Acetyl-Histone H3.1 (K56) Recombinant Monoclonal Antibody

What is histone H3 lysine 56 acetylation (K56Ac) and why is it significant in epigenetic research?

Histone H3 lysine 56 acetylation (K56Ac) is a post-translational modification that occurs in the helical core of histone H3, rather than on the N-terminal tail where most histone modifications occur. This modification is significant because it opens yeast chromatin and enables histone gene transcription, DNA replication, DNA repair, and prevents genomic instability. In mammals, K56Ac was initially difficult to detect, but has since been confirmed to exist at low levels (approximately 1% of total H3) in human cells including HeLa cells and human embryonic stem cells (hESCs) . The acetylation of H3 K56 in humans is mediated by the histone acetyltransferases CBP and p300 in concert with the histone chaperone ASF1 .

How does K56Ac distribution differ between yeast and human genomes?

While K56Ac is abundant and well-characterized in yeast, its presence in mammals was initially uncertain. In human embryonic stem cells (hESCs), K56Ac has been found at approximately 9.5-10.3% of promoter regions, corresponding to 0.38-0.42% of the genome (using a 500bp window analysis) . This distribution correlates with the low abundance (~1%) of K56Ac measured by mass spectrometry. Unlike yeast where K56Ac is widely distributed throughout the genome, human K56Ac appears to be enriched at specific promoter regions, particularly those associated with pluripotency and cell differentiation pathways in stem cells.

What are the technical challenges in detecting H3K56Ac in mammalian systems?

Detecting H3K56Ac in mammalian systems presents several challenges:

• Low abundance: K56Ac represents only about 1% of total H3 in human cells, compared to other H3 N-terminal acetylation sites which range from 3.8-22.9% .

• Antibody specificity issues: Many commercial antibodies cross-react with other acetylated lysines on histone H3 .

• Detection sensitivity: Early attempts using standard mass spectrometry failed to conclusively identify K56Ac in mammals, requiring the development of targeted mass spectrometry approaches .

• Competition with other modifications: K56 can also be methylated in a small fraction of mammalian histones, potentially complicating detection .

How should researchers validate the specificity of H3K56Ac antibodies for their experiments?

Researchers should implement a multi-step validation process for H3K56Ac antibodies:

What is the recommended procedure to test a new batch of H3K56Ac antibody?

When receiving a new batch of H3K56Ac antibody, researchers should perform the following validation procedures:

• Dot blot analysis with acetylated peptides representing multiple H3 lysine positions (especially K9, K27, and K56) to test for cross-reactivity.

• Western blot analysis using acid-extracted histones from cells expressing wild-type and K56R mutant H3 constructs. Try multiple antibody dilutions to identify conditions that maximize specificity.

• ChIP-qPCR at control regions known to be enriched or depleted for K56Ac to confirm expected patterns.

• Include appropriate negative controls in all experiments, such as IgG controls for ChIP and peptide competition controls for Western blots.

• Compare results with previously validated antibody batches when possible to identify potential changes in performance.

What is the recommended protocol for ChIP-seq analysis of H3K56Ac in human cells?

For ChIP-seq analysis of H3K56Ac in human cells, the following protocol is recommended based on successful approaches in the literature:

Sample Preparation and Chromatin Immunoprecipitation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature.

• Quench with 125mM glycine for 5 minutes.

• Lyse cells and sonicate chromatin to fragments of 200-500bp.

• Pre-clear chromatin with protein A/G beads.

• Immunoprecipitate with validated H3K56Ac antibody (preferably from Cell Signalling or Active Motif) overnight at 4°C .

• Include appropriate controls: input DNA, IgG control, and if possible, a spike-in control.

Library Preparation and Sequencing:

• Prepare libraries using standard protocols, with careful attention to amplification cycles to avoid PCR duplicates.

• Sequence to a depth of at least 20 million unique reads per sample to capture low-abundance modifications.

Data Analysis:

• Align reads to reference genome using BWA or Bowtie2.

• Call peaks using MACS2 with appropriate background control.

• Use a peak finding method similar to the Whitehead Neighborhood Model for identifying K56Ac-enriched regions .

• Consider the relatively low abundance (~1%) of K56Ac when interpreting results.

How can researchers distinguish between genuine H3K56Ac signal and antibody cross-reactivity in their experiments?

To distinguish genuine H3K56Ac signals from cross-reactivity, researchers should implement the following strategies:

• Include genetic controls: When possible, use cell lines expressing H3 K56R mutants as negative controls in ChIP experiments .

• Peptide competition: Perform parallel ChIP experiments with antibody pre-incubated with different acetylated peptides (K9Ac, K27Ac, K56Ac). Genuine K56Ac signal should be specifically competed by the K56Ac peptide .

• ASF1 knockdown validation: Since ASF1 is required for H3K56 acetylation in vivo, ASF1 knockdown should reduce genuine K56Ac signals. If signal persists after efficient ASF1 knockdown, it may indicate cross-reactivity .

• Correlation with known K56Ac patterns: In human embryonic stem cells, K56Ac is enriched at specific gene promoters. Signals that deviate significantly from these expected patterns should be scrutinized .

• Orthogonal validation: When possible, confirm key findings using mass spectrometry or other antibody-independent methods .

What are the optimal fixation and extraction conditions for preserving K56Ac epitopes in immunofluorescence experiments?

For optimal preservation of K56Ac epitopes in immunofluorescence experiments:

Fixation:

• Use freshly prepared 4% paraformaldehyde in PBS for 10-15 minutes at room temperature.

• Avoid over-fixation, which can mask epitopes.

• For some applications, methanol fixation (-20°C for 10 minutes) may provide better epitope accessibility.

Permeabilization:

• Use 0.1-0.2% Triton X-100 in PBS for 10 minutes at room temperature.

• Alternative: 0.5% Saponin can provide gentler permeabilization.

Epitope Retrieval:

• Heat-mediated antigen retrieval in citrate buffer (pH 6.0) may enhance epitope accessibility.

• Include 0.05% Tween-20 in wash buffers to reduce background.

Blocking:

• Block with 5% BSA or 5-10% normal serum (from the species in which the secondary antibody was raised).

• Include 0.1% Triton X-100 in blocking buffer to enhance nuclear penetration.

Antibody Dilution:

• For immunofluorescence, use Anti-Histone H3.1 (acetyl-K56) antibody at 1:30-1:200 dilution, optimizing for each application .

How does H3K56Ac dynamics correlate with transcriptional activation in human cells?

H3K56Ac dynamics show a complex relationship with transcriptional activation in human cells:

• In estrogen-responsive gene promoters, H3K56Ac levels increase upon gene induction, peaking around 60 minutes after stimulation, and then decrease. This pattern is inversely proportional to the removal and return of histone H3 at promoters that undergo chromatin disassembly (like pS2 and GREB1) .

• Interestingly, similar timing of H3K56Ac increase and decrease is observed at promoters that do not undergo chromatin disassembly during gene induction (such as PGR), suggesting that H3K56Ac is not required for chromatin disassembly from these human promoters .

• In human embryonic stem cells, approximately 9.5-10.3% of promoter regions show enrichment for K56Ac, corresponding to specific gene classes that may be important for stem cell function .

• Unlike in yeast, where K56Ac is clearly linked to nucleosome assembly during DNA replication and repair, the role of K56Ac in human transcriptional regulation appears to be more nuanced and context-dependent.

What are the key differences in H3K56Ac function between yeast and human systems?

Key differences in H3K56Ac function between yeast and human systems include:

These differences highlight the evolutionary specialization of histone modifications across species and caution against direct extrapolation of findings from yeast to human systems.

How should researchers interpret conflicting data between antibody-based detection and mass spectrometry analysis of H3K56Ac?

When faced with conflicting data between antibody-based detection and mass spectrometry analysis of H3K56Ac, researchers should:

• Prioritize mass spectrometry data: Mass spectrometry provides direct identification of modifications without relying on antibody specificity. When targeted mass spectrometry shows ~1% abundance of K56Ac , but antibody-based methods show stronger signals, suspect antibody cross-reactivity.

• Consider technical limitations: Standard mass spectrometry might miss low-abundance modifications, while targeted approaches are more sensitive. Earlier studies that failed to detect K56Ac by mass spectrometry may have lacked sufficient sensitivity .

• Evaluate antibody validation: If antibodies recognize H3 K56R mutants (which cannot be acetylated at position 56), this strongly suggests cross-reactivity with other modifications .

• Assess biological consistency: If treatments known not to affect K56Ac (by mass spectrometry) show changes in antibody signal, this suggests the antibody is detecting other modifications.

• Resolve with orthogonal approaches: When possible, combine multiple detection methods and genetic approaches (e.g., enzyme knockdowns, histone mutants) to build a consistent biological model.

What strategies can overcome the low abundance of K56Ac in human cells for reliable detection?

To overcome the low abundance (~1%) of K56Ac in human cells , researchers can implement these strategies:

• Targeted mass spectrometry approaches:

  • Use propionylation of histone H3 tryptic digests to improve peptide detection

  • Specifically interrogate samples for the presence of acetylated K56 peptides

  • Employ high-resolution mass spectrometry with multiple reaction monitoring (MRM)

• Enrichment techniques:

  • Use acid extraction of histones to concentrate the histone fraction

  • Consider chemical enrichment of acetylated peptides prior to analysis

  • For tagged histones, use tandem affinity purification to improve signal-to-noise ratio

• Signal amplification for antibody-based methods:

  • Employ tyramide signal amplification for immunofluorescence

  • Use highly sensitive detection systems for Western blots (e.g., femto-level chemiluminescence)

  • Consider proximity ligation assays to improve specificity and sensitivity

• Genetic approaches:

  • Temporarily inhibit relevant deacetylases to increase K56Ac levels

  • Express tagged versions of H3.1 to facilitate purification and detection

  • Use CRISPR activation of relevant acetyltransferases (CBP/p300) to boost K56Ac levels

How can researchers distinguish between H3.1 K56Ac and H3.3 K56Ac variants in their experiments?

Distinguishing between H3.1 K56Ac and H3.3 K56Ac variants requires specialized approaches:

• Variant-specific antibody selection:

  • Use antibodies specifically raised against H3.1 (acetyl-K56) vs. H3.3 (acetyl-K56)

  • Validate specificity using recombinant H3.1 and H3.3 proteins with K56Ac

• Mass spectrometry discrimination:

  • Exploit amino acid differences between H3.1 and H3.3 (they differ at positions 31, 87, 89, and 96)

  • Design targeted mass spectrometry that captures both the K56Ac modification and variant-specific residues

  • Use longer peptide fragments that span both K56 and variant-specific residues

• Genetic approaches:

  • Express tagged versions of H3.1 and H3.3 (FLAG-tagged or YFP-tagged)

  • Perform ChIP with variant-specific antibodies followed by K56Ac detection

  • Use H3.1 or H3.3 knockdown followed by K56Ac analysis to determine contribution of each variant

• Cell cycle analysis:

  • Since H3.1 is predominantly deposited during DNA replication while H3.3 is deposited throughout the cell cycle, cell synchronization can help distinguish their respective K56Ac patterns

  • Combine with FACS sorting to separate cells in different cell cycle phases

What are the most advanced methods for genome-wide mapping of K56Ac in relation to chromatin accessibility and other histone modifications?

The most advanced methods for genome-wide mapping of K56Ac in relation to chromatin features include:

• Integrated multi-omics approaches:

  • Sequential ChIP-seq for K56Ac followed by other modifications to identify co-occurring marks

  • ATAC-seq or DNase-seq paired with K56Ac ChIP-seq to correlate with chromatin accessibility

  • CUT&RUN or CUT&Tag for K56Ac, which offer improved signal-to-noise compared to conventional ChIP

• Single-cell epigenomic profiling:

  • Single-cell CUT&Tag for K56Ac to study cell-to-cell variability

  • Single-cell multiome approaches to simultaneously profile K56Ac and chromatin accessibility

  • Integration with single-cell transcriptomics to link K56Ac patterns with gene expression

• High-resolution genomic mapping:

  • ChIP-exo or ChIP-nexus for K56Ac to achieve near base-pair resolution of binding sites

  • Micro-C combined with K56Ac ChIP to relate modification to 3D chromatin structure

  • Advanced bioinformatic approaches like the Whitehead Neighborhood Model for peak calling

• Dynamic profiling:

  • Time-resolved ChIP-seq following stimulation or differentiation cues

  • SLAM-seq combined with K56Ac profiling to link nascent transcription with histone modification

  • Optogenetic approaches to induce targeted K56 acetylation and monitor genomic consequences

These advanced methodologies enable researchers to place K56Ac within the broader context of chromatin regulation and overcome the challenges posed by its low abundance in human cells.