Acetyl-HIST1H3A (K64) Antibody

What is the biological significance of H3K64 acetylation in chromatin regulation?

H3K64 acetylation represents a critical epigenetic modification located on the lateral surface of the histone octamer, in close proximity to the inner gyre of DNA. Unlike histone tail modifications, H3K64ac directly impacts nucleosome stability and dynamics. Research has demonstrated that this modification regulates chromatin accessibility by decreasing nucleosome stability, which facilitates nucleosome eviction and consequently promotes gene expression .

H3K64ac is particularly enriched at:

• Transcriptional start sites (TSS) of active genes

• Active enhancers, where it co-localizes with established enhancer marks such as H3K27ac, H3K4me1, and p300-binding

The functional significance of H3K64ac is highlighted by its mutually exclusive distribution with H3K64me3, a repressive mark associated with pericentromeric heterochromatin. At imprinted loci, the transcriptionally active alleles are specifically enriched in H3K64ac, whereas the inactive alleles are enriched in H3K64me3, suggesting these modifications define functionally opposing chromatin states .

How does H3K64ac differ from other histone H3 acetylation marks?

H3K64ac is distinct from commonly studied histone H3 acetylation marks in several key aspects:

The strategic location of H3K64ac on the lateral surface allows it to directly influence nucleosome biophysical properties, distinguishing it from tail modifications that primarily function through recruitment of effector proteins .

What are the recommended applications for Acetyl-HIST1H3A (K64) antibodies?

Based on the validation data, Acetyl-HIST1H3A (K64) antibodies have been successfully employed in the following applications:

When using these antibodies, it is critical to include appropriate controls to confirm specificity, particularly when investigating subtle changes in H3K64ac levels in different experimental conditions. The antibody's recognition of H3 is efficiently competed by K64ac immunizing peptide but not by other peptides containing acetylated, methylated, or unmodified histone regions .

How should I design ChIP-seq experiments to accurately profile H3K64ac genome-wide distribution?

When designing ChIP-seq experiments to profile H3K64ac, consider these critical methodological approaches:

Experimental Design Considerations:

• Crosslinking optimization: Since H3K64ac is located within the nucleosome core, optimize formaldehyde crosslinking time (8-10 minutes typically works well) to ensure adequate fixation without overfixation.

• Sonication parameters: Aim for fragments of 150-300bp to enhance resolution. Monitor fragmentation efficiency using a Bioanalyzer.

• Controls to include:

  • Input DNA control

  • IgG control for background binding

  • H3 ChIP for normalization (to distinguish changes in H3K64ac from changes in nucleosome occupancy)

  • Spike-in normalization with foreign chromatin (e.g., Drosophila) for quantitative comparisons

• Antibody validation: Perform peptide competition assays with K64-acetylated peptides versus other acetylated lysines to confirm specificity before ChIP-seq.

Data Analysis Guidelines:

• Correlate H3K64ac enrichment with:

  • RNA Polymerase II occupancy

  • Active histone marks (H3K9ac, H3K4me2)

  • Repressive marks (H3K27me3, H3K64me3) for anti-correlation validation

• Create meta-gene plots showing total H3 enrichment around TSSs grouped by expression level

• Analyze enhancer regions separately, with special focus on correlations with H3K27ac, H3K4me1, and p300-binding

From published studies, H3K64ac shows strong enrichment at TSSs of active genes, with a correlation coefficient of >0.8 with RNA Pol II occupancy. This enrichment is indicative of the steady-state mRNA level of the respective gene .

What experimental approaches can determine the functional consequences of H3K64ac on chromatin dynamics?

To investigate the functional impact of H3K64ac on chromatin dynamics, researchers have employed several sophisticated experimental approaches:

Biophysical and Biochemical Approaches:

• FRET measurements: Attach fluorescent dyes to specific sites on nucleosomal DNA (35bp from each end) and measure FRET signal changes during salt-dependent disruption. H3K64ac nucleosomes show decreased stability between ~0.5M and 1.0M NaCl concentrations.

• Single-molecule FRET: For more precise measurement of nucleosome stability at the single-molecule level, confirming lower stability of H3K64ac nucleosomes.

• ATP-dependent chromatin remodeling assays: Compare remodeling rates of H3K64ac versus unmodified nucleosomes with different remodeling enzymes (e.g., Chd1 shows faster repositioning of H3K64ac nucleosomes than unmodified ones, while RSC shows no difference).

• Site-specific incorporation of acetylated lysine: Use genetic approaches to incorporate acetyl-lysine directly at position 64 in recombinant histone H3 for in vitro studies.

Cellular and In Vivo Approaches:

• Expression of histone mutants: Compare wildtype H3.3, H3.3K64Q (acetylated lysine mimetic), and H3.3K64R (retains positive charge but is non-acetylatable) in cells.

• Gene expression analysis: Measure the effect of these mutants on gene expression, particularly for genes involved in specific pathways (e.g., TPA-dependent early-response genes).

• Chromatin accessibility assays: Use ATAC-seq or DNase-seq to determine if H3K64ac increases chromatin accessibility.

Data from such studies have shown that H3K64Q mutants can promote expression of certain genes (e.g., c-fos, Egr1, and c-myc) above levels obtained with wildtype H3.3 or acetylation-deficient H3.3K64R, demonstrating H3K64ac's intrinsic ability to impact transcription mechanisms in vivo .

How can I determine if p300/CBP is responsible for H3K64 acetylation in my experimental system?

To establish p300/CBP as H3K64 acetyltransferases in your experimental system, implement a multi-layered approach:

In Cellulo Approaches:

• Knockdown experiments: Systematically deplete candidate HATs from different families and assess changes in H3K64ac levels by Western blot. In published studies, knockdown of p300 and CBP, but not other HATs, decreased the steady-state levels of H3K64ac.

• ChIP analysis after knockdown: Perform ChIP with H3K64ac antibody after p300/CBP knockdown, focusing on p300/CBP-specific genomic target regions where the decrease should be most pronounced.

• Overexpression studies: Overexpress p300 in your system and measure H3K64ac levels. Published data show increased levels of H3K64ac upon p300 overexpression.

• Correlation analysis: Compare the genomic distribution of p300 binding sites with H3K64ac enrichment. Strong correlation would support a functional relationship.

In Vitro Biochemical Validation:

• HAT assays with recombinant proteins: Purify recombinant p300/CBP and incubate with:

  • Free histone H3

  • Recombinant nucleosomes

  • Native chromatin isolated from cells

• Mass spectrometry analysis: Confirm acetylation specifically at K64 rather than other residues by analyzing the products of the HAT reaction.

• Inhibitor studies: Use specific p300/CBP inhibitors (e.g., C646, A-485) to confirm the specificity of the acetylation reaction both in vitro and in cells.

Previous research has confirmed that p300 and CBP can acetylate H3K64 in vitro on both free H3 and within chromatin, and that p300 and H3K64ac distributions show a strong correlation genome-wide .

How should I address potential cross-reactivity when using Acetyl-HIST1H3A (K64) antibodies?

Cross-reactivity is a significant concern when using histone modification antibodies due to sequence similarities around different lysine residues. To address this issue:

Antibody Validation Approaches:

• Peptide competition assays: Test whether the antibody's recognition of H3 is competed by:

  • The immunizing K64ac peptide (should compete)

  • Other acetylated histone peptides (should not compete)

  • Methylated or unmodified histone regions (should not compete)

• Limited tryptic digestion: Perform limited tryptic digestion of native nucleosomes, which removes the H3 tails while leaving the DNA-protected H3 core region largely intact. A true H3K64ac antibody will still recognize the truncated H3 core (unlike antibodies to tail modifications like H3K9ac, H3K18ac, and H3K27ac).

• Knockout/mutation controls: Test the antibody on samples where:

  • H3K64 is mutated to arginine (K64R)

  • HATs responsible for H3K64ac (p300/CBP) are depleted or inhibited

Signs of Potential Cross-Reactivity:

• Detection of bands at unexpected molecular weights in Western blots

• Immunofluorescence patterns that don't match known H3K64ac distribution (nuclear with depletion from heterochromatin)

• ChIP-seq profiles that correlate better with other histone marks than expected

In published studies, properly validated H3K64ac antibodies specifically detect endogenous acetylated histone H3 and recognize a K64-acetylated peptide with high specificity compared to other H3 acetylated lysines .

How can I interpret changes in H3K64ac levels in relation to gene expression and other histone modifications?

Interpreting H3K64ac changes requires consideration of its functional context and relationship with other epigenetic marks:

Integration with Gene Expression Data:

• H3K64ac enrichment at TSSs positively correlates with gene expression levels.

• When analyzing changes in H3K64ac, group genes by expression level to assess if H3K64ac changes correspond to transcriptional changes.

• For genes showing discordant patterns (H3K64ac changes without expression changes), investigate compensatory mechanisms or additional regulatory factors.

Correlation with Other Histone Marks:
H3K64ac should be analyzed in relation to other marks:

Imprinted Loci Analysis:

• At imprinted control regions (ICRs), active alleles should be enriched in H3K64ac while inactive alleles should show H3K64me3 enrichment.

• Disruption of this pattern may indicate compromised imprinting regulation.

Developmental/Differentiation Context:

• During cellular differentiation, H3K64ac enrichment should shift from pluripotency genes to lineage-specific genes.

• In mouse embryonic stem (ES) cells, H3K64ac is enriched at pluripotency genes (e.g., Nanog, Pou5f1, Dppa3).

• After retinoic acid-induced differentiation, enrichment shifts towards differentiation-associated genes (e.g., Hoxb3, Hoxd3, Pax6).

When interpreting changes, consider that H3K64ac functions by directly affecting nucleosome stability rather than primarily through reader protein recruitment, distinguishing it from many other histone modifications .

What methodological considerations are important when analyzing H3K64ac in different cell types or disease models?

When analyzing H3K64ac across different biological contexts, consider these critical methodological aspects:

Cell Type Considerations:

• Cell cycle synchronization: H3K64ac levels may vary during cell cycle progression, particularly during S-phase when new nucleosomes are assembled. Consider synchronizing cells when making comparisons.

• Histone variant distribution: Since H3K64ac shows highest enrichment on H3.3 variant, cell types with different H3 variant compositions may show different baseline levels of H3K64ac.

• p300/CBP activity: Variations in p300/CBP activity or cofactor availability between cell types will affect H3K64ac levels. Measure these factors when comparing cell types.

Disease Model Considerations:

• Cancer models: Many cancers show altered p300/CBP activity or mutations. When analyzing H3K64ac in cancer models:

  • Account for copy number changes of histone genes

  • Consider competing modifications (H3K64me3)

  • Evaluate global versus gene-specific changes

• Developmental disorders: For disorders with known chromatin dysregulation:

  • Focus on loci with established developmental regulation

  • Consider imprinted regions where H3K64ac and H3K64me3 show allele-specific patterns

  • Analyze enhancer regions where H3K64ac may mark active enhancers

Technical Normalization Approaches:

• Internal normalization: Always normalize H3K64ac signal to total H3 levels to account for differences in nucleosome density.

• Spike-in controls: Use spike-in chromatin from a different species (e.g., Drosophila) for quantitative comparisons across samples.

• Multi-antibody validation: For critical findings, confirm results using independent antibodies against H3K64ac or orthogonal approaches like mass spectrometry.

Published research has shown that H3K64ac is present in various mouse and human cell lines and tissues, suggesting a ubiquitous function. HDAC-inhibitor treatment increases H3K64ac levels, which may be relevant when studying diseases treated with such inhibitors .

How can I optimize ChIP-qPCR protocols specifically for H3K64ac detection?

Optimizing ChIP-qPCR for H3K64ac requires attention to several parameters specific to this modification:

Sample Preparation Optimization:

• Crosslinking conditions: Test multiple formaldehyde concentrations (1-1.5%) and fixation times (8-12 minutes) to optimize crosslinking without affecting epitope accessibility.

• Chromatin preparation: Since H3K64 is within the nucleosome core and close to DNA, ensure thorough sonication to expose the epitope (verify fragment size of 200-300bp).

• Antibody concentration: Titrate antibody amounts (2-10μg per ChIP) to determine optimal concentration that maximizes signal-to-noise ratio.

Control Region Selection:

Protocol Adjustments:

• Washing stringency: Optimize salt concentration in wash buffers (150-500mM NaCl) to reduce background while maintaining specific binding.

• Elution conditions: For efficient elution of H3K64ac-bound chromatin, consider using SDS concentrations of 1-2% at 65°C.

• qPCR primer design: Design primers to amplify 80-150bp regions, avoiding regions with repetitive sequences.

Validation Strategy:

• Perform ChIP for total H3 in parallel to normalize H3K64ac signal

• Include IgG control to establish background levels

• Test regions with known H3K64ac enrichment (based on published ChIP-seq data)

• Compare results with other active marks (H3K9ac, H3K27ac) as correlation controls

In published studies, H3K64ac ChIP-qPCR has been successfully used to validate genome-wide data and confirm enrichment at active promoters and enhancers, with very low levels at repetitive elements .

What is the relationship between H3K64ac and nucleosome stability, and how can I study this experimentally?

H3K64ac has been shown to decrease nucleosome stability, facilitating DNA accessibility and nucleosome eviction. To investigate this relationship:

Biophysical Approaches to Measure Nucleosome Stability:

• Salt-dependent nucleosome stability assays: Reconstitute nucleosomes with recombinant H3K64ac (using genetic incorporation of acetyl-lysine) and measure stability across a salt gradient (0.2-2.0M NaCl). H3K64ac nucleosomes display decreased stability particularly between 0.5-1.0M NaCl.

• FRET-based nucleosome stability assays: Attach fluorescent dyes to nucleosomal DNA ends to monitor DNA unwrapping. Technical approach:

  • Label DNA at positions ~35bp from each end

  • Reconstitute nucleosomes with labeled DNA and H3K64ac or unmodified H3

  • Measure FRET signal changes during salt titration or thermal denaturation

• Single-molecule FRET: For higher resolution analysis of nucleosome dynamics:

  • Immobilize individual nucleosomes on microscope slides

  • Monitor real-time fluctuations in FRET efficiency

  • Compare "breathing" rates between H3K64ac and unmodified nucleosomes

Biochemical and Cellular Approaches:

• Nucleosome assembly/disassembly assays: Compare rates of assembly/disassembly between H3K64ac and unmodified nucleosomes.

• Chromatin remodeling assays: Test whether H3K64ac affects the activity of chromatin remodeling enzymes:

  • Reconstitute positioned nucleosomes with modified or unmodified H3

  • Incubate with remodelers like Chd1 or RSC

  • Analyze nucleosome repositioning by native PAGE

• Histone eviction in cells: Express H3K64Q (acetylated lysine mimetic) or H3K64R (non-acetylatable) and measure:

  • Histone turnover rates using SNAP-tag pulse-chase

  • Chromatin accessibility by ATAC-seq

  • Transcription factor binding by ChIP

Research has shown that H3K64ac decreases nucleosome stability by approximately 60mM in salt-dependent stability assays compared to unmodified nucleosomes. This effect is distinct from H3K56ac, which was reported not to significantly affect nucleosome stability under comparable conditions .

How can I integrate H3K64ac ChIP-seq data with other genomic datasets to understand chromatin state dynamics?

Integrating H3K64ac ChIP-seq with other genomic datasets requires systematic computational approaches:

Integration with Histone Modification Data:

• Correlation analysis: Calculate Pearson correlations between H3K64ac and other histone modifications:

  • Active marks (H3K4me3, H3K9ac, H3K27ac)

  • Enhancer marks (H3K4me1, H3K27ac)

  • Repressive marks (H3K27me3, H3K9me3, H3K64me3)

• Chromatin state prediction: Use hidden Markov model-based approaches (e.g., ChromHMM) to define chromatin states incorporating H3K64ac with other marks.

• Genomic feature analysis: Create meta-profiles of H3K64ac around:

  • Transcriptional start sites (grouped by expression level)

  • Enhancers (active vs. poised)

  • CTCF binding sites

  • Domain boundaries

Integration with Transcription Factor Binding and Accessibility Data:

• Co-localization analysis: Assess overlap between H3K64ac and:

  • Transcription factor binding sites (esp. p300/CBP)

  • Chromatin accessibility regions (DNase-seq, ATAC-seq)

  • CpG islands and other DNA features

• Motif enrichment: Identify transcription factor motifs enriched in H3K64ac peaks to identify potential regulatory mechanisms.

Integration with Expression Data:

• Correlation with gene expression: Group genes by expression levels and analyze H3K64ac patterns.

• Differential analysis: For treatment/condition comparisons:

  • Identify regions with differential H3K64ac

  • Correlate with differential gene expression

  • Analyze pathway enrichment for genes with concordant changes

Visualization and Analysis Tools:

• Genome browsers (e.g., UCSC, IGV) for visual inspection

• deepTools for generating heatmaps and profile plots

• Bioconductor packages for statistical analysis

• WashU Epigenome Browser for multi-sample comparisons

Research has demonstrated that H3K64ac clustering with H3K4me1 in correlation analyses indicates its role at enhancers. Additionally, when enhancers are defined by H3K4me1 peaks ±2kb away from TSSs, they can be clustered into three groups based on H3K64ac, H3K27ac, and H3K122ac patterns, revealing subclasses of regulatory elements with distinct properties .

What are emerging research areas regarding the function of H3K64ac in development and disease?

Several promising research directions are emerging regarding H3K64ac's role in biological processes:

Developmental Biology:

• Cell fate decisions: Investigating how H3K64ac dynamics contribute to developmental transitions and lineage commitment. Preliminary data suggest H3K64ac shifts from pluripotency genes to differentiation-specific genes during development.

• Epigenetic inheritance: Exploring whether H3K64ac patterns can be transmitted through cell divisions and contribute to epigenetic memory.

• Imprinting regulation: Further characterizing the role of H3K64ac/H3K64me3 in maintaining imprinted gene expression patterns, as these marks show allele-specific distribution at imprinting control regions.

Disease Implications:

• Cancer epigenetics: Investigating whether aberrant H3K64ac patterns contribute to oncogenic gene expression programs, particularly in cancers with p300/CBP mutations.

• Neurodevelopmental disorders: Examining H3K64ac in conditions linked to chromatin dysregulation (e.g., Rubinstein-Taybi syndrome, caused by mutations in CBP).

• Inflammatory diseases: Exploring the role of H3K64ac in rapid inflammatory gene activation, as p300/CBP are known regulators of inflammatory transcription factors.

Novel Mechanistic Questions:

• Reader proteins: Identifying potential proteins that specifically recognize H3K64ac, if any exist, despite challenges in detecting specific readers.

• Interplay with DNA methylation: Investigating how H3K64ac affects or is affected by DNA methylation patterns, particularly at regulatory elements.

• Chromatin higher-order structure: Determining how H3K64ac influences higher-order chromatin organization and nuclear compartmentalization.

• Crosstalk with other lateral surface modifications: Exploring functional relationships between H3K64ac and other lateral surface modifications like H3K56ac and H3K122ac.

Early findings show that H3K64ac functions without identified specific readers, suggesting a direct biophysical effect on chromatin structure. This mechanism is distinct from many histone tail modifications that function primarily through reader protein recruitment .

How might technological advances improve detection and functional analysis of H3K64ac?

Emerging technologies hold promise for advancing H3K64ac research:

Next-Generation Antibody Technologies:

• Recombinant antibodies: Development of recombinant monoclonal antibodies with enhanced specificity and lot-to-lot consistency.

• Nanobodies/single-domain antibodies: Smaller antibody fragments that may access H3K64ac more efficiently within compact chromatin structures.

• CUT&Tag/CUT&RUN adaptations: Optimizing these techniques specifically for H3K64ac to achieve higher resolution and sensitivity with less input material.

Mass Spectrometry Advances:

• Targeted MS approaches: Developing sensitive, targeted MS methods to quantify H3K64ac absolute levels across conditions.

• Middle-down MS: Analyzing larger histone fragments to understand combinatorial patterns of H3K64ac with other modifications.

• Crosslinking MS: Identifying proteins interacting with H3K64ac regions through crosslinking approaches.

Single-Cell Technologies:

• Single-cell ChIP-seq adaptations: Developing protocols to map H3K64ac in individual cells to understand heterogeneity.

• Multi-omics integration: Combining single-cell H3K64ac profiling with transcriptomics and other epigenetic marks.

• Live-cell imaging: Creating systems to visualize H3K64ac dynamics in living cells, potentially using engineered reader domains or antibody fragments.

Genome Engineering Approaches:

• Base editing technologies: Precise editing of H3K64 residue in endogenous histones to study functional consequences.

• Targeted modification systems: Developing tools to target acetyltransferases or deacetylases specifically to genomic loci to manipulate H3K64ac locally.

• Degron-based approaches: Controlling histone variant levels and turnover to study H3K64ac dynamics.

These technological advances would address current limitations in studying H3K64ac, including improved sensitivity for detecting low-abundance modifications, better understanding of combinatorial patterns, and enhanced spatial and temporal resolution of H3K64ac dynamics .

What is the current understanding of the interplay between H3K64ac and other lateral surface modifications?

The relationship between H3K64ac and other lateral surface modifications represents an emerging area of research:

Comparative Analysis of Lateral Surface Modifications:

Functional Interrelationships:

• Co-occurrence patterns: H3K64ac and H3K122ac frequently co-occur at active regulatory elements, particularly at enhancers and promoters, suggesting cooperative functions.

• Distinct mechanisms: Despite similar genomic distributions, these modifications affect nucleosome dynamics through different mechanisms:

  • H3K64ac: Located near the inner gyre of DNA, affecting DNA-histone contacts

  • H3K56ac: Located near DNA entry/exit points, affecting DNA unwrapping

  • H3K122ac: Located at the dyad axis, directly disrupting central histone-DNA contacts

• Differential effects on remodeling: H3K64ac enhances Chd1-mediated nucleosome repositioning but not RSC activity, suggesting specificity in remodeler interactions.

Combinatorial Effects:
Recent research suggests that combinations of lateral surface modifications may have synergistic effects on nucleosome dynamics. For example:

• H3K64ac+H3K122ac may destabilize nucleosomes more effectively than either modification alone

• The presence of multiple lateral surface acetylations may create a more permissive chromatin state than achievable by tail modifications

Biological Context Specificity:

• H3K64ac appears particularly important for developmental gene regulation

• H3K56ac is strongly linked to replication and DNA repair processes

• H3K122ac has been identified as marking a novel class of active enhancers lacking H3K27ac