Crotonyl-HIST1H3A (K18) Antibody

What is Crotonyl-HIST1H3A (K18) Antibody and what are its primary applications?

Crotonyl-HIST1H3A (K18) Antibody is a specialized immunological reagent that specifically recognizes histone H3.1 when it has been crotonylated at the lysine 18 position. This epigenetic modification plays a crucial role in regulating gene expression, particularly in active chromatin regions.

The antibody is typically available in polyclonal and monoclonal formats (such as EPR18773) with proven applications in:

• Western Blot (WB) at recommended dilutions of 1:1000

• Chromatin Immunoprecipitation (ChIP)

• Immunofluorescence (IF) at recommended dilutions of 1:20-1:200

• Peptide Array (PepArr)

• ELISA

For optimal results in IF applications, cells should be fixed in 4% formaldehyde, permeabilized using 0.2% Triton X-100, and blocked in 10% normal serum before overnight incubation with the antibody at 4°C .

How does histone crotonylation differ from histone acetylation?

Histone crotonylation and acetylation are both lysine acylations that occur on histone proteins, but they differ in several important ways:

These biophysical differences provide an important mechanism of specificity for protein interactions, allowing cells to regulate gene expression through distinct pathways . The extended hydrocarbon chain of the crotonyl group also increases the hydrophobicity and bulk of the lysine residue compared to acetylation, contributing to differential recognition by reader proteins .

How should Crotonyl-HIST1H3A (K18) Antibody be stored and handled?

For optimal long-term stability and activity:

• Store the antibody at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Many commercial formulations contain 50% glycerol and 0.03% Proclin 300 as a preservative in 0.01M PBS, pH 7.4

• Antibody should remain stable for at least 12 months when properly stored

• Allow the antibody to equilibrate to room temperature before opening the vial

• Working dilutions should be prepared fresh before use and can be stored at 4°C for up to one week

Proper storage and handling procedures are essential for maintaining antibody integrity and ensuring consistent experimental results across multiple studies .

What are the known "writer" and "eraser" enzymes for H3K18 crotonylation and how do they function?

The enzymatic regulation of H3K18 crotonylation involves specific writers and erasers:

Writers (Histone Crotonyltransferases, HCTs):

• p300/CBP: Though less efficient than its acetyltransferase activity, p300/CBP is a major histone crotonyltransferase in mammals. The active site of p300 can accommodate crotonyl-CoA, but with steric constraints requiring a conformational change where the crotonyl group is displaced into a "back hydrophobic pocket" .

• MYST family members: Human MOF and its yeast homolog Esa1 exhibit HCT activity, though weak in vitro, suggesting they function in cells as part of protein complexes .

• Gcn5: In budding yeast, Gcn5 catalyzes crotonylation at lysine residues 9, 14, 18, 23, and 27 of histone H3, as part of the ADA complex (Gcn5-Ada2-Ada3) .

Erasers (Histone Decrotonylases, HDCRs):

These enzymes operate within a complex regulatory network that balances crotonylation and decrotonylation in response to cellular signals and metabolic states, directly impacting gene expression patterns .

How does the YEATS domain function as a reader of histone crotonylation?

The YEATS domain has emerged as a specialized reader for histone crotonylation with significant implications for transcriptional regulation:

• YEATS domains have a preference for binding acyl chains longer than acetyl, with the strongest affinity for crotonyllysine (Kcr), showing a 2-7 fold higher affinity for Kcr compared to acetyllysine (Kac) .

• AF9 YEATS recognizes histone H3 crotonylation at K9, K18, and K27 with highest affinity for H3K9cr, while YEATS2 is selective for histone H3K27cr .

• In cellular contexts, AF9 binds nucleosomes marked by H3K9cr or H3K18cr in a YEATS-dependent manner, as demonstrated by co-immunoprecipitation experiments with wild-type AF9 but not with the F59A mutant that abolishes YEATS-Kcr binding .

• This specific recognition mechanism couples histone crotonylation to active transcription, as validated by nucleosome pulldown experiments with pre-modified nucleosomes .

This molecular recognition system represents a critical link between histone crotonylation and downstream functional outcomes, providing a mechanism for cells to translate this epigenetic mark into specific transcriptional responses .

What techniques can be used to validate the specificity of Crotonyl-HIST1H3A (K18) Antibody?

Validating antibody specificity is crucial for reliable experimental results. For Crotonyl-HIST1H3A (K18) Antibody, several complementary approaches can be employed:

Peptide Array Analysis:

• Antibodies can be tested against arrays containing multiple modified and unmodified histone peptides at varying concentrations

• For example, ab195475 was tested in a peptide array against 501 different modified and unmodified histone peptides, with each peptide printed at six concentrations in triplicate

• Binding affinity is calculated as the area under the curve when antibody binding values are plotted against peptide concentration

• Data visualization showing antigen-containing peptides as red circles and all other peptides as blue circles provides a comprehensive specificity profile

Western Blot with Controls:

• Positive controls: Use cell lines known to express crotonylated H3K18 (HeLa, NIH/3T3)

• Negative controls: Use peptide competition assays with crotonylated and non-crotonylated peptides

• Treatment controls: Compare samples from cells treated with HDAC inhibitors (increases crotonylation) or crotonylation-enhancing agents (sodium crotonylate)

Immunofluorescence with Specificity Controls:

• Compare staining patterns in cells with induced crotonylation versus control cells

• Perform parallel staining with other validated histone modification antibodies to confirm distinct localization patterns

• Include blocking peptide controls to demonstrate specific signal quenching

Chromatin Immunoprecipitation Controls:

• Include IgG control for non-specific binding

• Use cells treated with 30mM sodium crotonylate (4h) to enhance crotonylation signals

• Validate results with quantitative PCR using primers against known target regions, such as the β-Globin promoter

These validation approaches provide a comprehensive assessment of antibody specificity, ensuring reliable detection of H3K18 crotonylation in experimental settings .

How does metabolic regulation impact histone crotonylation levels?

Histone crotonylation is intimately connected to cellular metabolism through the availability of the metabolic substrate crotonyl-CoA:

Metabolic Pathways Affecting Crotonyl-CoA Levels:

• Crotonyl-CoA is generated during fatty acid oxidation and certain amino acid degradation pathways

• The ratio of crotonyl-CoA to acetyl-CoA affects the competition for sites of modification on histones

• Cellular metabolic status directly impacts the availability of crotonyl-CoA for histone modifications

Enzymatic Regulation of Crotonyl-CoA:

• CDYL (Chromodomain Y-like protein) functions as a crotonyl-CoA hydratase that converts crotonyl-CoA to β-hydroxybutyryl-CoA

• CDYL contains both a chromodomain and a CoA-binding pocket (CoAP) domain, allowing it to function at the intersection of chromatin binding and metabolic regulation

• The CoAP domain binds CoA, and both the chromodomain and CoAP domain are required for CDYL's negative regulation of histone crotonylation

• Importantly, CDYL's ability to bind HDAC1/2 abolishes its ability to bind CoA, suggesting a complex regulatory mechanism

This metabolic-epigenetic connection provides a mechanism by which cells can sense their metabolic environment and adjust gene expression programs accordingly through differential histone modifications . The competition between different acyl-CoA species for histone modification sites represents an important epigenetic mechanism that integrates metabolic signals with transcriptional control.

What experimental considerations are important when designing ChIP experiments with Crotonyl-HIST1H3A (K18) Antibody?

Chromatin Immunoprecipitation (ChIP) with Crotonyl-HIST1H3A (K18) Antibody requires careful experimental design:

Sample Preparation:

• For enhanced detection, cells can be treated with 30mM sodium crotonylate for 4 hours before harvest

• Samples should be treated with Micrococcal Nuclease followed by sonication to generate appropriately sized chromatin fragments

• Optimal chromatin fragmentation size should be 200-500bp for high-resolution mapping of H3K18cr sites

Immunoprecipitation Protocol:

• Use 5μg of anti-HIST1H3A Crotonyl K18 antibody per ChIP reaction

• Include a control normal rabbit IgG in parallel experiments

• Pre-clear chromatin with protein A/G beads to reduce background

• Allow overnight binding at 4°C with rotation for optimal antibody-antigen interaction

Analysis Approach:

• The resulting ChIP DNA should be quantified using real-time PCR with primers against regions of interest

• Primers against the β-Globin promoter have been validated for H3K18cr ChIP experiments

• For genome-wide analysis, ChIP-seq can be performed with appropriate sequencing depth (≥20 million reads)

• Data analysis should include comparison with other active histone marks (H3K4me3, H3K27ac) for context

Critical Controls:

• Input control (pre-immunoprecipitation chromatin)

• Mock IP control (no antibody)

• Species-matched IgG control

• Positive control loci known to be enriched for H3K18cr

Following these guidelines will help ensure robust and reproducible ChIP results when investigating H3K18 crotonylation genome-wide distribution patterns .

How does H3K18 crotonylation compare to other lysine acylations on the same residue?

H3K18 can undergo multiple acylation modifications, each with distinct properties and functions:

These different acylations can compete for the same lysine residue, creating a dynamic regulatory system that responds to cellular metabolic states. The rigid planar conformation of the crotonyl group due to its C-C π bond creates a unique recognition surface that distinguishes it from other acylations, allowing for specific reader protein interactions .

The differential recognition of these modifications by reader protein domains constitutes a molecular mechanism for translating the histone code into specific transcriptional responses. The competition between different acylations for the same site can serve as a metabolic sensing mechanism, where the relative abundance of different acyl-CoA donors in the cell determines which modification predominates .

What are the emerging therapeutic implications of targeting H3K18 crotonylation?

While still in early research phases, manipulating H3K18 crotonylation presents intriguing therapeutic possibilities:

Potential Therapeutic Approaches:

• HDAC inhibitors: Class I HDAC inhibitors like trichostatin A increase global histone crotonylation levels, including H3K18cr, potentially reprogramming gene expression in disease states

• Metabolic modulators: Compounds that alter cellular crotonyl-CoA levels, such as sodium crotonylate, could be used to manipulate crotonylation-dependent gene expression

• Reader domain inhibitors: Small molecules targeting the YEATS domain could specifically disrupt crotonylation-dependent transcriptional programs

Therapeutic Areas Under Investigation:

• Cancer: Since histone crotonylation is associated with active transcription, modulating this mark could potentially reactivate silenced tumor suppressor genes

• Tissue injury: Histone crotonylation has been implicated in tissue injury responses

• Spermatogenesis: H3K18cr plays important roles in germ cell development, suggesting potential applications in reproductive medicine

Challenges and Considerations:

• Specificity: Developing interventions that specifically target crotonylation without affecting other acylations remains challenging

• Tissue-specific effects: The effects of modulating H3K18cr likely vary across different tissues and cell types

• Metabolic integration: Any therapeutic approach must consider the broader metabolic network that regulates crotonyl-CoA availability

These emerging approaches highlight the importance of further research into the specific roles of H3K18 crotonylation in health and disease .