Butyrly-HIST1H2BC (K5) Antibody

What is Butyryl-HIST1H2BC (K5) and what is its biological significance?

Butyryl-HIST1H2BC (K5) refers to histone H2B type 1-C/E/F/G/I with butyrylation at the lysine 5 position. This post-translational modification occurs on histone H2B, which is a core component of nucleosomes that wrap and compact DNA into chromatin. H2B plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability by controlling DNA accessibility to cellular machineries . Butyrylation at K5 represents one of many modifications in the "histone code" that regulates chromatin structure and function, thereby influencing gene expression patterns . Recent research has revealed that histone butyrylation in the intestine is mediated by the microbiota and is associated with regulation of gene expression, suggesting important connections between gut microbes, epigenetic modifications, and host physiology .

How does H2B butyrylation differ from other histone modifications?

Histone butyrylation represents a distinct acyl modification involving the addition of a butyryl group (four-carbon chain) to lysine residues, compared to shorter modifications like acetylation (two-carbon chain) or longer modifications like crotonylation. While acetylation is primarily associated with transcriptional activation, butyrylation appears to have more complex and context-dependent effects on gene regulation . Interestingly, unlike the generally activating role of many histone acyl marks, genes affected by butyrylation are frequently downregulated, suggesting unique regulatory mechanisms . Different structural domains vary in their abilities to recognize lysine acylations; compared to bromodomains, YEATS and double PHD finger domains can better accommodate longer acyl side chains like butyrylation . This creates distinct recognition patterns and downstream effects compared to other modifications.

What are the common synonyms and designations for Butyryl-HIST1H2BC (K5)?

The Butyryl-HIST1H2BC (K5) antibody targets histone H2B butyrylated at lysine 5, which is alternatively designated by multiple synonyms in scientific literature and databases. These include: H2BK5bu, H2BC4, H2BFL, HIST1H2BC, H2BC6, H2BFH, HIST1H2BE, H2BC7, H2BFG, HIST1H2BF, H2BC8, H2BFA, HIST1H2BG, H2BC10, H2BFK, and HIST1H2BI . The protein may also be referred to as Histone H2B type 1-C/E/F/G/I, Histone H2B.1 A, Histone H2B.a, Histone H2B.g, Histone H2B.h, Histone H2B.k, Histone H2B.l, H2B/a, H2B/g, H2B/h, H2B/k, or H2B/l . When searching literature or databases, researchers should consider these alternative designations to ensure comprehensive results.

What experimental techniques can be used with Butyryl-HIST1H2BC (K5) antibody?

The Butyryl-HIST1H2BC (K5) polyclonal antibody is versatile and can be employed in multiple experimental techniques:

• Western Blotting (WB): Effective for detecting butyrylated H2B K5 in whole cell lysates, with demonstrated results in human cell lines including HeLa, HEK-293, A549, and HepG2 .

• Immunoprecipitation (IP): Can successfully pull down butyrylated H2B K5 from cell lysates, as demonstrated in HEK-293 cells .

• Chromatin Immunoprecipitation (ChIP): Suitable for investigating genomic localization of H2B K5 butyrylation, with validated protocols using micrococcal nuclease treatment and sonication of cells .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Effective for visualizing cellular localization of butyrylated H2B K5, with optimal results in fixed and permeabilized cells .

• ELISA: Can be used for quantitative detection of butyrylated H2B K5 in appropriate sample preparations .

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods based on the experimental context and sample type.

How should sample preparation be optimized for butyrylation analysis?

Optimal sample preparation for butyrylation analysis requires careful consideration of several factors:

For histone extraction and preservation of butyrylation marks:

• Use fresh samples whenever possible to minimize degradation of labile modifications.

• Include deacetylase inhibitors (e.g., sodium butyrate at 30mM) during sample preparation to preserve butyrylation marks. Treatment of cells with sodium butyrate for 4 hours significantly enhances detection of H2B K5 butyrylation in multiple cell lines .

• For mass spectrometry analysis, utilize chemical derivatization techniques with propionic anhydride to protect unmodified lysines and differentiate endogenous from artificial modifications .

For cell fixation in immunofluorescence:

• Fix cells in 4% formaldehyde.

• Permeabilize using 0.2% Triton X-100.

• Block in 10% normal goat serum before antibody incubation .

For chromatin immunoprecipitation:

• Treat cells with micrococcal nuclease.

• Sonicate to shear chromatin.

• Use 5 μg of antibody per immunoprecipitation with appropriate controls (e.g., normal rabbit IgG) .

These optimized protocols ensure maximal preservation and detection of butyrylation marks while minimizing background and non-specific signals.

What controls should be included when using Butyryl-HIST1H2BC (K5) antibody?

Rigorous experimental design for Butyryl-HIST1H2BC (K5) antibody applications should include the following controls:

• Positive controls:

  • Cell lysates treated with sodium butyrate (30mM for 4 hours), which enhances butyrylation levels, as demonstrated in HeLa, HEK-293, A549, and HepG2 cell lines .

  • Commercially available synthetic butyrylated H3K27bu nucleosomes for calibration curves in mass spectrometry experiments .

• Negative controls:

  • Untreated cell lysates showing baseline or reduced butyrylation levels .

  • Primary antibody omission controls for immunocytochemistry.

  • Normal rabbit IgG as an isotype control for immunoprecipitation and ChIP experiments .

• Specificity controls:

  • Peptide competition assays using the specific immunogen peptide.

  • Comparison with other histone modifications using antibodies against acetylation, propionylation, or unmodified H2B to ensure specificity .

• Quantitative controls:

  • For mass spectrometry, include titration curves of known quantities of acylated mono-nucleosomes (e.g., H3K9ac, H3K9bu, H3K27ac, H3K27bu) to calibrate quantification .

These controls ensure proper interpretation of results and help distinguish specific signals from experimental artifacts.

How does histone H2B butyrylation relate to transcriptional regulation?

The relationship between histone H2B butyrylation and transcriptional regulation is complex and context-dependent:

Paradoxical effects on gene expression:

• While many histone acyl marks activate transcription, genes affected by histone butyrylation are frequently downregulated, as demonstrated in mouse intestinal epithelium studies with tributyrin treatment .

• This contradicts the observation that H3K27bu levels positively correlate with gene expression, suggesting nuanced regulatory mechanisms .

Transcription elongation dynamics:

• H2B modifications impact RNA polymerase II transcription elongation. For instance, ubiquitylation of H2B controls RNA polymerase II transcription elongation by altering nucleosome assembly and chromatin dynamics .

• Similarly, butyrylation may affect chromatin accessibility during transcription, potentially influencing the distribution of RNA polymerase II and histones in gene coding regions .

Mechanistic considerations:

• The functional role of H2B butyrylation likely involves specific reader proteins—structural domains like YEATS and double PHD finger domains that can accommodate the longer butyryl side chains .

• Chromatin immunoprecipitation sequencing (ChIP-seq) data indicates that H3K27bu peaks in mouse cecal epithelial cells are enriched in genes related to oxidative stress and cellular adaptations, suggesting butyrylation may participate in stress response mechanisms .

These findings highlight the need for further investigation into the precise mechanisms by which butyrylation influences gene expression patterns in different cellular contexts.

What is known about the interplay between intestinal microbiota and histone butyrylation?

Recent research has revealed fascinating connections between intestinal microbiota and histone butyrylation:

Microbiota as a source of butyryl groups:

• Intestinal microbiota, particularly butyrate-producing bacteria, generate short-chain fatty acids including butyrate that can serve as substrates for histone butyrylation .

• Tributyrin treatment in mice can rescue gene expression patterns altered by microbiota depletion, demonstrating the functional significance of this relationship .

Physiological significance:

• Histone butyrylation mediated by microbiota appears to regulate gene expression in the intestinal epithelium, potentially affecting intestinal homeostasis and function .

• Butyrate and tributyrin treatment reduce oxidative stress, consistent with the enrichment of H3K27bu peaks in genes related to oxidative stress response .

Experimental approaches:

• Mass spectrometry analysis of histone post-translational modifications can be used to quantify butyrylation levels in intestinal epithelial cells under different microbiota conditions .

• ChIP-seq experiments help identify genomic regions enriched for histone butyrylation and correlate these with gene expression changes .

This emerging field represents an exciting frontier linking diet, microbiome, epigenetics, and host physiology, with potential implications for understanding inflammatory bowel diseases, metabolic disorders, and other conditions affected by microbiota dysbiosis.

How can mass spectrometry be optimized for detection of histone butyrylation?

Mass spectrometry (MS) approaches for histone butyrylation analysis require careful optimization:

Sample preparation procedures:

• Derivatize histones with propionic anhydride to protect unmodified lysines from trypsin cleavage .

• Use isotopically labeled propionic anhydride (D10, 98%) to differentiate endogenous (light) from artificial (heavy) histone propionylation .

• Mix 1 volume of 25% propionic anhydride in 2-propanol with 2 volumes of 0.1M ammonium bicarbonate containing histone extracts .

• Allow reaction to proceed for 15 minutes at 37°C, then dry samples in a speed-vac .

• Repeat derivatization once more before trypsin digestion .

• Digest with trypsin (1 μg per 20 μg histones) overnight at room temperature .

• Perform two additional rounds of derivatization to propionylate free N-termini .

• Desalt peptides with C18 stage tips before MS analysis .

Mass spectrometry analysis parameters:

• Consider specific peptide modifications including:

  • Artificial D5-propionyl (+61.057598 to K and N-termini)

  • Endogenous propionyl (+56.026215 to K)

  • Butyryl (+70.041865 to K)

  • Acetyl (+42.010565 to K)

• For quantification, construct titration curves using:

  • Unmodified nucleosomes as baseline

  • Acylated mono-nucleosomes (H3K9ac, H3K9bu, H3K27ac, H3K27bu) for standards

  • Create dilution series spanning several orders of magnitude (e.g., 3.3E-1 to 2E-7)

This detailed protocol enables reliable detection and quantification of histone butyrylation marks, facilitating comparative studies across different experimental conditions.

What role does Butyryl-HIST1H2BC (K5) play in cellular stress responses?

Emerging evidence suggests that histone H2B butyrylation at K5 may be involved in cellular stress response mechanisms:

Oxidative stress connection:

• ChIP-seq analysis of H3K27bu peaks in mouse cecal epithelial cells revealed enrichment in genes related to oxidative stress response and cellular adaptations .

• This aligns with observations that butyrate and tributyrin treatments can reduce oxidative stress in various experimental models .

Antimicrobial functions:

• Histone H2B has documented broad antibacterial activity and may contribute to the formation of functional antimicrobial barriers in the colonic epithelium .

• Butyrylation could potentially modulate this activity, representing a mechanism by which microbiota-derived butyrate influences host defense.

Stress-induced gene regulation:

• The association between butyrylation and gene downregulation suggests that this modification may participate in reprogramming gene expression during stress conditions .

• This could represent an adaptive mechanism to conserve cellular resources or activate specific stress response pathways.

Research studying the dynamics of butyrylation in response to various stressors (oxidative, inflammatory, metabolic) could provide valuable insights into this epigenetic regulation of stress responses and potential therapeutic approaches targeting these pathways.

How does H2B butyrylation interact with other histone modifications?

Histone modifications operate within a complex, interconnected network often referred to as the "histone code." H2B butyrylation interactions include:

Cross-talk with other modifications:

• Histone H2B ubiquitylation leads to methylation of histone H3 on specific lysine residues, establishing a conserved modification pathway .

• Similar cross-talk mechanisms may exist between butyrylation and other modifications, creating sequential or cooperative regulatory effects.

Functional divergence:

• Studies in fission yeast (Schizosaccharomyces pombe) have demonstrated significant functional divergence between ubiquitylation of H2B and methylation of Lys 4 on histone H3 .

• Similarly, butyrylation likely has distinct functions that may complement or antagonize other modifications in context-specific ways.

Chromatin distribution patterns:

• Different modifications show specific distribution patterns across gene bodies. For example, H3 distribution becomes skewed toward the 5' end in htb1-K119R mutants defective in H2B ubiquitylation .

• Understanding the genomic distribution of butyrylation in relation to other modifications will help elucidate its specific role in chromatin dynamics.

Methodological approaches for studying these interactions include sequential ChIP (re-ChIP), proteomics analysis of differently modified histones, and genetic studies manipulating enzymes responsible for various modifications to observe functional consequences.

What are the current technical challenges in studying histone butyrylation?

Researchers face several technical challenges when investigating histone butyrylation:

Antibody specificity issues:

• Ensuring antibody specificity among similar acyl modifications (acetylation, propionylation, butyrylation, crotonylation) requires rigorous validation .

• Cross-reactivity between modifications with similar chemical structures must be carefully assessed using competition assays and known standards.

Mass spectrometry limitations:

• Distinguishing between isomeric modifications that have the same mass but different structures can be challenging.

• Low abundance of butyrylation marks relative to more common modifications like acetylation creates detection sensitivity issues .

Functional characterization difficulties:

• The paradoxical relationship between butyrylation and gene expression (correlation with expression yet often associated with downregulation) complicates functional interpretation .

• Identifying specific "reader" proteins that recognize butyrylation and mediate its effects remains an ongoing challenge.

Temporal dynamics:

• Traditional RNA-seq and ChIP-seq provide static snapshots rather than dynamic information about the temporal sequence of butyrylation and transcriptional changes .

• Developing methods to track these modifications in real-time would advance understanding of their causal relationships.

Addressing these challenges requires multidisciplinary approaches combining advances in antibody development, mass spectrometry techniques, genetic engineering, and computational biology to fully unravel the complexities of histone butyrylation.

How can Butyryl-HIST1H2BC (K5) antibody be used to study disease mechanisms?

The Butyryl-HIST1H2BC (K5) antibody provides valuable tools for investigating disease mechanisms across several contexts:

Cancer research applications:

• Western blotting and immunofluorescence can be used to compare butyrylation levels between normal and cancer cell lines (HeLa, A549, HepG2) .

• ChIP-seq approaches can map genome-wide butyrylation differences in tumors versus normal tissues, potentially identifying dysregulated pathways.

Intestinal and inflammatory diseases:

• Given the connection between microbiota, butyrate production, and histone butyrylation, this antibody can help investigate epigenetic mechanisms in inflammatory bowel diseases .

• Immunohistochemistry of intestinal biopsies could reveal altered butyrylation patterns in diseased versus healthy tissues.

Metabolic disorders:

• As butyrate and tributyrin treatments affect oxidative stress responses, investigating butyrylation in metabolic disease models could reveal novel therapeutic targets .

• Correlation between metabolic parameters and histone butyrylation could establish biomarkers for disease progression or treatment response.

Neurodegenerative diseases:

• Histone modifications play critical roles in neuronal gene expression; butyrylation studies might reveal new mechanisms in conditions like Alzheimer's or Parkinson's disease.

These applications require careful experimental design with appropriate disease models, controls, and integration with other molecular and physiological data to establish meaningful connections to disease pathogenesis.

What are the emerging therapeutic implications of modulating histone butyrylation?

Modulating histone butyrylation represents an emerging area with potential therapeutic applications:

Microbiome-based interventions:

• Prebiotics that promote growth of butyrate-producing bacteria might enhance histone butyrylation and associated beneficial gene expression patterns .

• Probiotics containing butyrate-producing bacterial strains could directly influence the epigenetic landscape of intestinal epithelial cells.

Direct butyrate supplementation:

• Tributyrin treatment in mice rescues gene expression patterns altered by microbiota depletion, suggesting therapeutic potential .

• Various butyrate delivery systems could target specific tissues to modulate local histone butyrylation patterns.

Enzyme modulation:

• Identifying and targeting enzymes responsible for adding (writers) or removing (erasers) butyryl groups from histones could provide precise control over this modification.

• Inhibitors or activators of these enzymes might represent novel drug candidates for conditions with dysregulated histone butyrylation.

Reader protein targeting:

• Developing compounds that specifically interact with reader proteins for butyrylated histones could modulate downstream effects without altering the modification itself.

• YEATS and double PHD finger domains that recognize butyrylation represent potential therapeutic targets .

Research in this area is still developing, but the connections between butyrylation, gene expression, and cellular stress responses suggest promising therapeutic avenues worth further investigation.

What technological advances are needed to further our understanding of histone butyrylation?

Several technological advances would significantly enhance our understanding of histone butyrylation:

Improved antibody technologies:

• Development of monoclonal antibodies with even higher specificity for butyrylation versus other acyl modifications.

• Site-specific antibodies for different butyrylated residues beyond K5 to map the complete "butyrome."

Advanced imaging techniques:

• Super-resolution microscopy methods to visualize butyrylation patterns within nuclear architecture.

• Live-cell imaging approaches to track dynamic changes in butyrylation in response to various stimuli.

Enhanced mass spectrometry:

• More sensitive detection methods for low-abundance butyrylation marks.

• Techniques to distinguish between isomeric modifications with identical mass.

• Improvements in quantitative proteomics for precise measurement of butyrylation stoichiometry .

Functional genomics tools:

• CRISPR-based systems to precisely modulate butyrylation at specific genomic loci.

• High-throughput screening methods to identify enzymes and regulatory factors involved in butyrylation.

Single-cell technologies:

• Single-cell epigenomics approaches to capture cell-to-cell variability in butyrylation patterns.

• Integration of single-cell transcriptomics with epigenomic data to correlate butyrylation with gene expression at single-cell resolution.

These technological advances would address current limitations and open new avenues for understanding the complex roles of histone butyrylation in cellular function and disease.