:

  • Positive control: lysate from cells treated with sodium β-hydroxybutyrate

  • Negative control: untreated cell lysate

  • Loading control: total histone H4 antibody on parallel blot

Following these guidelines should yield clear detection of β-hydroxybutyrylated histone H4 at K5, with increased signal in treated samples compared to untreated controls .

How can researchers effectively use β-hydroxybutyryl-HIST1H4A (K5) antibody in ChIP experiments?

Chromatin immunoprecipitation (ChIP) with β-hydroxybutyryl-HIST1H4A (K5) antibody requires careful optimization. The following protocol outline provides guidance for successful experiments:

• Chromatin preparation:

  • Treat cells with sodium β-hydroxybutyrate (30-50mM for 4-72 hours) to induce β-hydroxybutyrylation

  • Cross-link DNA-protein complexes with 1% formaldehyde for 10 minutes

  • Sonicate chromatin to generate fragments of 200-500bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein G beads

  • Incubate cleared chromatin with β-hydroxybutyryl-HIST1H4A (K5) antibody (3-5μg) overnight at 4°C

  • Add protein G beads for 2-4 hours

  • Wash beads thoroughly with increasingly stringent buffers

• DNA recovery and analysis:

  • Reverse cross-links and purify DNA

  • Analyze by qPCR, sequencing, or other preferred methods

• Controls:

  • Input chromatin (10%)

  • IgG negative control

  • Parallel ChIP with total H4 antibody for normalization

  • ChIP with acetyl-H4K5 antibody for comparison

This approach allows researchers to map genomic regions associated with β-hydroxybutyrylated H4K5, providing insights into the regulatory role of this modification in gene expression .

What considerations are important for immunofluorescence experiments using β-hydroxybutyryl-HIST1H4A (K5) antibody?

Successful immunofluorescence (IF) experiments with β-hydroxybutyryl-HIST1H4A (K5) antibody require attention to several critical factors:

• Cell preparation and fixation:

  • Treat cells with sodium butyrate (30mM for 4 hours) to increase signal

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1-0.5% Triton X-100

• Antibody incubation:

  • Block with 5% BSA or normal serum

  • Use antibody at 1:50-1:200 dilution

  • Incubate overnight at 4°C or 2 hours at room temperature

• Visualization considerations:

  • Expected pattern: Nuclear staining with potential enrichment in euchromatic regions

  • Co-stain with DAPI for nuclear visualization

  • Consider co-staining with markers for specific nuclear compartments

• Controls and validation:

  • Compare treated vs. untreated cells

  • Include antibody specificity controls

  • Consider dual staining with other histone modification antibodies for colocalization studies

Following these guidelines should result in specific nuclear staining patterns that reflect the distribution of β-hydroxybutyrylated H4K5, with significantly higher signal intensity in cells treated with sodium butyrate .

How should researchers interpret inconsistent β-hydroxybutyrylation signals across different cell types?

Variations in β-hydroxybutyrylation signal intensity across different cell types can be attributed to several biological and technical factors:

When encountering inconsistent signals, researchers should systematically investigate these factors. For example, the search results show variability in baseline and induced β-hydroxybutyrylation across HEK-293, A549, K562, and HepG2 cells, suggesting cell-type-specific regulation of this modification .

What are the potential causes and solutions for background or non-specific signals when using β-hydroxybutyryl-HIST1H4A (K5) antibody?

Background and non-specific signals can significantly impact data quality. The following table outlines common issues and solutions:

Understanding these potential issues can help researchers troubleshoot unexpected results. For example, in the search results, unexpected bands at 52 and 85 kDa were noted in immunoprecipitation experiments, indicating potential non-specific interactions that should be controlled for .

How do experimental conditions affect β-hydroxybutyrylation levels, and what controls should be included?

The dynamic nature of β-hydroxybutyrylation requires careful consideration of experimental conditions:

• β-hydroxybutyrate treatment:

  • Concentration: 30-50mM sodium β-hydroxybutyrate or sodium butyrate is typically used

  • Duration: Treatment times range from 4-72 hours, with longer exposures potentially yielding stronger signals

  • Response curve: Consider establishing a time-course and dose-response to determine optimal conditions for each cell type

• Metabolic conditions affecting endogenous levels:

  • Serum starvation can increase endogenous β-hydroxybutyrate

  • Glucose availability affects β-hydroxybutyrate production

  • Cell confluence and growth phase influence metabolic state

• Essential controls:

  • Untreated cells (negative control)

  • HDAC inhibitor treatment (positive control) using NaBu, TSA, or FK228

  • Cells with p300 knockdown/knockout (should show reduced signal)

  • Cells with HDAC1/2 knockdown/knockout (should show increased signal)

• Standardization approaches:

  • Normalize to total histone H4

  • Include internal reference cell line in each experiment

  • Report signal relative to standard treatment conditions

These considerations ensure that observed changes in β-hydroxybutyrylation are attributable to the experimental variables rather than background fluctuations in cellular metabolism .

How can β-hydroxybutyryl-HIST1H4A (K5) antibody be used to study the relationship between metabolism and gene regulation?

The β-hydroxybutyryl-HIST1H4A (K5) antibody serves as a powerful tool for investigating the interface between metabolism and epigenetic regulation:

• Metabolic state manipulation experiments:

  • Compare β-hydroxybutyrylation patterns under various metabolic conditions (fasting, ketogenic diet, caloric restriction)

  • Analyze changes during metabolic stress (hypoxia, nutrient deprivation)

  • Measure β-hydroxybutyrylation in models of metabolic disease (diabetes, obesity)

• Integrated multi-omics approaches:

  • Combine ChIP-seq using β-hydroxybutyryl-HIST1H4A (K5) antibody with RNA-seq to correlate modification with gene expression changes

  • Integrate with metabolomics data to establish direct relationships between metabolite levels and epigenetic changes

  • Compare with other histone modification profiles to establish unique and overlapping regulatory regions

• Mechanistic studies:

  • Investigate the role of β-hydroxybutyrylation in transcriptional activation using reporter assays

  • Study the recruitment of specific reader proteins to β-hydroxybutyrylated histones

  • Examine the interplay between β-hydroxybutyrylation and other histone modifications

Research has already established that p300-dependent histone Kbhb can directly mediate in vitro transcription, suggesting a causal role in gene regulation. The comprehensive analysis identifying 3248 Kbhb sites on 1397 substrate proteins provides a foundation for further studies of this modification's regulatory roles .

What are the current challenges in quantifying global β-hydroxybutyrylation levels?

Accurate quantification of global β-hydroxybutyrylation presents several technical challenges:

• Technical limitations:

  • Western blot semi-quantitative nature limits precise measurement

  • Antibody affinity may vary between batches

  • Extraction efficiency of modified histones can be variable

  • Cross-reactivity with similar modifications may confound results

• Analytical approaches and their limitations:

  Method	Strengths	Limitations
  Western blot	Simple, accessible	Semi-quantitative, limited dynamic range
  Mass spectrometry	Precise, can identify multiple modifications	Expensive, requires specialized equipment
  ELISA	High throughput, quantitative	May have cross-reactivity issues
  ChIP-seq	Genome-wide distribution	Indirect measure of global levels

• Standardization issues:

  • Lack of universal standards for quantification

  • Variation in extraction protocols affects results

  • Different normalization approaches make cross-study comparisons difficult

• Biological variability:

  • Dynamic nature of the modification

  • Cell cycle dependence

  • Heterogeneity within cell populations

Researchers should consider these challenges when designing experiments to quantify β-hydroxybutyrylation and interpret results accordingly. Mass spectrometry-based approaches, though more complex, may provide the most accurate quantification of global levels and should be considered for definitive studies .

How does β-hydroxybutyrylation interact with other histone modifications in the epigenetic code?

The interplay between β-hydroxybutyrylation and other histone modifications represents a frontier in epigenetic research:

• Co-occurrence and mutual exclusivity:

  • β-hydroxybutyrylation occurs at lysine residues that can also be acetylated or methylated

  • The presence of one modification may preclude others at the same residue

  • Sequential or combinatorial modifications may create specific regulatory signatures

• Regulatory enzyme competition:

  • p300 catalyzes both acetylation and β-hydroxybutyrylation, suggesting potential competition based on metabolite availability

  • HDAC1/2 remove both acetyl and β-hydroxybutyryl groups, potentially creating regulatory feedback loops

  • The balance of writer and eraser activities likely depends on cellular metabolic state

• Functional consequences:

  • Different modifications may recruit distinct reader proteins

  • Modification patterns may determine chromatin structure and accessibility

  • Specific combinations may fine-tune gene expression in response to metabolic cues

• Research approaches:

  • Sequential ChIP (re-ChIP) to identify co-occurring modifications

  • Mass spectrometry to identify combinatorial patterns

  • Genetic manipulation of writer/eraser enzymes to study hierarchical relationships

This area represents a significant knowledge gap in the field. The finding that p300 and HDAC1/2 regulate β-hydroxybutyrylation suggests potential cross-talk with acetylation pathways, but comprehensive studies mapping the interrelationships between different modifications are still needed .

What role might β-hydroxybutyrylation play in disease pathogenesis and potential therapeutic approaches?

β-hydroxybutyrylation may have significant implications for disease mechanisms and treatment strategies:

• Metabolic disorders:

  • Altered β-hydroxybutyrylation patterns may contribute to transcriptional dysregulation in diabetes and obesity

  • The ketogenic diet, which increases β-hydroxybutyrate levels, may exert some of its beneficial effects through histone β-hydroxybutyrylation

  • Targeting the β-hydroxybutyrylation pathway could potentially restore metabolic homeostasis

• Neurological conditions:

  • β-hydroxybutyrate has been used to treat epilepsy, potentially acting through epigenetic mechanisms

  • Neurodegenerative diseases involving metabolic dysfunction might be influenced by aberrant β-hydroxybutyrylation

  • Brain-specific patterns of this modification could reveal new insights into neurological disorders

• Cancer biology:

  • β-hydroxybutyrate has been investigated as an adjuvant for cancer therapeutics

  • Altered metabolism is a hallmark of cancer, potentially affecting β-hydroxybutyrylation patterns

  • Targeting writer (p300) or eraser (HDAC1/2) enzymes represents a potential therapeutic strategy

• Therapeutic implications:

  • HDAC inhibitors that block de-β-hydroxybutyrylation activity (NaBu, TSA, FK228) may have therapeutic applications

  • Metabolic interventions that alter β-hydroxybutyrate levels could modulate gene expression in a targeted manner

  • Designer β-hydroxybutyrate analogs might enable selective targeting of specific gene programs

The connection between β-hydroxybutyrate metabolism and epigenetic regulation opens new avenues for understanding disease mechanisms and developing therapies that target this interface .

What methodological advances are needed to better characterize the β-hydroxybutyrylome?

Future progress in understanding β-hydroxybutyrylation depends on several methodological improvements:

• Antibody development needs:

  • Site-specific antibodies for different β-hydroxybutyrylated residues

  • Higher affinity and specificity reagents

  • Monoclonal antibodies for improved consistency

  • Antibodies compatible with a broader range of applications

• Mass spectrometry approaches:

  • Enhanced enrichment strategies for β-hydroxybutyrylated peptides

  • Improved fragmentation methods for modification-specific analysis

  • Quantitative approaches for comparing β-hydroxybutyrylation across conditions

  • Methods for analyzing low-abundance modifications

• Functional genomics tools:

  • CRISPR-based screens to identify readers, writers, and erasers

  • Development of β-hydroxybutyrylation-specific reader domain probes

  • Systems for site-specific installation of the modification

  • Methods to visualize the modification in living cells

• Computational resources:

  • Databases cataloging known β-hydroxybutyrylation sites

  • Prediction algorithms for potential modification sites

  • Tools for integrating β-hydroxybutyrylation data with other omics datasets

  • Pathway analysis specific to β-hydroxybutyrylation-regulated genes

While comprehensive analysis has identified 3248 Kbhb sites on 1397 substrate proteins, advancing these methodologies would enable deeper investigation of the biological significance and regulatory mechanisms of this modification .

How can researchers distinguish the direct effects of β-hydroxybutyrylation from indirect metabolic effects of β-hydroxybutyrate?

Separating direct epigenetic effects from indirect metabolic influences presents a significant challenge:

• Experimental approaches:

  • Site-specific mutations of lysine residues to prevent β-hydroxybutyrylation while maintaining protein function

  • Use of β-hydroxybutyrate analogs that cannot be used for protein modification

  • Selective inhibition or activation of writer/eraser enzymes without altering metabolite levels

  • In vitro transcription systems with defined β-hydroxybutyrylated chromatin templates

• Controls and comparisons:

  • Compare effects of β-hydroxybutyrate treatment with other metabolic interventions

  • Use p300 knockout/knockdown to block β-hydroxybutyrylation while maintaining β-hydroxybutyrate supplementation

  • Compare transcriptional changes from β-hydroxybutyrate treatment with ChIP-seq data for β-hydroxybutyrylated histones

• Time-course analyses:

  • Track the temporal relationship between β-hydroxybutyrate addition, histone modification, and transcriptional changes

  • Short-term vs. long-term effects may help distinguish direct epigenetic from indirect metabolic mechanisms

• Reader protein identification:

  • Identify proteins that specifically bind to β-hydroxybutyrylated histones

  • Map the recruitment of these readers to chromatin and correlate with transcriptional changes

  • Disrupt reader-modification interactions to test direct causality

The demonstration that p300-dependent histone Kbhb can directly mediate in vitro transcription provides important evidence for direct epigenetic effects, but further studies using these approaches will be necessary to fully characterize the relative contributions of direct and indirect mechanisms in different biological contexts .

What are the most promising future research directions for β-hydroxybutyrylation studies?

The study of β-hydroxybutyrylation is poised for significant advances in several key areas:

• Comprehensive mapping studies:

  • Cell type-specific β-hydroxybutyrylation patterns

  • Developmental dynamics of the modification

  • Changes in response to physiological and pathological conditions

  • Conservation and divergence across species

• Mechanistic investigations:

  • Identification of reader proteins that specifically recognize β-hydroxybutyrylated histones

  • Structural studies of writer/eraser/reader interactions with the modification

  • Crosstalk mechanisms with other epigenetic modifications

  • Sequence context preferences for modification sites

• Physiological significance:

  • Role in metabolic adaptation during fasting/feeding cycles

  • Function in exercise physiology and muscle adaptation

  • Involvement in aging processes

  • Contribution to intergenerational epigenetic inheritance

• Translational opportunities:

  • Development of targeted therapeutics modulating β-hydroxybutyrylation

  • Biomarker potential for metabolic health assessment

  • Applications in regenerative medicine

  • Implications for precision nutrition approaches

The field has established fundamental principles with the identification of regulatory enzymes (p300 as writer, HDAC1/2 as erasers) and thousands of substrate sites, creating a solid foundation for these future directions .

What considerations should guide the development of new tools and resources for β-hydroxybutyrylation research?

Advancing research in this field requires thoughtful development of new tools and resources:

• Reagent development principles:

  • Prioritize reproducibility and validation

  • Create tools applicable across multiple model systems

  • Focus on quantitative applications

  • Develop resources that enable single-cell analyses

• Data sharing and standardization:

  • Establish repositories for β-hydroxybutyrylation datasets

  • Develop standard protocols for modification analysis

  • Create common reference standards for quantification

  • Adopt unified nomenclature for modification sites

• Technology integration:

  • Combine imaging and omics approaches

  • Develop multiplexed detection methods for simultaneous analysis of multiple modifications

  • Create computational tools that integrate β-hydroxybutyrylation with metabolomics data

  • Design high-throughput screening platforms for modifier discovery

• Ethical and practical considerations:

  • Ensure accessibility of tools to diverse research communities

  • Consider environmental impact of reagent production

  • Address potential translational implications early in development

  • Engage multidisciplinary expertise in tool design

These considerations should guide the development of next-generation resources that will enable deeper understanding of this important epigenetic modification and its biological roles.

How might understanding β-hydroxybutyrylation impact other fields of biological research?

The study of β-hydroxybutyrylation has potential to influence numerous scientific disciplines:

• Evolutionary biology:

  • Insights into how metabolic regulation of gene expression evolved

  • Understanding of how epigenetic mechanisms adapt to environmental changes

  • Comparative studies across species may reveal conserved regulatory networks

• Developmental biology:

  • Role of metabolic-epigenetic coupling in cell fate decisions

  • Potential involvement in developmental programming by maternal nutrition

  • Implications for understanding developmental disorders with metabolic components

• Immunology:

  • Immune cell metabolism significantly affects function and β-hydroxybutyrate levels

  • Potential role in trained immunity and epigenetic memory in immune cells

  • Therapeutic possibilities for inflammatory conditions

• Neuroscience:

  • Brain utilizes β-hydroxybutyrate as an alternative fuel source

  • Potential role in neural plasticity and memory formation

  • Implications for neurodevelopmental and neurodegenerative conditions

• Aging research:

  • Metabolic interventions that extend lifespan may act partly through β-hydroxybutyrylation

  • Age-related changes in the writer/eraser enzyme balance

  • Connection to cellular senescence and age-related epigenetic drift