Succinyl-HIST1H3A (K56) Antibody
Description
Introduction to Succinyl-HIST1H3A (K56) Antibody
The Succinyl-HIST1H3A (K56) Antibody is a rabbit polyclonal antibody designed to detect the succinylated form of lysine 56 (K56) on the HIST1H3A protein, a variant of histone H3.1. Histones are core chromatin components, and post-translational modifications (PTMs) like succinylation regulate chromatin structure, gene expression, and cellular processes. Succinylation involves the addition of a succinyl group (−OOCCH₂CH₂COOH) to lysine residues, a modification increasingly recognized for its role in metabolism, epigenetics, and disease .
3.2. Applications of the Antibody
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ELISA: Quantification of succinylated H3.1 in cellular lysates.
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ICC: Visualization of subcellular localization of succinylated H3.1, particularly in nuclear regions .
4.1. Enzymatic Regulation
HAT1’s dual role as a succinyltransferase highlights the interplay between acetylation and succinylation:
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HAT1 Activity: Knockdown of HAT1 reduces histone H3 succinylation in HepG2 and pancreatic cancer cells, suggesting its involvement in succinylation .
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Succinyl-CoA Dependency: Succinylation requires succinyl-CoA, a metabolite linked to the tricarboxylic acid (TCA) cycle, implicating metabolic states in histone modification .
4.2. Functional Implications
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Genomic Stability: Analogous to acetylation, succinylation at K56 may facilitate chromatin assembly during replication or repair .
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Cancer Biology: Elevated succinylation in tumors could drive epigenetic reprogramming, as observed with HAT1 overexpression in pancreatic cancer .
Comparative Analysis: Succinylation vs. Acetylation at K56
Data synthesized from studies on H3K56 acetylation and HAT1 succinyltransferase activity .
Future Directions and Research Gaps
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Mechanistic Studies: Direct evidence linking K56 succinylation to chromatin dynamics or metabolic pathways remains sparse.
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Cancer Therapeutics: Exploring HAT1 inhibitors to target succinylation in tumors .
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Technical Challenges: Limited commercial antibodies for succinylation necessitate validation across cell types and conditions .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that epigenetic regulation in cancer involves the induction of 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 in determining whether the tumors are heterochronous. PMID: 29482987
- This study reports that JMJD5, a Jumonji C (JmjC) domain-containing protein, functions as a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions inducing a DNA damage response. PMID: 28982940
- These findings suggest that the Ki-67 antigen proliferative index has significant limitations, while phosphohistone H3 (PHH3) presents a viable alternative as a proliferative marker. PMID: 29040195
- These results identify cytokine-induced histone 3 lysine 27 trimethylation as a mechanism responsible for stabilizing gene silencing in macrophages. PMID: 27653678
- This data indicates that, during the early stages of human brain development, HIST1H3B constitutes the predominant proportion of H3.1 transcripts among H3.1 isoforms. PMID: 27251074
- In a series of 47 diffuse midline gliomas, histone H3-K27M mutation demonstrated mutual exclusivity with IDH1-R132H mutation and EGFR amplification. It 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 demonstrates that the histone chaperone HIRA co-localizes with viral genomes, binds to incoming viral particles, and deposits histone H3.3 onto these. PMID: 28981850
- These experiments demonstrated that PHF13 specifically binds 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
- This study provides the first description of 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). This mutation is correlated with poor prognosis and has been shown to influence EZH2 function. PMID: 27135271
- H3F3A K27M mutation in adult cerebellar HGG is not uncommon. PMID: 28547652
- Data suggests that lysyl oxidase-like 2 (LOXL2) acts as 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, while the H3K9me3 level was greatest during and immediately after replication. PMID: 27341472
- The SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. PMID: 27614073
- These findings suggest that the 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. This could represent an additional layer of regulation in epigenetic processes. PMID: 28341809
- These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate the 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 appear 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 suggests that nuclear antigen Sp100C acts as 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 genuine substrate for GzmA in vivo in Raji cells treated with staurosporin. PMID: 26032366
- These findings suggest that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet counts. PMID: 26232351
- Data shows 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 indicates that minichromosome maintenance protein 2 (MCM2) binding is not required for the incorporation of histone H3.1-H4 into chromatin but is important for the stability of H3.1-H4. PMID: 26167883
- Data suggests that histone H3 lysine methylation (H3K4me3) plays 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 indicates that lower-resolution mass spectrometry instruments can be utilized for histone post-translational modifications (PTMs) analysis. PMID: 25325711
- Data indicates that inhibition of lysine-specific demethylase 1 activity prevented IL-1beta-induced histone H3 lysine 9 (H3K9) demethylation at the 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 are regulated by a histone H3K9 acetyl/methyl balance. PMID: 22473132
Q&A
Antibody Specificity Validation: Addressing Cross-Reactivity Concerns
Question: How can researchers confirm the specificity of Succinyl-HIST1H3A (K56) antibodies, particularly given reports of nonspecific binding in acetylation-focused studies?
Answer:
Validation requires a multi-step approach to rule out cross-reactivity with acetylated or unmodified H3K56. Key methods include:
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Peptide Competition Assays: Incubate antibodies with synthetic peptides containing unmodified H3K56, acetylated H3K56 (H3K56ac), or succinylated H3K56 (H3K56succ). A reduction in signal only with the succinylated peptide confirms specificity .
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Mutant Cell Lines: Use cell lines expressing H3K56R (arginine substitution) to block succinylation. Absence of signal in Western blot/ChIP confirms target dependency .
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In Vitro Succinylation Assays: Test antibody reactivity against purified histone H3 treated with succinyltransferases (e.g., HAT1) and untreated controls. Specific binding to succinylated H3 validates antibody utility .
Table 1: Antibody Validation Strategies
| Method | Purpose | Key Controls |
|---|---|---|
| Peptide competition | Exclude acetylation cross-reactivity | H3K56ac peptide blocking |
| H3K56R mutant cells | Confirm site-specificity | Wild-type vs. mutant lysates |
| In vitro succinylation | Validate enzyme-dependent binding | HAT1 knockout vs. WT HAT1 |
Experimental Design for Functional Studies
Question: How to design experiments to study the role of H3K56 succinylation in chromatin dynamics or genome stability?
Answer:
Integrate epigenetic, genetic, and biochemical approaches:
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Succinylation Triggers:
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Stimulus-Response Models: Treat cells with DNA-damaging agents (e.g., MMS) or replication stressors (e.g., hydroxyurea) to induce succinylation, as seen in yeast H3K56ac studies .
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Enzyme Manipulation: Use HAT1 knockouts (CRISPR/Cas9) to deplete succinylation, then assess chromatin compaction or repair efficiency .
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Chromatin Disassembly Analysis:
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Genetic Interactions:
Interpreting Conflicting Data: Acetylation vs. Succinylation
Question: How to resolve discrepancies between studies attributing H3K56 modifications to acetylation or succinylation?
Answer:
Mechanistic and methodological distinctions are critical:
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Enzymatic Pathways:
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Detection Challenges:
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Antibody Specificity: Commercial H3K56ac antibodies often cross-react with other acetylated lysines (e.g., H3K9, H3K27) . Use succinyl-specific antibodies and validate via peptide competition .
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Mass Spectrometry: Confirm modification identity by LC-MS/MS, as immunoblot alone cannot distinguish acetylation/succinylation .
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Table 2: Key Differences in H3K56 Modifications
Advanced Applications: Integrating Succinylation with Other Epigenetic Marks
Question: How to investigate crosstalk between H3K56 succinylation and other histone modifications (e.g., acetylation, ubiquitination)?
Answer:
Multilayered approaches are required:
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Combinatorial ChIP-Seq:
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Perform sequential IP (ChIP-reChIP) to identify regions co-marked by H3K56succ and H3K27ac (enhancers) or H2BK120ub (DNA repair).
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Biochemical Interactions:
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In Vitro Assays: Test whether HAT1-mediated succinylation affects binding of acetyltransferases (e.g., GCN5) or deacetylases (e.g., HDACs) to H3K56.
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Metabolic Perturbation:
Troubleshooting: Low Signal in ChIP Experiments
Question: Why might Succinyl-HIST1H3A (K56) ChIP yield low signal, and how to optimize?
Answer:
Common issues and solutions:
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Fixation Efficiency:
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Antibody Concentration:
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Cellular Context:
Table 3: ChIP Optimization Parameters
| Parameter | Recommended Value | Rationale |
|---|---|---|
| Fixation time | 10–15 minutes | Preserves epitopes without over-crosslinking |
| Antibody dilution | 1:100–1:200 | Balances signal strength and background |
| Sonication conditions | 30% power, 30 sec cycles | Fragment DNA to 200–500 bp |
Comparative Analysis: Succinyl-HIST1H3A (K56) vs. Acetylated H3K56
Question: How to differentiate succinylation from acetylation at H3K56 in functional studies?
Answer:
Targeted experimental designs:
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Enzyme-Specific Inhibition:
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Mass Spectrometry:
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Genetic Models:
Future Directions: Emerging Methodologies
Question: What novel techniques could advance Succinyl-HIST1H3A (K56) research?
Answer:
Innovative approaches:
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Single-Cell Multiomics:
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scATAC-seq + scChIP: Profile chromatin accessibility and H3K56succ at single-cell resolution to link succinylation to transcriptional heterogeneity.
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Cryo-EM Structural Studies:
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Map H3K56succ interactions with nucleosome-binding proteins (e.g., histone chaperones) to elucidate chromatin remodeling mechanisms.
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Metabolome Integration:
