2-hydroxyisobutyryl-HIST1H3A (K4) Antibody
Description
Definition and Target Specificity
The 2-hydroxyisobutyryl-HIST1H3A (K4) antibody specifically recognizes the 2-hydroxyisobutyryl modification on lysine 4 of the HIST1H3A protein, a replication-dependent histone H3 variant. This modification is a recently characterized post-translational modification (PTM) associated with transcriptional regulation and chromatin dynamics .
Table 1: Supplier and Validation Data
Context Among Histone Modifications
2-hydroxyisobutyrylation is distinct from more studied PTMs like acetylation or methylation. It shares structural similarities with β-hydroxybutyrylation, a mark linked to metabolic states such as fasting or diabetic ketoacidosis .
Functional Insights
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Gene Regulation: Similar modifications (e.g., β-hydroxybutyrylation at H3K9 or H4K8) are enriched at active promoters, suggesting a role in transcriptional activation .
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Disease Associations: Metabolic stress-induced PTMs may influence chromatin states in pathologies like cancer or metabolic disorders, though direct evidence for 2-hydroxyisobutyryl-K4 is lacking .
Table 2: Comparative Analysis of H3K4-Targeting Antibodies
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ChIP-seq: Used to map histone marks genome-wide, though antibody specificity remains a concern .
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Western Blot/ICC: Detects global or subcellular localization of the modification .
Validation Challenges
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Specificity Issues: Studies highlight that >50% of commercial histone antibodies exhibit off-target binding, emphasizing the need for rigorous validation (e.g., peptide array or internally calibrated ChIP) .
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Quantitative Limitations: Unlike trimethylation (H3K4me3), 2-hydroxyisobutyrylation lacks established quantitative benchmarks .
Future Directions
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Mechanistic Studies: Define the enzymes responsible for adding/removing 2-hydroxyisobutyryl groups.
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Disease Models: Investigate its role in metabolic or neurodegenerative diseases using knockout models or chemical inhibitors.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates a mechanism for epigenetic regulation in cancer by inducing E3 ubiquitin ligase NEDD4-dependent histone H3 ubiquitination. PMID: 28300060
- The identification of increased expression of H3K27me3 during a patient's clinical course can be helpful for determining whether the tumors are heterochronous. PMID: 29482987
- Recent findings show that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. PMID: 28982940
- Data suggest that Ki-67 antigen proliferative index has significant limitations and phosphohistone H3 (PHH3) is a viable alternative proliferative marker. PMID: 29040195
- These results identify cytokine-induced histone 3 lysine 27 trimethylation as a mechanism that stabilizes gene silencing in macrophages. PMID: 27653678
- This data indicates that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. PMID: 27251074
- This series of 47 diffuse midline gliomas revealed that histone H3-K27M mutation was mutually exclusive with IDH1-R132H mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss, and monosomy 10. Among these K27M+ diffuse midline gliomas. PMID: 26517431
- Data show that histone chaperone HIRA co-localizes with viral genomes, binds to incoming viral and deposits histone H3.3 onto these. PMID: 28981850
- These experiments demonstrated that PHF13 binds specifically to DNA and to two types of histone H3 methyl tags (lysine 4-tri-methyl or lysine 4-di-methyl) where it functions as a transcriptional co-regulator. PMID: 27223324
- Hemi-methylated CpGs DNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. PMID: 27595565
- For the first time, we describe the MR imaging features of pediatric diffuse midline gliomas with histone H3 K27M mutation. PMID: 28183840
- Approximately 30% of pediatric high-grade gliomas (pedHGG) including GBM and DIPG harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3) which is correlated with poor outcome and was shown to influence EZH2 function. PMID: 27135271
- H3F3A K27M mutation in adult cerebellar HGG is not uncommon. PMID: 28547652
- Data show that lysyl oxidase-like 2 (LOXL2) is a histone modifier enzyme that removes trimethylated lysine 4 (K4) in histone H3 (H3K4me3) through an amino-oxidase reaction. PMID: 27735137
- Histone H3 lysine 9 (H3K9) acetylation was most prevalent when the Dbf4 transcription level was highest whereas the H3K9me3 level was greatest during and just after replication. PMID: 27341472
- SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. PMID: 27614073
- Data suggest that binding of the helical tail of histone 3 (H3) with PHD ('plant homeodomain') fingers of BAZ2A or BAZ2B (bromodomain adjacent to zinc finger domain 2A or 2B) requires molecular recognition of secondary structure motifs within the H3 tail and could represent an additional layer of regulation in epigenetic processes. PMID: 28341809
- The results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of the preinitiation complex. PMID: 27679476
- Histone H3 modifications caused by traffic-derived airborne particulate matter exposures in leukocytes. PMID: 27918982
- A key role of persistent histone H3 serine 10 or serine 28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response genes. PMID: 27996159
- hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in medulloblastoma. PMID: 27694758
- AS1eRNA-driven DNA looping and activating histone modifications promote the expression of DHRS4-AS1 to economically control the DHRS4 gene cluster. PMID: 26864944
- Data suggest that nuclear antigen Sp100C is a multifaceted histone H3 methylation and phosphorylation sensor. PMID: 27129259
- The authors propose that histone H3 threonine 118 phosphorylation via Aurora-A alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. PMID: 26878753
- Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its H3 histone recognition. PMID: 27045799
- Functional importance of H3K9me3 in hypoxia, apoptosis and repression of APAK. PMID: 25961932
- Taken together, the authors verified that histone H3 is a real substrate for GzmA in vivo in the Raji cells treated by staurosporin. PMID: 26032366
- We conclude that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet counts. PMID: 26232351
- Data show that double mutations on the residues in the interface (L325A/D328A) decreases the histone H3 H3K4me2/3 demethylation activity of lysine (K)-specific demethylase 5B (KDM5B). PMID: 24952722
- Data indicate that minichromosome maintenance protein 2 (MCM2) binding is not required for incorporation of histone H3.1-H4 into chromatin but is important for stability of H3.1-H4. PMID: 26167883
- Data suggest that histone H3 lysine methylation (H3K4me3) serves a crucial mechanistic role in leukemia stem cell (LSC) maintenance. PMID: 26190263
- PIP5K1A modulates ribosomal RNA gene silencing through its interaction with histone H3 lysine 9 trimethylation and heterochromatin protein HP1-alpha. PMID: 26157143
- Data indicate that lower-resolution mass spectrometry instruments can be utilized for histone post-translational modifications (PTMs) analysis. PMID: 25325711
- Data indicate that inhibition of lysine-specific demethylase 1 activity prevented IL-1beta-induced histone H3 lysine 9 (H3K9) demethylation at microsomal prostaglandin E synthase 1 (mPGES-1) promoter. PMID: 24886859
- The authors report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. PMID: 22473132
Q&A
What is 2-hydroxyisobutyrylation and how does it differ from other histone modifications?
Lysine 2-hydroxyisobutyrylation (Khib) is a relatively recently identified post-translational modification (PTM) that affects the association between histone and DNA. Unlike lysine acetylation, 2-hydroxyisobutyrylation is structurally and mechanistically distinct, showing quantitative responses to changes in glycolytic flux . Comprehensive analyses have identified 63 histone 2-hydroxyisobutyrylated lysine sites in human and mouse cells, with 27 of these sites being exclusive to 2-hydroxyisobutyrylation compared to acetylation and crotonylation . The stoichiometries of certain histone 2-hydroxyisobutyrylation marks (H3K79, H2BK108, H4K91, and H1K62) in synchronized G2/M HeLa cells are comparable to or even higher than those of many histone acetylation marks with known biological functions .
Which specific lysine residues on HIST1H3A undergo 2-hydroxyisobutyrylation?
Multiple lysine residues on HIST1H3A have been identified as targets for 2-hydroxyisobutyrylation. While the search results specifically mention a commercially available antibody for 2-hydroxyisobutyryl-HIST1H3A (K36) , comprehensive studies have identified several conserved 2-hydroxyisobutyrylation sites on histone H3 across plants, humans, and mice, including H3K56, H3K79, and H3K122 . Sequence alignment analysis has further confirmed that many of these modification sites are highly conserved across species, suggesting evolutionary importance of this PTM .
How is 2-hydroxyisobutyrylation linked to cellular metabolism?
Histone 2-hydroxyisobutyrylation represents a critical link between metabolism and epigenetic regulation. β-hydroxybutyrate (BHB) functions as a substrate for histone lysine β-hydroxybutyrylation (Kbhb), which then activates gene expression independent of acetylation . Research has demonstrated that histone Kbhb marks are dramatically induced in response to elevated BHB levels in cultured cells and in livers of mice subjected to prolonged fasting or streptozotocin (STZ)-induced diabetic ketoacidosis . Cellular experiments have confirmed that histone Kbhb levels are elevated in a BHB dose-dependent manner, establishing a direct connection between metabolic state and histone modification patterns .
What are the recommended applications for 2-hydroxyisobutyryl-HIST1H3A antibodies?
Based on product information, 2-hydroxyisobutyryl-HIST1H3A antibodies are primarily recommended for ELISA and immunofluorescence (IF) applications . For immunofluorescence, the recommended dilution range is 1:50-1:200 . These antibodies are typically polyclonal, produced in rabbit hosts, and recognize the peptide sequence around the site of 2-hydroxyisobutyryl-Lys (36) derived from Human Histone H3 . While these represent the primary validated applications, researchers may need to optimize conditions for other experimental contexts.
What controls should be implemented when using 2-hydroxyisobutyryl-HIST1H3A antibodies?
For rigorous experimental design, the following controls should be considered:
| Control Type | Purpose | Implementation |
|---|---|---|
| Positive Control | Verify antibody functionality | Samples with known 2-hydroxyisobutyrylation (e.g., cells treated with BHB) |
| Negative Control | Assess background signal | Samples with depleted 2-hydroxyisobutyrylation (e.g., treated with HDAC1-3) |
| Peptide Competition | Confirm antibody specificity | Pre-incubation of antibody with 2-hydroxyisobutyryl-modified peptides |
| Cross-Reactivity Check | Determine modification specificity | Comparison with other histone modifications (acetylation, succinylation) |
These controls help distinguish true biological signals from technical artifacts and ensure experimental reproducibility.
How should researchers store and handle 2-hydroxyisobutyryl-HIST1H3A antibodies?
Proper storage and handling are critical for maintaining antibody functionality. According to product information, 2-hydroxyisobutyryl-HIST1H3A antibodies should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity . The antibodies are typically provided in liquid form with a buffer containing preservatives (0.03% Proclin 300) and constituents (50% Glycerol, 0.01M PBS, pH 7.4) . When working with these antibodies, researchers should follow standard antibody handling protocols, including minimizing exposure to room temperature.
How do enzymatic "writers" and "erasers" regulate 2-hydroxyisobutyrylation?
The dynamic regulation of 2-hydroxyisobutyrylation involves specific enzymes that add (writers) or remove (erasers) this modification:
Writers:
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The acyltransferase p300 acts as a histone Kbhb "writer," catalyzing the addition of BHB to lysine residues
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Histone acetyltransferase Esa1p in budding yeast and its homologue TIP60 in humans can trigger K hib reactions both in vitro and in vivo
Erasers:
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Histone deacetylases HDAC1-3, SIRT1, and SIRT2 have demonstrated significant de-β-hydroxybutyrylation activity against core histones in vitro through high-performance liquid chromatography (HPLC) analysis
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In cellular contexts, studies have shown that primarily HDAC1 and HDAC2 function as histone Kbhb deacetylases
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In yeast models, Rpd3p and Hos3p have been identified as potential regulatory enzymes for lysine de-2-hydroxyisobutyrylation reactions
Research indicates that the level of histone Kbhb at sites including H3K9, H3K18, H3K27, and H4K8 decreases in response to p300 knockdown, with some of these sites showing greater sensitivity to p300 knockdown than corresponding Kac sites .
How does 2-hydroxyisobutyrylation interact with other histone modifications?
Histone 2-hydroxyisobutyrylation exists within a complex landscape of post-translational modifications. Comprehensive comparative analyses of modification patterns reveal both unique and overlapping features:
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While many PTMs occupy distinct lysine residues, suggesting unique functions, some sites can accommodate multiple modifications
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Comparative analysis of rice acetylome, succinylome, and 2-hydroxyisobutyrylome found that 99 2-hydroxyisobutyrylated sites could be modified by any of the three PTMs, while 8,924 sites were unique to K hib
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The preference of different chiral Kbhb for deacetylases suggests structural specificity in the recognition and processing of these modifications
These patterns of overlap and exclusivity likely contribute to a histone code that finely regulates chromatin structure and gene expression in response to cellular conditions.
What genomic distribution patterns are associated with HIST1H3A 2-hydroxyisobutyrylation?
The genomic distribution of histone 2-hydroxyisobutyrylation provides insights into its functional roles. Research has revealed that:
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The H3K9bhb mark localizes in gene promoters and is significantly elevated in mouse liver during starvation
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Pathways associated with genes marked by elevated H3K9bhb include amino acid catabolism, circadian rhythms, redox balance, PPAR signaling pathways, and oxidative phosphorylation
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Unlike some other histone modifications, K hib has shown dramatic changes in genomic distribution during male germ cell differentiation
These distribution patterns suggest that 2-hydroxyisobutyrylation plays important roles in metabolic adaptation, developmental processes, and tissue-specific gene regulation.
What mass spectrometry approaches are most effective for studying HIST1H3A 2-hydroxyisobutyrylation?
Mass spectrometry represents a powerful approach for identifying and characterizing 2-hydroxyisobutyrylation:
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Antibody-based affinity enrichment coupled with nano-HPLC/MS/MS analyses has successfully identified thousands of 2-hydroxyisobutyryl lysine sites across various proteomes
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Affinity-directed HPLC/MS/MS analysis specifically on histones has identified 63 2-hydroxyisobutyrylation sites from HeLa cells and mouse testis cells
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These approaches can be complemented with functional annotation analyses to connect modification patterns with biological processes
When designing mass spectrometry experiments, researchers should consider enrichment strategies, digestion protocols, and data analysis pipelines optimized for detecting and quantifying 2-hydroxyisobutyrylated peptides.
How can site-specific 2-hydroxyisobutyrylation be distinguished from other modifications?
Distinguishing between various histone modifications represents a significant challenge. Several approaches can help address this challenge:
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Site-specific incorporation of ε-N-2-Hydroxyisobutyryl-lysine into bacterial and mammalian cells using amber suppression-mediated strategies provides a controlled system for studying specific modifications
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Sequence motif analysis has revealed that negative charged amino acids, D and E, are strongly preferred around 2-hydroxyisobutyrylated sites, providing a distinctive signature
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Structural feature analysis shows that 2-hydroxyisobutyryllysine is less surface accessible than unmodified lysine and has a greater propensity to be found in regions of intrinsic disorder and coils
These distinctive features can be leveraged to develop more specific detection methods and to interpret experimental results more accurately.
What experimental designs best reveal the functional consequences of HIST1H3A 2-hydroxyisobutyrylation?
To elucidate the functional roles of 2-hydroxyisobutyrylation, researchers should consider multi-faceted experimental approaches:
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Metabolic manipulation studies: Altering BHB levels through fasting protocols or direct supplementation can reveal how metabolic states influence 2-hydroxyisobutyrylation patterns and subsequent gene expression
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Enzyme modulation experiments: Manipulating the activity or expression of writers (p300, TIP60) and erasers (HDAC1-3) can help establish causality between 2-hydroxyisobutyrylation and biological outcomes
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Comparative multi-omics approaches: Integrating proteomics, transcriptomics, and metabolomics data can provide comprehensive insights into how 2-hydroxyisobutyrylation coordinates cellular responses
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Site-specific mutagenesis: Converting key lysine residues to non-modifiable amino acids can directly test the functional significance of specific 2-hydroxyisobutyrylation sites
By combining these approaches, researchers can develop a more complete understanding of how 2-hydroxyisobutyrylation contributes to cellular physiology and disease states.
