β-hydroxybutyryl-HIST1H1D (K75) Antibody

What is β-hydroxybutyryl-HIST1H1D (K75) and why is it significant in epigenetic research?

β-hydroxybutyryl-HIST1H1D (K75) refers to a specific histone modification where a β-hydroxybutyryl group is attached to the lysine 75 residue of histone H1.3 (HIST1H1D). This post-translational modification is particularly significant in epigenetic research as it represents a direct link between cellular metabolism and gene regulation.

Histone H1.3 (HIST1H1D) functions as a linker histone that binds to DNA between nucleosomes, facilitating the formation of higher-order chromatin structures. It plays crucial roles in the condensation of nucleosome chains and regulates gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . The β-hydroxybutyrylation of this histone represents a mechanism by which metabolic states can influence gene expression patterns.

Studies have shown that histone β-hydroxybutyrylation levels are tightly associated with elevated β-hydroxybutyrate concentrations that occur during prolonged fasting and in disease models such as Type 1 Diabetes Mellitus (T1DM) . Unlike histone acetylation which shows minimal changes under these conditions, histone β-hydroxybutyrylation responds dynamically to metabolic shifts, making it a valuable marker for investigating metabolism-epigenome relationships.

How does β-hydroxybutyrylation differ from other histone modifications such as acetylation?

β-hydroxybutyrylation represents a distinct histone modification with unique regulatory patterns compared to more extensively studied modifications like acetylation:

What are the recommended applications for β-hydroxybutyryl-HIST1H1D (K75) antibodies?

β-hydroxybutyryl-HIST1H1D (K75) antibodies have been validated for several key research applications:

• Western Blot (WB): Useful for detecting and quantifying β-hydroxybutyrylated HIST1H1D in cell or tissue lysates. Recommended dilutions typically range from 1:100 to 1:1000 .

• Enzyme-Linked Immunosorbent Assay (ELISA): Enables high-throughput quantitative analysis of β-hydroxybutyrylation levels. Common dilutions range from 1:2000 to 1:10000 .

• Immunocytochemistry (ICC): Allows visualization of the cellular and subcellular distribution of β-hydroxybutyrylated histones. Typically used at dilutions of 1:10 to 1:100 .

• Chromatin Immunoprecipitation (ChIP): Though not explicitly mentioned in the search results, this antibody can theoretically be used for ChIP experiments to identify genomic regions associated with β-hydroxybutyrylated histones.

When using these antibodies, researchers should verify reactivity with their specific samples, as most available antibodies are validated for human samples . Positive controls can include cell lysates from cell lines treated with sodium β-hydroxybutyrate, which has been shown to increase β-hydroxybutyrylation levels in a dose-dependent manner .

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

The relationship between metabolism and histone β-hydroxybutyrylation represents a direct mechanistic link between cellular energetic state and epigenetic regulation. This relationship is characterized by several key findings:

• β-hydroxybutyrate as both metabolite and epigenetic regulator: Beyond its canonical role as an energy source during fasting or ketosis, β-hydroxybutyrate serves as a substrate for histone β-hydroxybutyrylation, directly connecting metabolic state to chromatin modification .

• Dose-dependent relationship: Studies have demonstrated that histone β-hydroxybutyrylation levels increase in a dose-dependent manner in response to rising cellular β-hydroxybutyrate concentrations. This has been confirmed through immunoblot analysis of protein lysates from cells treated with sodium β-hydroxybutyrate .

• Specificity of the response: While β-hydroxybutyrylation shows dramatic changes in response to β-hydroxybutyrate levels, other histone modifications like acetylation show minimal changes. This suggests a specific rather than general effect on histone modifications .

• Physiological relevance: In physiological states characterized by elevated β-hydroxybutyrate levels, such as prolonged fasting or type 1 diabetes, significant increases in histone β-hydroxybutyrylation can be observed. In the streptozotocin (STZ)-induced T1DM mouse model, a 10-fold elevation in blood β-hydroxybutyrate levels corresponded with dramatically elevated histone β-hydroxybutyrylation in liver tissue .

• Isotopic labeling confirmation: The direct metabolic incorporation of β-hydroxybutyrate into histone modifications has been confirmed through isotopic labeling experiments. When cells were cultured with isotopically labeled β-hydroxybutyrate ([13C]2), mass spectrometry analysis detected histone peptides modified by the isotopic β-hydroxybutyryl group, confirming the direct metabolic route from β-hydroxybutyrate to histone modification .

This metabolic-epigenetic relationship provides a mechanism by which cells can adapt gene expression in response to changing metabolic conditions, potentially allowing for rapid transcriptional responses to energy status.

What are the optimal experimental protocols for detecting β-hydroxybutyryl-HIST1H1D (K75) in various experimental systems?

Optimal detection of β-hydroxybutyryl-HIST1H1D (K75) requires careful consideration of sample preparation, antibody selection, and detection methods. Here are detailed protocols for common applications:

Western Blot Protocol:

• Sample Preparation:

  • Extract histones using an acid extraction method to enrich for histone proteins.

  • For cell culture: Treat cells with 30mM sodium butyrate for 4 hours to increase β-hydroxybutyrylation signal if studying induction .

  • For animal tissue: Flash-freeze samples immediately after collection to preserve modification status.

• Gel Electrophoresis and Transfer:

  • Use 15-18% SDS-PAGE gels for optimal separation of histone proteins.

  • Transfer to PVDF membrane (preferred over nitrocellulose for histone proteins).

• Antibody Incubation:

  • Block with 5% non-fat dry milk or BSA in TBST.

  • Incubate with primary β-hydroxybutyryl-HIST1H1D (K75) antibody at 1:100-1:1000 dilution .

  • Use goat anti-rabbit IgG as secondary antibody (typically at 1:5000-1:50000 dilution) .

• Detection and Controls:

  • Include lysates from 293, A549, or K562 cell lines treated with sodium butyrate as positive controls .

  • Expected band size: approximately 23 kDa .

  • Compare with untreated samples to demonstrate specificity and induction.

Immunocytochemistry Protocol:

• Cell Preparation:

  • Culture cells on coverslips or chamber slides.

  • Fix with 4% paraformaldehyde for 10 minutes at room temperature.

  • Permeabilize with 0.2% Triton X-100 for 10 minutes.

• Antibody Staining:

  • Block with 1-3% BSA in PBS for 1 hour.

  • Incubate with primary β-hydroxybutyryl-HIST1H1D (K75) antibody at 1:10-1:100 dilution .

  • Use fluorophore-conjugated secondary antibody suitable for fluorescence microscopy.

• Imaging Considerations:

  • Include DAPI staining to visualize nuclei.

  • Consider co-staining with other histone markers to study co-localization patterns.

  • Always include negative controls (secondary antibody only) and positive controls (sodium butyrate-treated cells).

Mass Spectrometry Detection:

For definitive characterization of β-hydroxybutyrylation sites:

• Sample Preparation:

  • Extract histones using acid extraction.

  • Perform trypsin digestion to generate peptide fragments.

• Analysis:

  • Use HPLC/MS/MS for peptide analysis.

  • Look for peptides with a mass shift corresponding to β-hydroxybutyrylation (+86.03679 Da).

  • For confirmation studies, use isotopically labeled β-hydroxybutyrate ([13C]2) which will produce a modification with an additional mass shift of 2 Da .

These protocols should be optimized for each specific experimental system, paying particular attention to antibody dilutions and incubation conditions based on the specific manufacturer's recommendations.

How can researchers distinguish between β-hydroxybutyrylation and other similar histone modifications?

Distinguishing between β-hydroxybutyrylation and other similar histone modifications (particularly acylations) presents a significant challenge in epigenetic research. Here are methodological approaches to ensure specificity:

• Antibody Specificity Validation:

  • Perform peptide competition assays using modified and unmodified peptides to confirm antibody specificity.

  • Test antibody cross-reactivity against peptides containing similar modifications (acetylation, butyrylation, crotonylation, etc.).

  • Validate antibody specificity using histones from cells where β-hydroxybutyrylation is induced (via β-hydroxybutyrate treatment) versus controls .

• Mass Spectrometry Approaches:

  • High-resolution mass spectrometry can differentiate between modifications based on precise mass differences.

  • β-hydroxybutyrylation adds a mass of 86.03679 Da, which is distinct from acetylation (42.01057 Da) and butyrylation (70.04187 Da).

  • Fragmentation patterns in MS/MS can further distinguish between isomeric or near-isobaric modifications.

  • Isotopic labeling with [13C]2-β-hydroxybutyrate provides definitive confirmation of β-hydroxybutyrylation by creating a specific mass shift of 2 Da in the modified peptides .

• Metabolic Regulation Pattern:

  • β-hydroxybutyrylation shows a distinctive response pattern to metabolic changes that differentiates it from other modifications.

  • Unlike acetylation, which shows minimal changes in response to fasting or β-hydroxybutyrate treatment, β-hydroxybutyrylation increases dramatically under these conditions .

  • This differential regulation can be used to distinguish the modifications in functional studies.

• Multiple Detection Methods:

  • Employ complementary techniques including western blotting, immunofluorescence, and mass spectrometry.

  • Consistent results across multiple platforms provide stronger evidence for specific modification identification.

• Site-Specific Analysis:

  • Use site-specific antibodies (such as those targeting β-hydroxybutyryl-HIST1H1D (K75) specifically) rather than pan-modification antibodies when possible .

  • Compare the patterns of modification at specific residues, as β-hydroxybutyrylation may occur at sites that overlap with, but are not identical to, other modifications.

By combining these approaches, researchers can confidently distinguish β-hydroxybutyrylation from other similar histone modifications and ensure the accuracy of their experimental findings.

What are the impacts of histone β-hydroxybutyrylation on gene expression and chromatin structure?

Histone β-hydroxybutyrylation has significant impacts on gene expression and chromatin structure, serving as a mechanistic link between metabolism and epigenetic regulation:

• Chromatin Structure Modulation:

  • Histone H1 proteins, including HIST1H1D, bind to linker DNA between nucleosomes to form higher-order chromatin structures .

  • β-hydroxybutyrylation of HIST1H1D at K75 may alter the binding affinity to DNA, potentially affecting chromatin compaction.

  • As a linker histone modification, changes in HIST1H1D β-hydroxybutyrylation likely impact higher-order chromatin organization rather than just local nucleosome structure.

• Transcriptional Regulation:

  • Histone H1 acts as a regulator of individual gene transcription through multiple mechanisms including chromatin remodeling, nucleosome spacing, and DNA methylation .

  • β-hydroxybutyrylation of H1 histones likely influences these regulatory functions, potentially altering accessibility of transcription machinery to DNA.

  • Studies in bovine models have shown that β-hydroxybutyrate treatment alters the transcriptome in cells, suggesting a direct impact of this modification on gene expression patterns .

• Metabolic-Transcriptional Coupling:

  • Elevated β-hydroxybutyrate levels during fasting or in diabetic conditions lead to increased histone β-hydroxybutyrylation .

  • This creates a direct mechanism by which metabolic state can influence gene expression, allowing for adaptive transcriptional responses to changing energy availability.

  • The modification may serve as a metabolic sensor that triggers specific transcriptional programs appropriate to fasting or ketotic states.

• Epigenetic Programming:

  • In models of metabolic disease such as T1DM, dramatic increases in histone β-hydroxybutyrylation suggest this modification may contribute to epigenetic reprogramming associated with pathological conditions .

  • This epigenetic mechanism may help explain how transient metabolic changes can lead to persistent alterations in gene expression patterns.

• Distinction from Acetylation Effects:

  • While histone acetylation generally promotes gene activation by neutralizing positive charges on histones, β-hydroxybutyrylation contains a hydroxyl group that may create different physical and chemical properties.

  • The differential regulation of β-hydroxybutyrylation versus acetylation under metabolic stress suggests these modifications may regulate distinct sets of genes or genomic regions .

Understanding these impacts is vital for researchers investigating the role of metabolism in gene regulation and how metabolic dysregulation may contribute to disease through epigenetic mechanisms.

What are the most common technical challenges when working with β-hydroxybutyryl-HIST1H1D (K75) antibodies?

Researchers working with β-hydroxybutyryl-HIST1H1D (K75) antibodies often encounter several technical challenges that can impact experimental outcomes. Here are the most common issues and recommended solutions:

• Low Signal Intensity:

  • Challenge: β-hydroxybutyrylation may be present at lower abundance than modifications like acetylation, resulting in weak signals.

  • Solution: Pre-treat samples with sodium β-hydroxybutyrate (20-30mM for 4 hours) to increase β-hydroxybutyrylation levels . Optimize antibody concentration by testing a range of dilutions from 1:100 to 1:1000 for Western blot and 1:10 to 1:100 for ICC .

• Specificity Concerns:

  • Challenge: Cross-reactivity with similar histone modifications (acetylation, butyrylation).

  • Solution: Include appropriate controls in experiments, such as comparing β-hydroxybutyrate-treated samples with untreated samples. Perform peptide competition assays to confirm antibody specificity.

• Sample Preservation Issues:

  • Challenge: β-hydroxybutyrylation may be sensitive to sample handling and storage conditions.

  • Solution: Use freshly prepared samples when possible. For storage, maintain samples at -20°C or -80°C and avoid repeated freeze-thaw cycles . Include deacetylase and general HDAC inhibitors in lysis buffers to prevent post-lysis modification removal.

• Background Signal:

  • Challenge: High background in immunostaining or Western blot applications.

  • Solution: Optimize blocking conditions using 5% BSA rather than milk for Western blots. For ICC, extend blocking time to 1-2 hours and include thorough washing steps. Consider using antigen affinity-purified antibodies which generally have lower background .

• Lot-to-Lot Variability:

  • Challenge: Polyclonal antibodies may show variation between production lots.

  • Solution: Validate each new antibody lot against a previous lot or with known positive controls. Consider purchasing larger quantities of a single lot for long-term projects.

• Limited Species Reactivity:

  • Challenge: Most available antibodies are validated only for human samples .

  • Solution: When working with non-human samples, perform preliminary validation experiments to confirm cross-reactivity. Consider using conserved regions of the histone sequence to predict potential cross-reactivity.

By anticipating these challenges and implementing the suggested solutions, researchers can improve the reliability and reproducibility of experiments using β-hydroxybutyryl-HIST1H1D (K75) antibodies.

How can researchers effectively validate the specificity of β-hydroxybutyryl-HIST1H1D (K75) antibodies?

Thorough validation of antibody specificity is critical for ensuring reliable experimental results. Here is a comprehensive approach to validating β-hydroxybutyryl-HIST1H1D (K75) antibodies:

• Peptide Competition Assays:

  • Pre-incubate the antibody with excess β-hydroxybutyrylated peptide (corresponding to the K75 site of HIST1H1D).

  • Run parallel Western blots with competed and non-competed antibody.

  • Specific signals should be abolished or significantly reduced in the competed samples.

  • Include both modified and unmodified peptides to confirm modification specificity.

• Metabolic Manipulation Controls:

  • Compare samples from cells treated with increasing concentrations of sodium β-hydroxybutyrate (5mM, 10mM, 20mM, 30mM) to untreated controls.

  • A dose-dependent increase in signal intensity strongly supports antibody specificity for β-hydroxybutyrylation .

  • Parallel assessment of other histone modifications (e.g., acetylation) can confirm the specificity of the response.

• Genetic Knockdown/Knockout Validation:

  • Use siRNA or CRISPR approaches to reduce expression of HIST1H1D.

  • The antibody signal should decrease correspondingly if it is truly specific to HIST1H1D.

  • This approach validates the histone variant specificity but not necessarily the modification specificity.

• Mass Spectrometry Correlation:

  • Perform parallel analysis of samples using the antibody of interest and mass spectrometry.

  • MS/MS can definitively identify β-hydroxybutyrylation at specific lysine residues.

  • Correlation between antibody signal intensity and MS quantification of β-hydroxybutyrylation provides strong validation.

  • Isotopic labeling with [13C]2-β-hydroxybutyrate can provide additional confirmation of modification specificity .

• Cross-Reactivity Testing:

  • Test the antibody against peptides containing other acyl modifications at the K75 position (acetylation, butyrylation, crotonylation).

  • Perform Western blot analysis on samples known to contain these alternative modifications.

  • A truly specific antibody should show minimal cross-reactivity with other modifications.

• Reproducibility Across Methods:

  • Validate the antibody using multiple techniques (Western blot, ICC, ChIP).

  • Consistent results across different methodologies strengthen confidence in antibody specificity.

  • Compare results from different antibodies targeting the same modification if available.

Following this comprehensive validation strategy ensures that experimental results obtained with β-hydroxybutyryl-HIST1H1D (K75) antibodies accurately reflect the biology of this histone modification.

How can β-hydroxybutyryl-HIST1H1D (K75) antibodies be utilized in metabolic disease research?

β-hydroxybutyryl-HIST1H1D (K75) antibodies offer valuable tools for investigating the intersection of metabolism and epigenetics in disease contexts. Here are methodological approaches for their application in metabolic disease research:

• Diabetes Research Applications:

  • Use these antibodies to assess histone β-hydroxybutyrylation levels in tissues from diabetic models like streptozotocin (STZ)-induced T1DM mice.

  • Compare β-hydroxybutyrylation patterns with metabolic parameters such as blood glucose and β-hydroxybutyrate levels .

  • Investigate whether altered histone β-hydroxybutyrylation contributes to the dysregulated gene expression observed in diabetic complications.

  • Methodology: Combine immunoblotting for global changes with ChIP-seq to identify specifically affected genomic regions.

• Fasting and Ketotic States:

  • Apply these antibodies to study how nutritional interventions such as intermittent fasting, ketogenic diets, or caloric restriction affect histone β-hydroxybutyrylation.

  • Correlate changes in histone modification with physiological adaptations to these metabolic states.

  • Methodology: Time-course studies combining tissue-specific Western blotting, ChIP-seq, and RNA-seq to connect epigenetic changes with transcriptional outcomes.

• Metabolic Syndrome and Obesity Research:

  • Investigate whether disruptions in β-hydroxybutyrylation patterns contribute to metabolic syndrome pathophysiology.

  • Compare β-hydroxybutyrylation levels in tissues from obese versus lean subjects to identify potential epigenetic mechanisms of metabolic dysfunction.

  • Methodology: Tissue microarrays with immunohistochemistry using these antibodies can provide high-throughput screening of multiple samples.

• Therapeutic Intervention Studies:

  • Monitor changes in histone β-hydroxybutyrylation in response to metabolic therapeutics.

  • Test whether β-hydroxybutyrate supplementation can normalize aberrant epigenetic patterns in metabolic disease models.

  • Methodology: Combine antibody-based detection with functional genomics to assess whether epigenetic changes correlate with improved physiological outcomes.

• Cellular Metabolism-Epigenome Integration:

  • Use these antibodies in conjunction with metabolic tracers to map how cellular metabolism directly influences the epigenome.

  • Apply in models of altered mitochondrial function to assess the relationship between energy metabolism and histone modifications.

  • Methodology: Combine isotopic labeling of β-hydroxybutyrate with immunoprecipitation and mass spectrometry to track metabolite-to-modification pathways .

This antibody provides a unique window into how metabolic states directly impact the epigenome, potentially revealing novel therapeutic targets at the intersection of metabolism and gene regulation in disease contexts.

What emerging research directions are utilizing β-hydroxybutyryl-HIST1H1D (K75) antibodies?

The study of histone β-hydroxybutyrylation represents a rapidly evolving field with several promising research directions that utilize β-hydroxybutyryl-HIST1H1D (K75) antibodies:

• Single-Cell Epigenomics:

  • Emerging Application: Integration of β-hydroxybutyryl-HIST1H1D (K75) antibodies into single-cell techniques to explore cell-to-cell variation in histone modifications.

  • Methodological Approach: Adaptation of CUT&Tag or CUT&RUN protocols for single-cell applications with these antibodies, allowing examination of β-hydroxybutyrylation patterns at single-cell resolution.

  • Research Potential: This approach could reveal how metabolic heterogeneity within tissues translates to epigenetic heterogeneity, with implications for understanding cellular subpopulations in disease states.

• Developmental Epigenetics:

  • Emerging Application: Investigation of β-hydroxybutyrylation dynamics during embryonic and fetal development.

  • Methodological Approach: Time-course studies of development combining ChIP-seq using these antibodies with RNA-seq and metabolic profiling.

  • Research Potential: This work could uncover how maternal metabolic state influences offspring epigenetic programming, potentially explaining metabolic imprinting phenomena observed in epidemiological studies.

• Neural Function and Metabolism:

  • Emerging Application: Study of β-hydroxybutyrylation in neural tissues, particularly relevant given the brain's utilization of ketone bodies.

  • Methodological Approach: Region-specific analysis of brain tissue using these antibodies combined with functional studies.

  • Research Potential: This research could illuminate how metabolic shifts affect neural function through epigenetic mechanisms, with relevance to neurological disorders and cognitive performance during ketosis.

• Aging and Longevity Research:

  • Emerging Application: Investigation of how age-related changes in metabolism impact histone β-hydroxybutyrylation patterns.

  • Methodological Approach: Longitudinal studies comparing young and aged tissues using ChIP-seq with these antibodies.

  • Research Potential: This work could reveal whether interventions that extend lifespan (such as caloric restriction) act in part through modulation of histone β-hydroxybutyrylation.

• Integration with Spatial Transcriptomics:

  • Emerging Application: Combining β-hydroxybutyryl-HIST1H1D (K75) immunostaining with spatial transcriptomics.

  • Methodological Approach: Multiplex imaging using these antibodies alongside RNA in situ hybridization techniques.

  • Research Potential: This integration could map how local metabolic environments within tissues create epigenetic microenvironments that influence gene expression patterns.

• Circadian Rhythm and Metabolic Cycling:

  • Emerging Application: Examination of how circadian metabolic cycles influence histone β-hydroxybutyrylation patterns.

  • Methodological Approach: Time-course sampling combined with ChIP-seq using these antibodies.

  • Research Potential: This research could elucidate how time-restricted feeding and other chronobiological interventions affect health through epigenetic mechanisms.

These emerging research directions highlight the versatility of β-hydroxybutyryl-HIST1H1D (K75) antibodies as tools for exploring the complex interplay between metabolism and epigenetic regulation across diverse biological contexts.

What are the key considerations for researchers beginning work with β-hydroxybutyryl-HIST1H1D (K75) antibodies?

Researchers initiating studies with β-hydroxybutyryl-HIST1H1D (K75) antibodies should consider several critical factors to ensure experimental success and meaningful data interpretation:

• Antibody Selection and Validation:

  • Choose antibodies that have been specifically validated for your application of interest (WB, ICC, ChIP).

  • Verify the antibody's specificity through preliminary experiments including peptide competition assays.

  • Consider the clonality - most available antibodies are polyclonal, which may provide better signal but potentially lower specificity than monoclonal alternatives .

• Experimental Design Considerations:

  • Include appropriate biological controls, particularly samples with elevated β-hydroxybutyrate levels (e.g., cells treated with sodium β-hydroxybutyrate).

  • Plan for time-course experiments when studying dynamic changes in β-hydroxybutyrylation.

  • Consider parallel assessment of other histone modifications (especially acetylation) to distinguish specific effects.

• Technical Protocol Optimization:

  • Optimize antibody dilutions for each application (typical ranges: 1:100-1:1000 for WB, 1:10-1:100 for ICC) .

  • Pay careful attention to sample preparation, particularly histone extraction methods.

  • Consider pre-treating samples with β-hydroxybutyrate to enhance signal detection when studying induction.

• Metabolic Context Awareness:

  • Monitor and report relevant metabolic parameters (e.g., glucose levels, β-hydroxybutyrate concentrations) alongside epigenetic data.

  • Consider the nutritional and metabolic state of experimental models, as fasting or ketosis will significantly impact β-hydroxybutyrylation levels .

  • Be aware that cell culture media composition may influence baseline β-hydroxybutyrylation levels.

• Data Integration Approaches:

  • Plan for multi-omics integration combining epigenomic data (from ChIP-seq) with transcriptomics and metabolomics.

  • Consider computational approaches for correlating β-hydroxybutyrylation patterns with gene expression changes.

  • Develop hypotheses that connect metabolic state, epigenetic modification, and functional outcomes.