Butyrly-HIST1H2BC (K20) Antibody

What is the biological significance of histone H2B butyrylation?

Histone H2B butyrylation represents a post-translational modification that contributes to chromatin structure regulation. As a core component of nucleosomes, histone H2B helps wrap and compact DNA into chromatin, which limits DNA accessibility to cellular machineries. Butyrylation of specific lysine residues in H2B modifies this DNA-histone interaction, thereby influencing transcription regulation, DNA repair, DNA replication, and chromosomal stability. This modification is part of the broader "histone code" that dynamically regulates DNA accessibility through complex post-translational modifications and nucleosome remodeling .

How does butyryl-lysine incorporation differ from other histone modifications?

Butyrylation represents one of several acylation modifications that can occur on histones, distinguishing itself from more common modifications like acetylation by its longer four-carbon chain. Researchers have successfully incorporated butyryl-lysine into histones using specifically developed synthetases that charge tRNAs with this modified amino acid. This approach allows for the production of custom histones with precise modifications. The butyryl modification, compared to acetyl groups, creates different electronic and steric properties that can potentially recruit distinct reader proteins or affect chromatin structure differently .

What detection methods are most suitable for studying histone H2B butyrylation?

Based on experimental validations, several techniques have proven effective for butyrylated histone detection. Western blotting (WB), immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and immunocytochemistry/immunofluorescence (ICC/IF) have all been successfully employed using specific antibodies against butyrylated H2B. For example, anti-Histone H2B (butyryl K5) antibody has been validated for all these applications with human samples. When performing western blot analysis, researchers typically observe bands at approximately 14 kDa, corresponding to the predicted size of histone H2B .

How can ChIP-seq be optimized for butyrylated histone H2B detection?

Optimizing ChIP-seq for butyrylated histone H2B requires careful consideration of several methodological factors. Based on established protocols, cells should be treated with appropriate stimuli to enhance butyrylation signals - sodium butyrate (30 mM for 4 hours) has been demonstrated to effectively increase butyrylation levels in multiple cell lines including HeLa, HEK-293, A549, and HepG2. For chromatin preparation, treat approximately 10^6 cells with Micrococcal Nuclease followed by sonication to generate appropriately sized chromatin fragments. Immunoprecipitation should be performed with 5 μg of specific anti-butyryl antibody per sample, alongside a control normal rabbit IgG. The resulting ChIP DNA can be quantified using real-time PCR with appropriate primers before proceeding to sequencing. When analyzing the data, compare enrichment patterns with other histone modifications to identify unique genomic distribution patterns of butyrylation .

What are the structural mechanisms by which reader proteins recognize butyrylated histones?

Reader proteins recognize butyrylated histones through specialized structural domains, particularly bromodomains (BRDs). These domains contain a hydrophobic pocket that accommodates acylated lysine residues. While bromodomains primarily evolved to recognize acetylated lysines, certain BRD variants can also bind butyrylated lysines. Structural studies have identified key features that determine specificity: (1) a hydrophobic binding pocket formed by aromatic residues, (2) specific side chain interactions between the histone tail and residues from the reader protein, and (3) recognition of amino acids adjacent to the modified lysine. For example, researchers have discovered a gain-of-function mutation in BRD1 that conferred affinity for butyryl marks without losing the intrinsic acetyl binding capacity. This structural flexibility allows certain reader domains to accommodate the larger butyryl group while maintaining specificity for the histone context .

How can synthetic biology approaches be used to study the functional consequences of histone butyrylation?

Synthetic biology offers powerful strategies to investigate histone butyrylation functions. Researchers have successfully employed recoded mRNA translation systems to produce pre-modified histones in mammalian cells. By replacing specific lysine codons with amber stop codons and introducing synthetases that charge tRNAs with butyryl-lysine, researchers can generate histones with site-specific butyrylation. This approach enables precise control over the modification state of histones expressed in cells. Additionally, engineered fusion proteins combining butyryl-lysine reader domains (such as adapted bromodomains) with effector domains (activators or repressors) can be used to target specific genomic loci marked by butyrylation. These synthetic systems allow researchers to distinguish the direct effects of butyrylation from secondary consequences, providing mechanistic insights into how this modification regulates gene expression .

What controls are essential when validating butyryl-specific antibodies?

Validating butyryl-specific antibodies requires rigorous controls to ensure specificity and minimize false-positive results. Essential controls include: (1) Treatment/non-treatment comparisons - testing antibody reactivity in cells treated with sodium butyrate (30 mM for 4 hours) versus untreated cells, as demonstrated in multiple cell lines including HeLa, HEK-293, A549, and HepG2; (2) Peptide competition assays - pre-incubating the antibody with synthetic butyrylated peptides to confirm epitope specificity; (3) Cross-reactivity assessment - testing against other acylated histone modifications (acetylation, propionylation, crotonylation) to ensure modification-specificity; and (4) Immunoprecipitation validation - performing IP followed by western blot analysis with alternative detection methods. Western blot results should show enrichment of the target modification in treated samples compared to untreated controls, with bands at the expected molecular weight (approximately 14 kDa for histone H2B) .

How can researchers address technical challenges when analyzing histone butyrylation in different cell types?

Different cell types present varied challenges for histone butyrylation analysis. Based on experimental data from multiple cell lines (HeLa, HEK-293, A549, HepG2), researchers should consider these technical approaches: (1) Optimization of butyrylation induction - while 30 mM sodium butyrate for 4 hours works efficiently across multiple cell lines, some cell types may require adjusted treatment durations or concentrations; (2) Extraction protocol modifications - different cell types may require adjusted lysis conditions to efficiently extract histones while preserving butyrylation; (3) Background reduction - for immunofluorescence analysis, optimize fixation (4% formaldehyde) and permeabilization (0.2% Triton X-100) protocols, followed by blocking with 10% normal goat serum to minimize non-specific binding; and (4) Signal amplification strategies - for cells with lower butyrylation levels, consider using more sensitive detection systems such as tyramide signal amplification or proximity ligation assays. These approaches should be systematically tested and validated for each new cell type under investigation .

What are the similarities and differences between various reader domains that recognize histone acylations?

The table above compares different reader domains that recognize various histone modifications. While bromodomains predominantly recognize acetylated and sometimes butyrylated lysines through a hydrophobic pocket within a 4-helix bundle structure, other domains have evolved to recognize different modifications. For example, chromodomains contain an aromatic cage that specifically accommodates methylated lysines. The structural differences between these domains determine their specificity and affinity for different modifications. Importantly, bromodomains exhibit a degree of promiscuity that allows some variants to recognize larger acyl modifications like butyrylation, although typically with different binding affinities compared to acetylation .

How might synthetic biology approaches advance our understanding of histone butyrylation?

Emerging synthetic biology techniques hold significant promise for deciphering histone butyrylation functions. Recent advances in genetic code expansion have enabled site-specific incorporation of butyryl-lysine into histones in living cells. Researchers have successfully used synthetases that charge tRNAs with butyryl-lysine to incorporate this modification at specific positions within histones. This approach can be further developed to create designer chromatin with precisely positioned butyrylation marks, allowing researchers to dissect the causal relationships between specific butyrylation events and downstream effects. Additionally, engineered reader domains with enhanced specificity for butyrylated lysines could be fused to various effector domains to manipulate gene expression at butyrylated loci. The development of light-inducible or chemically-inducible butyrylation systems would further enable temporal control over this modification, providing insights into the dynamics of butyrylation-mediated processes .

What technological developments might enhance detection and functional analysis of histone butyrylation?

Several technological frontiers could significantly advance histone butyrylation research. Mass spectrometry methods optimized for acyl-modification detection would enable more comprehensive mapping of butyrylation sites across the genome and improve quantification of butyrylation stoichiometry. Development of antibodies with enhanced specificity for butyrylated histones at different lysine residues would expand the researcher's toolkit for studying this modification. Single-molecule imaging techniques adapted for tracking butyrylation dynamics in living cells could reveal temporal aspects of this modification during cellular processes. CRISPR-based epigenome editing tools specifically designed to add or remove butyrylation marks at targeted genomic loci would allow for precise functional analysis. Integration of these approaches with computational modeling of chromatin structural dynamics could further elucidate how butyrylation alters nucleosome properties and affects DNA accessibility .

What are the critical experimental design considerations when studying histone butyrylation?

When designing experiments to investigate histone butyrylation, researchers should implement several critical practices: (1) Include appropriate positive controls, such as sodium butyrate treatment (30 mM for 4 hours), which has been validated to increase butyrylation levels in multiple cell lines; (2) Employ stringent antibody validation using peptide competition assays and cross-reactivity tests; (3) Utilize multiple complementary detection techniques (Western blot, ChIP, immunofluorescence) to corroborate findings; (4) Include parallel analysis of related histone modifications (acetylation, propionylation) to establish modification-specific effects; and (5) Validate key findings using orthogonal approaches such as mass spectrometry. Following these experimental principles will enhance the reliability and reproducibility of histone butyrylation research, advancing our understanding of this important epigenetic modification .