Butyrly-HIST1H4A (K16) Antibody

What is the difference between butyrylation and acetylation at H4K16?

Histone H4K16 can undergo both acetylation and butyrylation, which are distinct post-translational modifications with potentially different functional consequences. Acetylation at H4K16 involves the addition of an acetyl group (2-carbon acyl chain) to the lysine residue, while butyrylation involves the addition of a butyryl group (4-carbon acyl chain). These modifications affect chromatin structure differently due to their distinct chemical properties. Acetylation at H4K16 is well-established to neutralize the positive charge of lysine, weakening histone-DNA and nucleosome-nucleosome interactions, thereby promoting chromatin accessibility and transcriptional activation . In contrast, butyrylation at H4K16, while similarly neutralizing the positive charge, may recruit different effector proteins or reader domains due to the longer acyl chain, potentially leading to distinct downstream effects on chromatin structure and gene regulation .

What are the recommended applications for Butyryl-HIST1H4A (K16) antibodies?

Butyryl-HIST1H4A (K16) antibodies have been validated for multiple research applications, with varying protocols and optimization requirements:

For optimal results, researchers should validate antibody specificity using appropriate positive controls, such as cells treated with sodium butyrate (30mM for 4 hours), which increases global histone butyrylation levels .

How can specificity of Butyryl-HIST1H4A (K16) antibody be verified?

Verifying antibody specificity is critical for reliable research findings. For Butyryl-HIST1H4A (K16) antibodies, several validation approaches are recommended:

• Peptide Competition Assay: Pre-incubate the antibody with synthetic butyrylated H4K16 peptides before application. Signal reduction confirms specificity for the modified epitope .

• Cross-reactivity Testing: Test against acetylated H4K16 peptides to ensure the antibody distinguishes between butyrylation and acetylation at the same residue. Commercial antibodies are typically raised against synthetic peptides corresponding to human histone H4 with the specific butyryl-K16 modification .

• Knockout/Knockdown Controls: Use cells with HDAC inhibition (increased butyrylation) versus cells with butyryl-transferase knockdown (decreased butyrylation) .

• Western Blot Analysis: Compare signal between sodium butyrate-treated cells (enhanced butyrylation) and untreated cells. As demonstrated in the technical data, HEK-293 and K562 cell lines show significantly increased H4K16bu signals after sodium butyrate treatment .

How do sample preparation methods affect Butyryl-HIST1H4A (K16) detection efficiency?

Sample preparation significantly impacts the detection of histone butyrylation at H4K16. Several critical factors must be considered:

Fixation Effects: For immunohistochemistry and immunofluorescence applications, overfixation with formaldehyde can mask epitopes and reduce antibody accessibility to the butyryl-K16 modification. Optimal fixation involves 4% paraformaldehyde for 10-15 minutes, followed by permeabilization with 0.1-0.5% Triton X-100 .

Extraction Methods: For western blot and ChIP applications, histone extraction methods significantly impact butyrylation detection. Acid extraction (e.g., 0.2N HCl) is preferable to detergent-based methods as it better preserves post-translational modifications. During extraction, adding deacetylase and debutyrylase inhibitors (such as sodium butyrate, trichostatin A, or nicotinamide) is crucial to prevent loss of the butyryl mark .

Chromatin Preparation: For ChIP analysis, chromatin fragmentation methodology affects epitope accessibility. Micrococcal nuclease digestion followed by mild sonication preserves the butyryl-K16 modification while generating appropriately sized chromatin fragments (200-500 bp) for immunoprecipitation. Complete solubilization of chromatin is essential for quantitative recovery of modified histones .

Butyrylation Preservation: Butyrylation is a dynamic modification that can be lost during sample preparation. Including 5-10 mM sodium butyrate in all buffers helps maintain butyrylation levels during experimental procedures .

What are the key differences in experimental design when studying Butyryl-HIST1H4A (K16) versus Acetyl-HIST1H4A (K16)?

When designing experiments to study butyrylation versus acetylation at H4K16, researchers must consider several critical differences:

Enzyme Modulators: Different enzymes regulate these modifications. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate acetylation, while specific acyltransferases like p300/CBP and class I HDACs may regulate butyrylation. Experiment designs should include appropriate enzyme inhibitors or activators specific to the modification being studied .

Metabolic Influences: Butyrylation is more directly linked to cellular metabolism, particularly to butyryl-CoA levels, which are influenced by fatty acid metabolism. Experimental designs should account for metabolic state and nutrients available to cells. When comparing acetylation versus butyrylation, standardized metabolic conditions are essential .

Antibody Selection: For comparative studies, antibody specificity is paramount. Validation should confirm that anti-butyryl-K16 antibodies do not cross-react with acetyl-K16, and vice versa. Sequential immunoprecipitation or dual labeling strategies with stringent controls are recommended for co-localization studies .

Functional Readouts: Different downstream effects may require different functional assays. While both modifications generally promote chromatin accessibility, they may recruit different reader proteins. Experiments should include reader domain binding assays or proteomics approaches to identify modification-specific interacting partners .

How can ChIP-seq protocols be optimized for Butyryl-HIST1H4A (K16) antibodies?

Optimizing ChIP-seq protocols for Butyryl-HIST1H4A (K16) requires several critical modifications to standard procedures:

Antibody Validation: Before ChIP-seq, validate antibody specificity using dot blots or western blots with synthetic peptides containing various histone modifications. This ensures the antibody specifically recognizes butyryl-K16 without cross-reactivity to acetyl-K16 or unmodified H4 .

Chromatin Preparation: Optimal chromatin fragmentation is crucial. Based on experimental data, a combination of micrococcal nuclease digestion (1000 gel units for 10 minutes at 37°C) followed by mild sonication (10 cycles of 30 seconds on/30 seconds off) provides ideal fragment sizes (200-400 bp) while preserving the butyryl modification .

Immunoprecipitation Conditions: For successful butyryl-K16 ChIP-seq:

• Use 5 μg of validated antibody per IP reaction

• Maintain 4°C throughout the IP procedure to minimize enzymatic removal of butyryl groups

• Include 5 mM sodium butyrate in all buffers to inhibit debutyrylase activity

• Extend incubation time to overnight for optimal antigen-antibody binding

• Implement stringent washing protocols (at least 5 washes) to reduce background

Library Preparation Considerations: ChIP-seq libraries from butyryl-K16 immunoprecipitations typically yield less material than those from more abundant modifications like H3K4me3. Therefore, optimize PCR cycles during library preparation to prevent over-amplification while ensuring sufficient material for sequencing. Input normalization is essential for accurate peak calling .

Data Analysis Parameters: For butyryl-K16 ChIP-seq data analysis, implement broad peak calling algorithms rather than narrow peak detection, as butyrylation patterns tend to span broader genomic regions compared to transcription factor binding sites .

What are common causes of false positives or negatives in Butyryl-HIST1H4A (K16) detection?

Several technical factors can lead to false results when working with Butyryl-HIST1H4A (K16) antibodies:

False Positives:

• Cross-reactivity with acetylation: Many butyryl-K16 antibodies may cross-react with acetyl-K16 due to structural similarities. Always perform specificity tests using competing peptides with different modifications .

• Non-specific binding: Secondary antibodies may bind non-specifically to endogenous immunoglobulins in the sample. Include isotype controls and perform blocking with appropriate reagents (5% BSA is often more effective than milk for phospho and acyl modifications) .

• Buffer contamination: Trace amounts of sodium butyrate or other HDAC inhibitors in buffers can artificially increase butyrylation signals. Prepare fresh buffers for control samples to avoid this issue .

False Negatives:

• Epitope masking: The butyryl modification may be masked by protein-protein interactions or adjacent modifications. Sample preparation with appropriate denaturing conditions can help expose the epitope .

• Modification instability: Butyrylation is dynamically regulated and can be lost during sample processing. Always include debutyrylase inhibitors in lysis and wash buffers .

• Insufficient sensitivity: Some applications require signal amplification methods. For difficult-to-detect samples, consider using higher antibody concentrations (1:50 dilution) or signal enhancement systems like tyramide signal amplification for immunohistochemistry .

How can researchers distinguish between site-specific and non-specific signals in ChIP experiments?

Distinguishing specific from non-specific ChIP signals requires rigorous experimental design and appropriate controls:

Essential Controls:

• IgG Control: Always perform parallel ChIP with matched isotype IgG to establish background signal levels. The signal-to-noise ratio should be at least 5-fold for confident peak calling .

• Peptide Competition: Pre-incubate the butyryl-K16 antibody with excess butyrylated peptide prior to ChIP. Specific signals should be substantially reduced or eliminated .

• Spike-in Controls: Include exogenous chromatin (e.g., Drosophila) with a species-specific antibody as an internal normalization control to account for technical variations between samples .

Validation Approaches:

• Sequential ChIP: For loci with suspected multiple modifications, perform sequential ChIP (first with butyryl-K16 antibody, then with antibodies against other modifications) to determine co-occurrence of marks .

• Genetic Validation: Compare ChIP signals between wild-type cells and cells with altered butyrylation (e.g., HDAC inhibitor treatment or butyryl-transferase knockdown). Specific signals should show expected changes corresponding to modification levels .

• Correlation with Functional Outcomes: Validate ChIP-seq findings by correlating butyryl-K16 peaks with gene expression data (RNA-seq) or chromatin accessibility (ATAC-seq). Genuine butyryl-K16 sites should correlate with expected functional outcomes (typically increased accessibility and expression) .

What approaches are recommended for multiplex detection of different histone modifications alongside Butyryl-HIST1H4A (K16)?

Investigating the interplay between butyrylation and other histone modifications requires sophisticated multiplex approaches:

Immunofluorescence Multiplex Strategies:

• Sequential Immunostaining: For co-detection of butyryl-K16 with other modifications, implement sequential staining with complete antibody stripping between rounds. This prevents cross-reactivity between antibodies. Effective stripping can be achieved with glycine-HCl buffer (pH 2.5) followed by re-blocking .

• Species-Distinct Primary Antibodies: Select primary antibodies raised in different host species (e.g., rabbit anti-butyryl-K16 paired with mouse anti-H3K27me3) to enable simultaneous detection with species-specific secondary antibodies .

• Spectral Unmixing: When utilizing fluorophores with overlapping emission spectra, employ spectral unmixing algorithms during image acquisition and processing to resolve distinct modification patterns .

Biochemical Multiplex Methods:

• Sequential ChIP (Re-ChIP): For analyzing co-occurrence of butyryl-K16 with other modifications on the same nucleosomes, implement sequential ChIP protocols. First, perform ChIP with butyryl-K16 antibody, then re-ChIP the eluted material with antibodies against other modifications .

• Mass Spectrometry Approaches: For unbiased detection of multiple modifications, combine antibody-based enrichment of butyrylated histones with mass spectrometry analysis. This enables identification of combinatorial modification patterns without antibody bias .

• CUT&RUN or CUT&Tag Multiplexing: These newer chromatin profiling techniques can be adapted for multiplex detection with lower input requirements than traditional ChIP. By using antibodies with different tags (e.g., protein A vs. protein G) or sequential epitope retrieval, multiple modifications can be profiled from the same sample .

What are the known genomic distribution patterns of Butyryl-HIST1H4A (K16) in comparison to Acetyl-HIST1H4A (K16)?

The genomic distribution of butyryl-H4K16 shows both overlapping and distinct patterns compared to acetyl-H4K16, reflecting their potentially different functional roles:

Comparative Distribution Patterns:

ChIP-seq experiments have revealed that while both modifications are generally associated with active chromatin, their precise distribution patterns differ. Butyryl-H4K16 tends to have broader distribution patterns and may be more specifically regulated in response to metabolic conditions, reflecting its connection to butyryl-CoA availability .

How does butyrylation at H4K16 impact chromatin structure and gene expression?

Butyrylation at H4K16 exerts multi-faceted effects on chromatin architecture and gene regulation through several mechanisms:

Structural Effects:

• Nucleosome Stability: The addition of the butyryl group (4-carbon acyl chain) to lysine 16 neutralizes the positive charge and adds steric bulk, destabilizing nucleosome structure more significantly than acetylation. This leads to decreased inter-nucleosomal interactions and reduced chromatin compaction .

• Linker DNA Accessibility: Butyrylation at H4K16 particularly affects the interaction between the H4 tail and linker DNA, potentially creating more accessible regions for transcription factor binding. This effect appears more pronounced than with acetylation due to the longer hydrocarbon chain of the butyryl group .

Regulatory Impacts:

• Transcriptional Enhancement: Genes marked by H4K16 butyrylation typically show higher expression levels. ChIP-seq analysis coupled with RNA-seq data demonstrates positive correlation between butyrylation levels and transcriptional activity, particularly for metabolism-related genes .

• Reader Protein Recruitment: Butyryl-H4K16 recruits a partially distinct set of reader proteins compared to acetyl-H4K16. Proteomic analyses have identified specific bromodomain-containing proteins that preferentially bind to butyrylated histones, potentially leading to distinct downstream effects .

• Metabolic Regulation: H4K16 butyrylation levels respond dynamically to cellular metabolic states, particularly to the availability of butyryl-CoA derived from fatty acid metabolism. This creates a potential mechanism for metabolism-epigenome crosstalk .

What is the role of Butyryl-HIST1H4A (K16) in disease states and therapeutic implications?

Emerging evidence suggests important roles for H4K16 butyrylation in various disease contexts:

Pathological Associations:

• Cancer Biology: Altered H4K16 butyrylation patterns have been observed in multiple cancer types. In colorectal cancer tissues, immunohistochemistry with butyryl-H4K16 antibodies reveals decreased global butyrylation compared to adjacent normal tissues, correlating with disease progression. This suggests potential tumor suppressor functions of this modification .

• Metabolic Disorders: As H4K16 butyrylation is linked to cellular metabolism, dysregulation occurs in metabolic diseases. Experimental models of diabetes show altered butyrylation patterns in hepatic tissue, potentially contributing to aberrant gene expression in glucose homeostasis pathways .

• Neurological Conditions: In neurodegenerative disease models, decreased H4K16 butyrylation has been observed in affected brain regions. This correlates with reduced expression of neuroprotective genes, suggesting potential roles in neurodegeneration .

Therapeutic Implications:

• HDAC Inhibitors: Class I HDACs remove butyryl groups from histones. HDAC inhibitors like sodium butyrate increase global levels of histone butyrylation, including at H4K16. This contributes to their therapeutic effects in cancer and inflammatory conditions .

• Metabolic Modulators: Compounds that increase cellular butyryl-CoA levels can enhance histone butyrylation. Butyrate-producing gut microbiota or dietary interventions that increase short-chain fatty acids may exert beneficial effects partly through increased histone butyrylation .

• Targeted Epigenetic Therapies: Understanding the specific reader proteins and enzymes that regulate H4K16 butyrylation opens possibilities for more targeted epigenetic therapies. Inhibitors specific to butyryl-lysine reader domains represent an emerging therapeutic approach .

What emerging technologies are enhancing the study of Butyryl-HIST1H4A (K16)?

Recent technological innovations are revolutionizing how researchers study histone butyrylation:

Single-Cell Approaches:

• Single-Cell CUT&Tag: This technique allows butyryl-H4K16 profiling at single-cell resolution, revealing cell-to-cell heterogeneity in butyrylation patterns that was previously masked in bulk analyses. The method requires optimization of antibody concentration and incubation conditions specifically for butyryl-K16 detection .

• Mass Cytometry (CyTOF): By using metal-conjugated antibodies against butyryl-H4K16, researchers can now quantify this modification alongside dozens of other cellular parameters at single-cell resolution. This enables correlation of butyrylation levels with cell state and identity markers .

Genome Engineering Tools:

• Locus-Specific Modification: CRISPR-based epigenetic editing systems using catalytically dead Cas9 fused to butyryl-transferase domains now enable targeted butyrylation at specific genomic loci. This allows causal determination of butyrylation effects on gene expression .

• Engineered Reader Domains: Synthetic proteins containing engineered reader domains specific for butyryl-lysine can be used to visualize or manipulate butyrylated chromatin regions in living cells .

Chemical Biology Approaches:

• Click Chemistry for Butyrylation Tracking: Azide-alkyne click chemistry with butyryl-azide analogs allows pulse-chase experiments to track newly added butyryl groups on histones, providing insights into butyrylation dynamics .

• Photo-crosslinking Probes: Butyryl-lysine analogs with photo-crosslinking capabilities enable covalent capture of proteins that interact with butyrylated histones, facilitating more comprehensive identification of reader proteins .

How do butyrylation and acetylation at H4K16 interact with other histone modifications?

Histone modifications function as an integrated system, with complex interactions between different marks:

Modification Crosstalk Patterns:

Regulatory Mechanisms:

• Sequential Modification: Certain modifications may serve as prerequisites for others. For example, H3K4 methylation may precede and facilitate H4K16 butyrylation at some genomic loci, creating an ordered modification pathway .

• Enzymatic Interactions: The enzymes that add or remove butyryl groups may be influenced by neighboring modifications. For instance, some histone butyryl-transferases show reduced activity on H3K9-methylated substrates .

• Reader Protein Competition: Reader proteins for different modifications may compete or cooperate for binding to modified nucleosomes. The presence of multiple modifications creates combinatorial binding patterns that fine-tune chromatin regulation .

What are the recommended experimental designs for studying butyrylation dynamics in response to metabolic changes?

Investigating the interplay between metabolism and histone butyrylation requires carefully designed experiments:

Metabolic Manipulation Strategies:

• Nutrient Modulation: Design experiments with precisely controlled media compositions, varying carbon sources (glucose, fatty acids) to alter cellular butyryl-CoA pools. Monitor changes in H4K16 butyrylation using validated antibodies in western blot or ChIP-seq assays .

• Isotope Tracing: Implement 13C-labeled butyrate or fatty acid precursors combined with mass spectrometry to track the flow of carbon atoms into histone butyrylation, establishing direct metabolic links .

• Enzymatic Perturbations: Manipulate enzymes in butyryl-CoA metabolism (e.g., acyl-CoA dehydrogenases) through genetic knockdown or chemical inhibition, then measure effects on histone butyrylation using antibody-based approaches .

Time-Course Experimental Design:
For optimal detection of butyrylation dynamics, implement time-course experiments with these parameters:

Integrated Multi-Omics Approach:
For comprehensive understanding, combine:

• ChIP-seq with butyryl-H4K16 antibodies

• RNA-seq for transcriptional outcomes

• Metabolomics for butyryl-CoA and related metabolites

• Proteomics for butyrylation-responsive chromatin proteins

This integrated approach provides correlative evidence linking metabolic state, histone butyrylation, and gene regulation .

What are the recommended validation standards for publications involving Butyryl-HIST1H4A (K16) antibodies?

For research involving butyryl-H4K16 antibodies to meet current publication standards, the following validation practices are essential:

Antibody Validation Requirements:

• Specificity Testing: Demonstrate specificity using peptide competition assays with both butyrylated and acetylated H4K16 peptides. Include dot blot or western blot data showing selective recognition of the butyrylated epitope .

• Knockout/Knockdown Controls: Include appropriate genetic controls, such as cells with knockdown of butyryl-transferase enzymes, showing reduced signal. Alternatively, use cells treated with HDAC inhibitors to increase signal .

• Cross-Platform Validation: Confirm findings using at least two independent techniques (e.g., western blot plus ChIP-qPCR or immunofluorescence) to strengthen confidence in antibody specificity .

• Lot-to-Lot Consistency: When using commercial antibodies across extended studies, document lot numbers and perform consistency checks between lots to ensure reproducibility .

Experimental Design Standards:

• Appropriate Controls: Include IgG controls for all IP-based assays, untreated versus treated samples, and competitor peptide controls in all experiments .

• Quantification Methods: Employ quantitative methods with statistical analysis rather than showing only representative images. For western blots, include densitometry from multiple biological replicates .

• Biological Relevance: Demonstrate the biological significance of butyryl-H4K16 findings by correlating with functional readouts such as gene expression, chromatin accessibility, or phenotypic outcomes .

How should researchers integrate butyrylation data with other epigenomic and transcriptomic datasets?

Effective integration of butyrylation data with other datasets requires specific analytical approaches:

Data Integration Strategies:

• Common Reference Genome: Ensure all datasets are aligned to the same reference genome build to enable accurate integration. For human studies, GRCh38/hg38 is currently recommended .

• Normalization Procedures: Implement appropriate normalization methods before integration, such as quantile normalization for ChIP-seq data or rlog transformation for RNA-seq, to make datasets comparable .

• Peak Overlap Analysis: For integrating butyryl-H4K16 ChIP-seq with other histone modification data, use tools like BEDTools or DiffBind to identify regions of overlap or mutual exclusion .

• Correlation Analysis: Calculate genome-wide correlation coefficients (Pearson or Spearman) between butyrylation signals and other epigenetic marks or expression levels to identify relationships .

Visualization and Interpretation:

• Genome Browser Visualization: Generate browser tracks displaying butyryl-H4K16 alongside other modifications at specific loci of interest. Include input-normalized signal tracks rather than simply peak calls .

• Heatmap Clustering: Create heatmaps of butyrylation patterns centered on genomic features (TSS, enhancers) clustered with other modifications to identify co-occurrence patterns .

• Network Analysis: Construct gene regulatory networks incorporating butyrylation data with transcription factor binding and expression data to identify regulatory circuits affected by this modification .

What future research directions should be prioritized for understanding Butyryl-HIST1H4A (K16) function?

Several promising research avenues should be prioritized to advance understanding of butyryl-H4K16 function:

Mechanistic Investigations:

• Reader Protein Identification: Comprehensive identification of proteins that specifically recognize butyryl-H4K16 using proteomics approaches such as SILAC-based quantitative mass spectrometry with synthetic butyrylated nucleosomes as bait .

• Regulatory Enzyme Characterization: Identification and characterization of enzymes specifically responsible for adding (writers) and removing (erasers) butyryl groups at H4K16, including determination of their substrate preferences and regulation .

• Structural Biology: Crystal or cryo-EM structures of nucleosomes containing butyryl-H4K16 to determine precise structural impacts on chromatin fiber organization compared to acetylation .

Physiological and Disease Relevance:

• Tissue-Specific Profiling: Comprehensive mapping of butyryl-H4K16 across different tissues under various physiological and pathological conditions using validated antibodies to identify tissue-specific regulation .

• Metabolic Disease Models: Investigation of butyryl-H4K16 dynamics in metabolic disease models, particularly focusing on tissues involved in metabolic regulation (liver, adipose tissue, pancreas) .

• Therapeutic Targeting: Development of small molecules that can specifically modulate butyryl-H4K16 levels or its reader protein interactions for potential therapeutic applications .

Technological Developments:

• Improved Antibodies: Development of higher-specificity monoclonal antibodies against butyryl-H4K16 with reduced cross-reactivity to acetyl-K16 .

• Single-Molecule Approaches: Application of single-molecule imaging techniques to visualize butyrylation dynamics in living cells in real-time .

• CRISPR-Based Locus-Specific Modulation: Refinement of CRISPR-based systems for locus-specific manipulation of butyrylation to establish causative relationships between this modification and gene regulation .