Acetyl-Histone H4 (K16) Recombinant Monoclonal Antibody

What is the biological significance of histone H4 lysine 16 acetylation?

Histone H4 lysine 16 acetylation (H4K16ac) serves as a critical switch in the transition of chromatin from a repressive to a transcriptionally active state. Unlike other histone modifications, H4K16ac directly impacts chromatin structure by inhibiting the formation of compact 30-nanometer chromatin fibers and preventing fiber-fiber interactions, effectively decondensing the chromatin structure . This structural alteration facilitates transcriptional activation by making DNA more accessible to transcription machinery. Studies have demonstrated that H4K16ac is uniquely positioned to influence genome-wide chromatin dynamics, making it a vital epigenetic modification for gene transcription regulation .

How does H4K16 acetylation differ from other histone modifications in its functional impact?

H4K16 acetylation stands apart from other histone modifications in several significant ways:

• Direct structural impact: H4K16ac is the only histone modification definitively linked to direct changes in chromatin folding and higher-order structure .

• Boundary maintenance: In organisms like Saccharomyces cerevisiae, H4K16ac is essential for maintaining proper boundaries between transcriptionally active euchromatin and repressed heterochromatin at silent loci, including mating-type loci, telomeres, and rDNA arrays .

• Species-specific roles: H4K16ac serves in dosage compensation in fruit flies and is frequently lost in human cancer cells, suggesting its role as a potential cancer biomarker .

• Enzyme interaction: Beyond structural effects, H4K16ac modulates functional interactions with chromatin remodeling complexes, specifically inhibiting the activity of ATP-utilizing chromatin assembly and remodeling enzymes like ACF .

What are the key functional domains and protein interactions affected by H4K16 acetylation?

H4K16 acetylation affects several key functional domains and protein interactions:

These interactions demonstrate how a single modification can orchestrate complex changes in chromatin architecture and function .

What are the validated applications for Acetyl-Histone H4 (K16) Recombinant Monoclonal Antibody?

The Acetyl-Histone H4 (K16) Recombinant Monoclonal Antibody has been validated for multiple research applications, enabling comprehensive investigation of this epigenetic mark. According to manufacturer specifications, this antibody has been successfully employed in the following techniques:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative assessment of H4K16ac levels in protein samples .

• Western Blot (WB): For detection of H4K16ac in cell and tissue lysates, allowing protein size confirmation .

• Immunocytochemistry (ICC): For cellular localization studies of H4K16ac .

• Immunofluorescence (IF): For visualization of H4K16ac distribution within cellular compartments .

• Flow Cytometry (FC): For quantitative analysis of H4K16ac levels in individual cells within populations .

Each application requires specific optimization parameters including antibody dilution, incubation conditions, and detection methods to ensure reliable results.

How should researchers select between different antibody clones for H4K16ac detection in specific experimental contexts?

When selecting an optimal Acetyl-Histone H4 (K16) antibody for specific experimental applications, researchers should consider several critical factors:

• Epitope specificity: Confirm that the antibody specifically recognizes acetylated K16 without cross-reactivity to other acetylated lysine residues on H4 (such as K5, K8, or K12). This is particularly important as these marks often co-occur in active chromatin .

• Species cross-reactivity: Verify the antibody's compatibility with your model organism. The recombinant monoclonal antibody against H4K16ac from Biomatik recognizes human antigens, but validation for other species should be confirmed before use .

• Application compatibility: Select antibodies validated for your specific application. While some antibodies perform well across multiple techniques, others may be optimized for specific applications like ChIP or IF .

• Monoclonal vs. polyclonal: Monoclonal antibodies offer higher specificity but might recognize a single epitope, while polyclonal antibodies provide broader epitope recognition but potential batch-to-batch variability.

• Published validation: Review literature citations supporting the antibody's use in your specific application to gauge reliability and performance.

What controls should be included when using Acetyl-Histone H4 (K16) antibodies in chromatin immunoprecipitation (ChIP) experiments?

For robust ChIP experiments using Acetyl-Histone H4 (K16) antibodies, the following controls are essential:

• Input DNA control: A sample of chromatin before immunoprecipitation to normalize for DNA abundance and technical variations.

• Isotype control: An antibody of the same isotype but irrelevant specificity to assess non-specific binding.

• Positive control regions: Genomic loci known to be enriched for H4K16ac (e.g., actively transcribed genes).

• Negative control regions: Genomic loci known to lack H4K16ac (e.g., silent heterochromatic regions).

• Acetylation inhibitor control: Cells treated with histone deacetylase inhibitors (e.g., trichostatin A) to increase global H4K16ac levels as a positive biological control.

• Deacetylation verification: Samples where H4K16ac has been enzymatically removed to confirm antibody specificity.

• Spike-in control: Addition of chromatin from a different species for normalization across samples when comparing different experimental conditions.

Inclusion of these controls ensures reliable interpretation of ChIP data and helps distinguish true biological signals from technical artifacts.

How should researchers optimize immunofluorescence protocols for detecting H4K16ac in different cell types?

Optimizing immunofluorescence protocols for H4K16ac detection requires careful consideration of fixation, permeabilization, and antibody conditions:

• Fixation optimization:

  • For most cell types, 4% paraformaldehyde for 10-15 minutes at room temperature preserves nuclear structure while maintaining epitope accessibility.

  • Avoid overfixation, which can mask the H4K16ac epitope through excessive protein crosslinking.

  • For some sensitive cell types, methanol fixation (-20°C for 10 minutes) may provide better results.

• Permeabilization parameters:

  • Use 0.1-0.5% Triton X-100 for 5-10 minutes to allow antibody access to nuclear antigens.

  • For dense chromatin, consider additional permeabilization with 0.5% SDS for 5 minutes to improve epitope accessibility.

• Blocking and antibody incubation:

  • Implement extended blocking (1-2 hours) with 5% BSA or 10% normal serum to reduce background.

  • Optimize antibody concentration through titration experiments (typically 1:100 to 1:500 dilutions).

  • Consider overnight incubation at 4°C for improved signal-to-noise ratio.

• Signal amplification considerations:

  • For cells with low H4K16ac levels, employ tyramide signal amplification systems.

  • Use high-sensitivity detection systems like quantum dots for quantitative analyses.

• Cell-type specific considerations:

  • Highly proliferative cells often exhibit higher H4K16ac levels during S-phase.

  • Primary cells may require gentler permeabilization than established cell lines.

Thorough validation through appropriate controls, including H4K16ac-depleted samples and competitive blocking with acetylated peptides, ensures specificity of the observed signals .

What are the recommended approaches for quantifying global changes in H4K16ac levels across experimental conditions?

Several complementary approaches can be employed to quantify global H4K16ac changes:

For optimal results, researchers should:

• Establish a standard curve using recombinant acetylated histones for quantitative methods.

• Normalize H4K16ac signals to total H4 protein to account for variations in histone levels.

• Include biological replicates (minimum n=3) and technical replicates for statistical validity.

• Consider cell cycle synchronization as H4K16ac levels fluctuate during different cell cycle phases .

How can the specificity of Acetyl-Histone H4 (K16) antibody be validated in experimental systems?

Rigorous validation of Acetyl-Histone H4 (K16) antibody specificity requires multiple complementary approaches:

• Peptide competition assays: Pre-incubate the antibody with acetylated H4K16 peptides before application to samples. Specific binding should be blocked, resulting in signal reduction.

• Acetylation-deficient mutants: Test the antibody against samples from cells expressing H4K16R or H4K16A mutants (which cannot be acetylated at position 16) to confirm absence of signal.

• Enzymatic manipulation:

  • Treat samples with histone deacetylases specific for H4K16ac to reduce signals.

  • Conversely, increase acetylation using HDAC inhibitors or by overexpressing acetylation enzymes like MOF/MYST1 that target H4K16.

• Cross-reactivity testing: Evaluate antibody response to peptide arrays containing various histone modifications, particularly other acetylated lysines on H4 (K5, K8, K12).

• Orthogonal method verification: Compare results with alternative detection methods like mass spectrometry to confirm specificity.

• Knockout validation: When possible, use genetic knockout/knockdown of enzymes responsible for H4K16 acetylation (like MOF/KAT8) to demonstrate reduction in signal.

Documentation of these validation steps enhances experimental reliability and supports the interpretation of results obtained with Acetyl-Histone H4 (K16) antibodies .

How should researchers interpret H4K16ac changes in the context of broader histone modification patterns?

Interpreting H4K16ac changes requires consideration of the broader epigenetic landscape:

• Histone modification co-occurrence patterns:

  • H4K16ac frequently co-occurs with other active marks like H3K4me3 and H3K27ac at promoters and enhancers.

  • An unexpected dissociation from these patterns may indicate unique regulatory mechanisms.

  • Create correlation matrices of multiple histone marks across experimental conditions to identify coordinated or divergent responses.

• Chromatin state transitions:

  • H4K16ac changes should be analyzed in relation to chromatin compaction state.

  • Increases in H4K16ac without corresponding transcriptional activation may indicate competing repressive mechanisms.

  • Decreases in H4K16ac at previously active regions suggest establishment of heterochromatin boundaries .

• Genomic context dependencies:

  • H4K16ac has different interpretations depending on genomic location:

    • At telomeres: anti-silencing function and boundary element role

    • At gene bodies: transcriptional elongation support

    • At promoters: transcription initiation facilitation

• Temporal dynamics:

  • Rapid H4K16ac changes may indicate immediate-early gene responses

  • Stable, long-term changes suggest established epigenetic reprogramming

• Cross-talk analysis:

  • Changes in H4K16ac should be evaluated alongside modifications that antagonize or synergize with it

  • Consider enzyme competition, where HATs and HDACs targeting H4K16 may be redirected to different genomic locations under experimental conditions .

What are the methodological considerations for studying the relationship between H4K16ac and chromatin structure?

Investigating the relationship between H4K16ac and chromatin structure requires specialized approaches:

• In vitro reconstitution systems:

  • Use chemically ligated H4 homogeneously acetylated at K16 to assemble nucleosomal arrays.

  • Apply biophysical techniques such as analytical ultracentrifugation to measure sedimentation coefficients that reflect chromatin compaction states.

  • Employ electron microscopy to directly visualize structural differences between acetylated and non-acetylated chromatin fibers .

• Micrococcal nuclease (MNase) accessibility assays:

  • Compare the MNase digestion patterns between regions with high and low H4K16ac levels.

  • Analyze the correlation between H4K16ac enrichment and nucleosome positioning/occupancy maps.

  • Quantify differences in chromatin accessibility using MNase-seq or similar techniques.

• Chromosome conformation capture techniques:

  • Apply 3C/4C/Hi-C methodologies to assess how H4K16ac impacts long-range chromatin interactions.

  • Compare chromatin interaction frequencies between regions with differential H4K16ac under experimental conditions.

  • Identify topologically associating domains (TADs) whose boundaries correlate with H4K16ac enrichment.

• ATAC-seq and DNase-seq integration:

  • Correlate H4K16ac ChIP-seq profiles with chromatin accessibility data.

  • Analyze how experimental manipulation of H4K16ac levels affects genome-wide accessibility patterns.

  • Develop quantitative models relating H4K16ac density to chromatin openness.

• Live-cell imaging approaches:

  • Use H4K16ac-specific antibody fragments or acetylation-sensitive chromatin readers fused to fluorescent proteins.

  • Apply super-resolution microscopy to visualize chromatin compaction states in relation to H4K16ac.

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure chromatin dynamics in regions with varying H4K16ac levels .

How can researchers investigate the interplay between H4K16ac and chromatin remodeling complexes?

Investigating the interplay between H4K16ac and chromatin remodeling complexes requires multifaceted approaches:

• Biochemical interaction assays:

  • Perform pull-down experiments using acetylated and non-acetylated H4K16 peptides as bait.

  • Conduct co-immunoprecipitation studies to identify differential interactions of remodeling complexes with H4K16ac-enriched chromatin.

  • Use quantitative proteomics to identify proteins that preferentially bind to or are excluded from H4K16-acetylated nucleosomes.

• Functional enzyme assays:

  • Measure ATPase activity of remodeling enzymes like ACF in the presence of H4K16ac-containing nucleosomes.

  • Assess nucleosome sliding rates using positioned nucleosome templates with or without H4K16ac.

  • Quantify remodeling complex residence time on differentially modified chromatin templates.

• Genomic co-localization analysis:

  • Perform ChIP-seq for both H4K16ac and chromatin remodelers to identify regions of co-occupancy or mutual exclusion.

  • Analyze how experimental manipulation of H4K16ac levels affects genomic distribution of remodeling complexes.

  • Create genome-wide maps of remodeler binding in wild-type versus H4K16 mutant backgrounds.

• In vitro reconstitution systems:

  • Reconstitute chromatin with specifically modified histones to test remodeling complex activity.

  • Use single-molecule techniques to visualize how H4K16ac affects remodeler binding and activity in real-time.

  • Implement FRET-based assays to measure conformational changes in chromatin induced by remodelers in the context of H4K16ac.

Research has shown that H4K16ac specifically inhibits the activity of the ACF chromatin remodeling complex, suggesting a mechanism by which this modification maintains open chromatin states by preventing remodeler-mediated compaction .

How should researchers address inconsistent or contradictory results in H4K16ac detection across different experimental methods?

When faced with discrepancies in H4K16ac detection across methods, consider these systematic troubleshooting approaches:

• Methodological sensitivity differences:

  • Western blotting may show global changes that are diluted in ChIP-seq analyses due to genomic averaging.

  • Immunofluorescence may detect nuclear subcompartment-specific changes missed by bulk methods.

  • Establish detection limits for each method and determine if discrepancies fall within these technical constraints.

• Epitope occlusion considerations:

  • In ChIP experiments, protein complexes may mask the H4K16ac epitope in specific genomic contexts.

  • In fixed samples, crosslinking can reduce epitope accessibility differently across preparation methods.

  • Test alternative fixation/extraction protocols to uncover potentially masked epitopes.

• Antibody-specific artifacts:

  • Different antibody clones may have varying specificities for the acetylated epitope.

  • Validate results using multiple independent antibodies targeting the same modification.

  • Perform peptide competition assays with all antibodies to confirm specificity.

• Context-dependent effects:

  • H4K16ac may have different stability in various chromatin environments.

  • Neighboring modifications might influence antibody recognition.

  • Map the complete modification landscape at discrepant regions.

• Temporal dynamics:

  • Inconsistencies may reflect different temporal dynamics captured by different methods.

  • Implement time-course experiments with consistent sampling across all methods.

• Biological heterogeneity:

  • Cell population heterogeneity may be detected differently by population-averaging versus single-cell methods.

  • Consider cell sorting or single-cell approaches to resolve population-based inconsistencies .

What are the key considerations when designing experiments to study the causal relationship between H4K16ac and gene expression?

Establishing causality between H4K16ac and gene expression requires carefully designed experiments:

• Targeted manipulation approaches:

  • Deploy site-specific acetyltransferases (e.g., dCas9-p300 fusion) to induce H4K16ac at specific loci.

  • Use deacetylases (e.g., dCas9-HDAC fusion) for targeted removal of H4K16ac.

  • Implement inducible systems to control the timing of acetylation changes.

• Genetic manipulation strategies:

  • Generate H4K16R or H4K16Q mutants (mimicking unacetylated or acetylated states, respectively).

  • Create conditional knockouts of enzymes responsible for H4K16 acetylation/deacetylation.

  • Employ RNA interference or CRISPR approaches to modulate expression of acetylation machinery.

• Temporal resolution considerations:

  • Implement time-course experiments following acetylation state changes.

  • Use rapid induction systems (e.g., auxin-inducible degradation) to determine immediate effects.

  • Distinguish between direct and indirect effects through early time point analyses.

• Controls for specificity:

  • Include manipulations of other histone modifications as comparative controls.

  • Test effects on genes known to be responsive or non-responsive to chromatin state changes.

  • Use genome-wide approaches to identify all affected loci and common features.

• Functional readouts beyond expression:

  • Assess changes in chromatin accessibility (ATAC-seq, DNase-seq).

  • Measure RNA polymerase II occupancy and phosphorylation status.

  • Analyze transcription initiation versus elongation effects.

• Mechanistic dissection:

  • Identify proteins recruited to or displaced from H4K16ac regions.

  • Determine if H4K16ac effects require specific reader proteins.

  • Test whether chromatin structural changes are necessary for expression effects .

How can researchers distinguish direct effects of H4K16ac from indirect consequences in multi-omics datasets?

Distinguishing direct from indirect effects of H4K16ac in multi-omics datasets requires sophisticated analytical approaches:

• Temporal analysis frameworks:

  • Implement densely sampled time-course experiments after targeted H4K16ac manipulation.

  • Apply mathematical modeling to identify immediate versus delayed responses.

  • Use clustering algorithms to group genes by response kinetics.

• Spatial correlation assessment:

  • Analyze the spatial relationship between H4K16ac changes and alterations in other genomic features.

  • Calculate distance dependencies between H4K16ac sites and expression changes.

  • Implement genomic neighborhood analyses to identify local versus distant effects.

• Integrative data analysis strategies:

  • Construct conditional dependency networks from multi-omics data.

  • Apply causal inference algorithms like directed acyclic graphs (DAGs).

  • Use mediation analysis to identify molecular intermediaries between H4K16ac and expression changes.

• Perturbation validation approaches:

  • Systematically inhibit potential mediators of H4K16ac effects.

  • Create genetic backgrounds lacking specific reader proteins.

  • Deploy orthogonal techniques to verify computational predictions.

• Data integration visualization:

  • Generate multi-layer genome browsers displaying aligned datasets.

  • Implement dimensionality reduction to visualize relationships between different data types.

  • Develop quantitative metrics for direct versus indirect effect likelihood.

• Feature importance quantification:

  • Apply machine learning algorithms to rank features predicting expression changes.

  • Calculate information gain from H4K16ac in predictive models.

  • Perform bootstrapping analyses to estimate confidence in feature importance rankings .

What emerging technologies might enhance the study of H4K16ac dynamics and functions?

Several cutting-edge technologies are poised to revolutionize H4K16ac research:

• Single-molecule imaging techniques:

  • Super-resolution microscopy approaches like STORM and PALM can visualize individual H4K16ac marks in the nuclear context.

  • Single-molecule tracking can reveal the dynamic establishment and removal of H4K16ac in living cells.

  • Correlative light and electron microscopy can connect H4K16ac patterns with chromatin ultrastructure.

• Mass spectrometry innovations:

  • Top-down proteomics can analyze intact histone proteins with multiple modifications simultaneously.

  • Crosslinking mass spectrometry can identify proteins in proximity to H4K16ac in native chromatin.

  • Imaging mass spectrometry could map H4K16ac distribution across tissue sections with subcellular resolution.

• Single-cell multi-omics:

  • Integration of single-cell ChIP-seq, RNA-seq, and ATAC-seq can reveal cell-specific relationships between H4K16ac and gene expression.

  • Single-cell proteomics approaches could quantify H4K16ac alongside hundreds of other chromatin factors.

  • Spatial transcriptomics combined with H4K16ac detection can map territorial organization of acetylation patterns.

• Synthetic biology approaches:

  • Optogenetic tools for rapid, reversible modulation of H4K16ac at specific genomic loci.

  • Synthetic chromatin systems with designer histone modifications for mechanistic studies.

  • Engineered reader domains with tunable specificity for H4K16ac to probe functional consequences.

• Computational methods:

  • Deep learning algorithms to predict H4K16ac patterns from DNA sequence and other epigenetic marks.

  • Network modeling approaches to understand H4K16ac in the context of broader epigenetic circuits.

  • Multi-scale simulations connecting molecular dynamics of acetylated chromatin to higher-order structures .

How might understanding of H4K16ac contribute to therapeutic developments for chromatin-related disorders?

H4K16ac research holds significant potential for therapeutic innovations in chromatin-related disorders:

• Cancer therapeutics:

  • Loss of H4K16ac is a hallmark of many cancers, suggesting restoration strategies could have anti-tumor effects.

  • Selective inhibitors of histone deacetylases targeting H4K16ac could reactivate silenced tumor suppressor genes.

  • Diagnostic applications using H4K16ac as a biomarker might enable early detection or classification of cancer subtypes .

• Neurodevelopmental disorders:

  • Mutations in enzymes regulating H4K16ac (like MOF/KAT8) have been linked to intellectual disability syndromes.

  • Targeted modulation of H4K16ac readers could potentially correct aberrant gene expression in neurodevelopmental conditions.

  • H4K16ac profiling might identify subtypes of autism spectrum disorders with distinct epigenetic signatures.

• Aging-related conditions:

  • H4K16ac changes during cellular senescence and organismal aging suggest intervention points for age-related diseases.

  • Compounds that maintain youthful H4K16ac patterns could potentially delay aspects of aging.

  • Integration with other epigenetic clocks may improve understanding of biological versus chronological aging.

• Metabolic disorders:

  • H4K16ac responds to metabolic states, suggesting therapeutic approaches linking metabolism and epigenetics.

  • Dietary interventions affecting acetyl-CoA availability might modulate H4K16ac in metabolic tissues.

  • Engineering synthetic readers to detect metabolic-epigenetic imbalances could enable personalized interventions.

• Drug development platforms:

  • High-throughput screening systems to identify compounds affecting H4K16ac at specific genomic loci.

  • Reporter systems to monitor H4K16ac dynamics in response to therapeutic candidates.

  • Predictive frameworks to identify patient populations likely to respond to H4K16ac-targeting therapies .

What are the key challenges and potential solutions in achieving site-specific manipulation of H4K16ac for mechanistic studies?

Precise manipulation of H4K16ac faces significant challenges, with several emerging solutions:

• Challenge: Genomic specificity

  • Current limitations: Traditional HDAC inhibitors or HAT activators affect H4K16ac genome-wide.

  • Emerging solutions:

    • CRISPR-dCas9 fused to acetyltransferases (KAT8/MOF) or deacetylases (SIRT1) for locus-specific targeting.

    • Zinc finger or TALE nucleases linked to modifying enzymes for alternative targeting strategies.

    • RNA-guided recruitment of modifying enzymes using Cas13 systems to achieve transcript-associated H4K16ac changes.

• Challenge: Temporal control

  • Current limitations: Constitutive expression systems cannot reveal immediate versus adaptive responses.

  • Emerging solutions:

    • Optogenetic systems enabling light-controlled induction of H4K16ac changes.

    • Chemical-inducible proximity systems for rapid and reversible enzyme recruitment.

    • Temperature-sensitive degrons fused to relevant enzymes for temporal regulation.

• Challenge: Modification specificity

  • Current limitations: Enzymes often modify multiple residues beyond H4K16.

  • Emerging solutions:

    • Engineered enzymes with enhanced substrate specificity through rational design or directed evolution.

    • Orthogonal acyl-transferase systems recognizing artificial histone sequences inserted at specific loci.

    • Split-enzyme complementation strategies requiring multiple targeting events for activity.

• Challenge: Readout sensitivity

  • Current limitations: Detecting consequences of single-locus H4K16ac changes is technically difficult.

  • Emerging solutions:

    • Single-molecule imaging of transcription at targeted loci.

    • Massively parallel reporter assays to test thousands of regulatory elements simultaneously.

    • Live-cell biosensors of chromatin compaction states to visualize structural consequences.

• Challenge: Off-target effects

  • Current limitations: Manipulating one modification often affects others through crosstalk.

  • Emerging solutions:

    • Comprehensive epigenome profiling after targeted manipulation to identify and account for secondary changes.

    • Combinatorial targeting of multiple modifications simultaneously to parse individual contributions.

    • Computational modeling to predict and mitigate off-target consequences .