Butyrly-HIST1H3A (K122) Antibody

What is HIST1H3A and what role does K122 butyrylation play in chromatin regulation?

HIST1H3A (Histone H3.1) is a core component of nucleosomes, the fundamental units of chromatin packaging in eukaryotic cells. Nucleosomes wrap and compact DNA, significantly influencing DNA accessibility to cellular machinery involved in transcription, replication, and repair . Butyrylation at lysine 122 (K122) represents a specific post-translational modification within the "histone code" that regulates chromatin structure and function. This modification occurs near the C-terminal region of the histone and likely affects nucleosome stability and DNA-histone interactions, potentially facilitating transcriptional activation by loosening histone-DNA contacts at this critical position .

How do antibodies against Butyryl-HIST1H3A (K122) differ from antibodies targeting other histone modifications?

Antibodies against Butyryl-HIST1H3A (K122) are specifically designed to recognize the butyryl modification at the K122 position, distinguishing it from similar modifications (such as acetylation or formylation) at the same position, as well as from identical modifications at different lysine residues (such as K9 butyrylation) . These antibodies typically employ unique epitopes surrounding the K122 position to ensure specificity. Unlike antibodies targeting more common modifications like acetylation or methylation, butyrylation-specific antibodies may require additional validation steps to confirm their specificity against closely related acylations such as β-hydroxybutyrylation, which can also occur at K122 .

What are the recommended applications for Butyryl-HIST1H3A (K122) antibodies?

Butyryl-HIST1H3A (K122) antibodies are validated for multiple research applications including Western blotting (WB), immunocytochemistry (ICC), immunohistochemistry (IHC), and chromatin immunoprecipitation (ChIP) . In Western blotting, these antibodies typically detect a band of approximately 15-17 kDa corresponding to histone H3.1 . For immunostaining applications, recommended dilutions generally range from 1:100 to 1:1000 for Western blotting and 1:10 to 1:100 for ICC, though optimal concentrations should be determined experimentally for each specific application and tissue type .

How should Butyryl-HIST1H3A (K122) antibodies be stored and handled?

For optimal performance and shelf life, Butyryl-HIST1H3A (K122) antibodies should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided by preparing small aliquots for single use. Most commercial preparations are supplied in buffer containing preservatives (such as 0.03% Proclin 300) and stabilizers (such as 50% glycerol in PBS, pH 7.4) . When working with these antibodies, centrifugation is recommended if the solution is not completely clear after standing at room temperature. For long-term storage, maintaining aliquots at -20°C or below is essential for preserving antibody activity.

What controls should be included when using Butyryl-HIST1H3A (K122) antibodies for ChIP experiments?

When performing ChIP experiments with Butyryl-HIST1H3A (K122) antibodies, several controls are essential for result validation. First, input controls (non-immunoprecipitated chromatin) should be included to normalize for variations in starting chromatin amounts. Second, IgG negative controls using normal rabbit IgG should be performed in parallel to assess non-specific binding . Third, positive controls targeting known butyrylated regions (such as the β-Globin promoter) can verify immunoprecipitation efficiency . For additional specificity validation, comparing ChIP results between untreated cells and cells treated with histone deacetylase inhibitors or butyryl-CoA donors (such as sodium butyrate) can demonstrate enrichment of butyrylation-specific signals .

How can researchers induce and enhance histone butyrylation for experimental detection?

To enhance histone butyrylation for experimental detection, researchers commonly treat cells with sodium butyrate (typically at 30mM concentration for 4-6 hours) . This treatment increases intracellular butyrate levels, promoting histone butyrylation at various lysine residues including K122. Alternative approaches include metabolic manipulation to increase butyryl-CoA production, such as supplementation with short-chain fatty acids or modulation of fatty acid oxidation pathways. Inhibition of histone deacetylases (HDACs), particularly class I and class II HDACs, can also prevent the removal of butyryl groups, resulting in accumulation of this modification. For studying position-specific effects, researchers may employ cell systems expressing mutant histones (K122R) as negative controls to confirm antibody specificity.

What are the recommended procedures for sample preparation when detecting Butyryl-HIST1H3A (K122)?

Optimal sample preparation for Butyryl-HIST1H3A (K122) detection begins with proper cell lysis and histone extraction. For Western blotting applications, acid extraction methods using 0.2N HCl or commercially available histone extraction kits are recommended to enrich for histones while minimizing the presence of contaminating proteins . For immunocytochemistry, cells should be fixed in 4% formaldehyde and permeabilized with 0.2% Triton X-100, followed by blocking with 10% normal serum to reduce background . For ChIP applications, optimal crosslinking (typically 1% formaldehyde for 10 minutes) followed by chromatin fragmentation via sonication or micrococcal nuclease digestion to achieve fragments of 200-500bp is crucial for successful immunoprecipitation of Butyryl-HIST1H3A (K122) .

How does Butyryl-HIST1H3A (K122) functionally differ from other acylation modifications at the same position?

The K122 position of histone H3.1 can undergo various acylation modifications including acetylation, butyrylation, β-hydroxybutyrylation, and formylation . Each modification imparts distinct functional consequences due to their different chemical properties. Butyrylation at K122 features a four-carbon acyl chain, which is longer than acetylation (two-carbon) but lacks the hydroxyl group present in β-hydroxybutyrylation . This structural difference affects the modification's impact on nucleosome stability and protein-protein interactions. While acetylation at K122 is known to destabilize the nucleosome by disrupting histone-DNA contacts, butyrylation likely produces a more pronounced effect due to its larger hydrophobic moiety. Unlike formylation, which may serve as a cellular stress signal, butyrylation appears to be more closely linked to metabolic state and energy production via butyryl-CoA levels, potentially connecting cellular metabolism directly to chromatin regulation through distinct signaling pathways.

What are the optimal ChIP-seq parameters for studying genome-wide distribution of Butyryl-HIST1H3A (K122)?

For successful ChIP-seq analysis of Butyryl-HIST1H3A (K122), several parameters require optimization. Chromatin should be fragmented to 200-300bp using either sonication or enzymatic digestion, with fragment size verified by gel electrophoresis. Immunoprecipitation typically requires 3-5μg of Butyryl-HIST1H3A (K122) antibody per 25-30μg of chromatin . For sequencing library preparation, 10-20ng of immunoprecipitated DNA is recommended, with 15-25 million paired-end reads (50-75bp) providing sufficient coverage for genome-wide analysis. Data analysis should include normalization to input controls and comparison with IgG backgrounds. Peak calling algorithms such as MACS2 with a q-value cutoff of 0.01 are commonly employed, followed by annotation to genomic features. Integration with transcriptomic data and other histone modifications (particularly activating marks like H3K27ac) provides contextual information about the regulatory functions of Butyryl-HIST1H3A (K122) across the genome.

How do metabolic fluctuations affect Butyryl-HIST1H3A (K122) levels and what techniques can monitor these changes?

Histone butyrylation is intimately connected to cellular metabolism through fluctuations in butyryl-CoA levels, which are influenced by processes such as fatty acid oxidation and amino acid catabolism. To monitor how metabolic changes affect Butyryl-HIST1H3A (K122) levels, researchers can employ a multi-omics approach integrating:

• Metabolomics to measure intracellular butyryl-CoA concentrations using LC-MS/MS

• Western blotting with Butyryl-HIST1H3A (K122) antibodies to quantify global modification levels

• ChIP-seq to identify genome-wide changes in Butyryl-HIST1H3A (K122) distribution

• RNA-seq to correlate transcriptional changes with altered butyrylation patterns

Metabolic intervention experiments can include glucose or oxygen deprivation, fatty acid supplementation, or inhibition of specific metabolic pathways. Time-course experiments combining these techniques can reveal the kinetics of butyrylation response to metabolic fluctuations, with measurements taken at intervals ranging from 30 minutes to 24 hours after metabolic perturbation to capture both rapid signaling and sustained epigenetic responses.

How can researchers verify the specificity of Butyryl-HIST1H3A (K122) antibodies?

Verifying antibody specificity is crucial for accurate interpretation of experimental results. For Butyryl-HIST1H3A (K122) antibodies, a multi-layered validation approach is recommended:

• Peptide competition assays: Pre-incubating the antibody with butyrylated K122 peptides should abolish signal detection, while non-modified peptides or peptides with different modifications should not affect antibody binding .

• Comparative testing: Parallel analysis of samples from cells treated with/without sodium butyrate (30mM for 4 hours) should show differential signal intensity corresponding to increased butyrylation levels .

• Knockout/knockdown validation: Using CRISPR-Cas9 to generate K122R mutants or employing siRNA against enzymes responsible for butyrylation can provide additional specificity confirmation.

• Cross-reactivity assessment: Testing against peptide arrays containing similar modifications (acetylation, propionylation, crotonylation) at the K122 position as well as identical modifications at different lysine residues can confirm modification and position specificity .

• Mass spectrometry verification: LC-MS/MS analysis of immunoprecipitated histones can independently confirm the presence of butyrylation at K122.

What are common causes of false positive or false negative results when using Butyryl-HIST1H3A (K122) antibodies?

False positive results may arise from:

• Cross-reactivity with similar modifications (particularly β-hydroxybutyrylation at K122) or identical modifications at different lysine residues

• Non-specific antibody binding, especially in samples with high protein concentration

• Insufficient blocking or washing steps in immunoblotting or immunostaining protocols

• Degraded samples leading to exposure of non-specific epitopes

False negative results commonly stem from:

• Low abundance of the butyrylation mark, which may require enrichment techniques or signal amplification

• Epitope masking due to protein-protein interactions or other post-translational modifications

• Improper sample preparation, particularly inadequate cell lysis or histone extraction

• Antibody degradation from improper storage or repeated freeze-thaw cycles

• Use of fixatives or buffers that disrupt the butyryl epitope structure

Researchers should systematically evaluate these factors when troubleshooting unexpected results.

How does Butyryl-HIST1H3A (K122) interact with other histone modifications in the broader epigenetic landscape?

Understanding the interplay between Butyryl-HIST1H3A (K122) and other histone modifications represents a frontier in epigenetic research. Evidence suggests that butyrylation at K122 may functionally interact with other nearby modifications, particularly those occurring in the globular domain of histone H3. For instance, modifications at sites like H3K64, H3K79, and H3K115 may collectively influence nucleosome stability and dynamics. Sequential ChIP (ChIP-reChIP) experiments can reveal co-occurrence patterns of these marks, while nucleosome stability assays comparing single versus combinatorial modifications can determine synergistic or antagonistic effects. Mass spectrometry-based approaches including Middle-Down and Top-Down proteomics are particularly valuable for identifying combinatorial patterns on the same histone tail. Emerging evidence suggests that K122 butyrylation may preferentially associate with active enhancers marked by H3K27ac and H3K4me1, potentially serving as part of a broader "acylation signature" that regulates enhancer activity in response to metabolic signals.

What are the enzymes responsible for writing and erasing Butyryl-HIST1H3A (K122) modifications?

The enzymatic regulation of histone butyrylation remains incompletely characterized compared to modifications like acetylation and methylation. Current research indicates that some histone acetyltransferases (HATs) possess promiscuous activity that allows them to catalyze butyrylation, including p300/CBP and potentially PCAF. These enzymes likely utilize butyryl-CoA as a substrate rather than acetyl-CoA, with preference influenced by the relative intracellular concentrations of these metabolites. For "erasing" this modification, several histone deacetylases (HDACs) have demonstrated debutyrylase activity, particularly SIRT1, SIRT2, and HDAC3. Emerging research techniques to study these enzymatic activities include:

• In vitro enzyme assays with recombinant enzymes and synthetic histone peptides

• Chemical genetics approaches using enzyme inhibitors with varying specificity

• CRISPR-Cas9 screening to identify novel enzymes affecting butyrylation levels

• Proteomics to identify proteins that specifically bind to butyrylated K122

The context-dependent activity of these enzymes across different cell types and physiological conditions remains an active area of investigation.

How do research findings from Butyryl-HIST1H3A (K122) compare with other lysine butyrylation sites on histone H3?

Butyrylation has been identified at multiple lysine residues on histone H3, including K9, K14, K18, K23, K56, K79, and K122, each with potentially distinct functional implications . Comparative analysis reveals important differences in regulatory patterns:

K122 butyrylation appears to be particularly important for enhancer activation and likely functions by directly affecting histone-DNA interactions due to its position at the dyad axis of the nucleosome. Unlike K9 butyrylation, which shows significant overlap with acetylation sites, K122 butyrylation may have more specialized functions related to metabolic sensing or specific developmental programs. ChIP-seq studies demonstrate that while K9 butyrylation broadly correlates with active transcription, K122 butyrylation shows more specific enrichment patterns at certain enhancer subsets, suggesting context-dependent regulation.

What bioinformatic pipelines are optimal for integrating Butyryl-HIST1H3A (K122) ChIP-seq data with other omics datasets?

For comprehensive interpretation of Butyryl-HIST1H3A (K122) ChIP-seq data in the context of other omics datasets, researchers should consider the following integrated analytical pipeline:

• Primary ChIP-seq analysis:

  • Quality control using FastQC and trimming with Trimmomatic

  • Alignment to reference genome using Bowtie2 or BWA

  • Peak calling with MACS2 (parameters: --broad --broad-cutoff 0.1 --qvalue 0.01)

  • Signal visualization using deepTools

• Multi-omics integration:

  • Correlation with RNA-seq using tools like BETA or GSEA

  • Integration with other histone modifications using ChromHMM or EpiSig

  • Correlation with chromatin accessibility (ATAC-seq) using DiffBind

  • Motif enrichment analysis using HOMER or MEME suite

• Metabolomics integration:

  • Correlation of butyryl-CoA levels with butyrylation patterns

  • Pathway analysis using MetaboAnalyst or KEGG

  • Machine learning approaches to identify metabolites predictive of butyrylation patterns

• Visualization and interpretation:

  • Integrated genome browser views (IGV, WashU Epigenome Browser)

  • Network analysis using Cytoscape

  • R packages for statistical analysis (DESeq2, limma)

This integrated approach can reveal regulatory networks connecting metabolism, epigenetics, and transcription, providing insights into the functional significance of Butyryl-HIST1H3A (K122) in various biological contexts.