Acetyl-Histone H4 (K5) Recombinant Monoclonal Antibody

What is the biological significance of H4K5 acetylation in chromatin regulation?

H4K5 acetylation is a post-translational modification occurring at lysine 5 of the histone H4 N-terminal tail that serves multiple critical functions in genome regulation. This modification predominantly promotes chromatin decondensation, contributing to transcriptional activation by making DNA more accessible to transcription machinery . H4K5 acetylation is also intimately involved in DNA repair processes and chromatin assembly pathways .

Particularly noteworthy is that H4K5 acetylation, along with H4K12 acetylation, forms a specific diacetylation pattern in newly assembled histones. This diacetylation is established by histone acetyltransferases (HATs) in predeposition complexes before incorporation into chromatin . While not strictly required for histone assembly, these modifications appear to stimulate nuclear import of new histones and contribute to recovery from replication block-mediated DNA damage . The modification also participates in a complex regulatory code with other histone marks that collectively fine-tune gene expression patterns .

How does H4K5 acetylation differ from other acetylation sites on histone H4?

Histone H4 contains multiple lysine residues that can be acetylated (K5, K8, K12, and K16), each with distinct distribution patterns and functional implications. While all these acetylations generally promote euchromatin formation and transcriptional activation, they exhibit specialized roles in cellular processes .

H4K5 acetylation, particularly when paired with H4K12 acetylation, creates a signature diacetylation pattern that marks newly synthesized histones before their incorporation into chromatin . This pattern can be distinguished from hyperacetylated H4, where K5, K8, K12, and K16 are all acetylated . In contrast, H4K16 acetylation is specifically associated with DNA damage repair and cellular senescence pathways .

ChIP-seq analysis has revealed that H4K8 and H4K16 acetylation are particularly enriched around transcription start sites, highlighting their direct involvement in gene activation . Meanwhile, H4K5 acetylation shows characteristic distribution patterns that can be visualized through immunofluorescence, often presenting enhanced signals in specific cell populations such as elongating spermatids .

How do acetylation and butyrylation at H4K5 compete in regulatory functions?

Recent research has uncovered an intriguing competitive relationship between acetylation and butyrylation at H4K5, revealing a sophisticated regulatory mechanism in chromatin biology. Both modifications occur at active gene transcription start sites and directly stimulate transcription, likely through similar mechanisms involving chromatin decondensation .

Mass spectrometry analysis of histones from mouse testis has identified ten butyrylation sites, including H4K5bu, occurring either separately or in combination with H4K8bu . Immunohistochemistry studies demonstrate that H4K5 and K8 butyrylation patterns are enhanced in elongating spermatids, similar to the previously observed patterns for H4K5 and K8 acetylation .

The functional distinction between these modifications becomes apparent in protein recognition patterns: H4K5 butyrylation prevents binding of the testis-specific gene expression-driver Brdt, while H4K5 acetylation permits this interaction . Additionally, H4K5K8 butyrylation is associated with delayed histone removal in spermatogenic cells compared to acetylation, suggesting distinct roles in chromatin dynamics during spermatogenesis . This alternating pattern of H4 acetylation and butyrylation appears to maintain direct gene activation while regulating dynamic bromodomain binding, potentially impacting the final male epigenome features .

What methods should be used to validate the specificity of an H4K5ac antibody?

Robust validation of H4K5ac antibodies is essential for obtaining reliable experimental results. A comprehensive validation approach should employ multiple complementary techniques:

• Peptide-based validation: ELISA assays using synthetic peptides harboring specific modifications or amino acid substitutions provide initial specificity assessment . Researchers should test against H4 peptides with acetylation at different positions (K5, K8, K12, K16) and with various combinations of modifications to evaluate cross-reactivity.

• Immunoblotting validation: Using recombinant proteins that harbor specific modifications or amino acid substitutions allows for evaluation of antibody specificity in a format similar to common experimental applications .

• Genetic validation: Testing antibodies against histone mutations in chromatin immunoprecipitation (ChIP) assays provides a stringent validation approach that accounts for the chromatin context . This method has been employed for highly specific antibody sets targeting acetylation sites in histones.

• Immunofluorescence characterization: Visualization of characteristic distribution patterns via immunofluorescence confirms expected nuclear localization and distribution patterns .

• Binding affinity measurements: Surface plasmon resonance (SPR) measurements can determine antibody affinity for target epitopes, allowing comparison between different antibodies and assessment of specificity quantitatively .

As an example of thorough validation, the H4K5 acetylation (H4K5ac)-specific antibody CMA405 was characterized to react with K5ac only when the neighboring K8 was unacetylated, providing a unique tool to distinguish newly assembled H4 (diacetylated at K5 and K12) from hyperacetylated H4 (where K5, K8, K12, and K16 are all acetylated) .

Why do neighboring modifications affect antibody recognition of H4K5ac?

The recognition of H4K5 acetylation by antibodies can be significantly influenced by the modification status of neighboring residues, a phenomenon with important methodological implications. The structural proximity of lysine residues in the H4 tail (K5, K8, K12, K16) means that modifications at one site can alter the local conformation, charge distribution, or steric accessibility of nearby epitopes .

A clear example of this is the H4K5ac-specific antibody CMA405, which recognizes K5ac only when the neighboring K8 is unacetylated . This specificity provides a valuable tool for distinguishing newly assembled H4 (diacetylated at K5 and K12) from hyperacetylated H4 (acetylated at K5, K8, K12, and K16), but also highlights the importance of understanding antibody dependencies on neighboring modifications.

The impact of neighboring modifications reflects the complex three-dimensional structure of the histone tail when modified. NMR studies and molecular dynamics simulations have shown that acetylation significantly alters the chemical environment of the histone H4 tail, leading to conformational changes that can affect antibody binding . These structural changes include tail compaction effects, where acetylated H4 tails favor more compact conformers compared to unacetylated tails .

For researchers, this means that antibody specificity must be thoroughly validated against combinations of modifications, not just against the target modification in isolation. The broader implication is that the biological "reading" of histone modifications by nuclear proteins likely involves recognition of modification patterns rather than individual marks.

What advantages do recombinant monoclonal antibodies offer for H4K5ac detection compared to polyclonal antibodies?

Recombinant monoclonal antibodies provide several significant advantages for H4K5ac detection in epigenetic research over traditional polyclonal antibodies:

• High batch-to-batch consistency: Recombinant production ensures identical antibodies in each batch, eliminating the variability inherent to polyclonal antibodies generated in different animals or at different times . This consistency is crucial for longitudinal studies and for comparing results across experiments.

• Improved specificity: Monoclonal antibodies target a single epitope, reducing the risk of cross-reactivity with similar histone modifications (particularly important given the sequence similarity around different acetylated lysines in the H4 tail) .

• Defined recognition site: The precise epitope recognized by a monoclonal antibody can be characterized in detail, allowing researchers to understand exactly what modification pattern is being detected . This is crucial when neighboring modifications can affect antibody recognition.

• Reproducibility for quantitative applications: The consistent binding properties of recombinant monoclonal antibodies make them better suited for quantitative applications where precise comparisons between samples are required .

• Renewable source: Unlike polyclonal antibodies that require continued animal immunization, recombinant antibodies can be produced indefinitely from the same genetic sequence, ensuring long-term availability of identical reagents .

The production of recombinant monoclonal antibodies against H4K5ac typically involves cloning genes encoding both heavy and light chains, integrating these into expression vectors, transfecting host cells, followed by culture, purification via affinity chromatography, and comprehensive validation across multiple applications . This rigorous process yields antibodies with defined characteristics that support reliable and reproducible detection of H4K5 acetylation.

What are the optimal conditions for using H4K5ac antibodies in chromatin immunoprecipitation (ChIP) experiments?

Optimizing chromatin immunoprecipitation (ChIP) protocols for H4K5ac detection requires careful attention to several critical parameters:

• Crosslinking parameters: Most protocols recommend 1% formaldehyde for 10-15 minutes at room temperature to effectively capture histone-DNA interactions without overfixing, which can mask epitopes .

• Chromatin fragmentation: Sonication should be optimized to generate fragments of 200-500 bp, which provides sufficient resolution while maintaining epitope integrity. Parameters must be empirically determined for each cell type and sonicator model .

• Antibody concentrations: Typically 2-5 μg per ChIP reaction, though this should be titrated for each antibody to identify the optimal concentration that maximizes specific signal while minimizing background .

• Washing stringency: Multiple washes with increasing salt concentrations (typically from 150mM to 500mM NaCl) help reduce non-specific binding. For H4K5ac, which can have cross-reactivity with other acetylation sites, washing stringency is particularly important .

• Controls: Include input samples (pre-immunoprecipitation chromatin), IgG controls (non-specific antibody), and positive control regions known to be enriched for H4K5ac to validate each experiment .

• Antibody validation: Before proceeding to genome-wide ChIP-seq, confirm enrichment at expected regions using ChIP-qPCR .

ChIP-seq studies have revealed that H4K5 acetylation, similar to H4K8 and H4K16 acetylation, is enriched around transcription start sites of active genes . In heterochromatin regions, such as telomeric and silent mating locus heterochromatin, H4K5 along with other acetylation sites is typically hypoacetylated . These characteristic patterns can serve as useful positive and negative controls when establishing ChIP protocols.

How can H4K5ac antibodies be effectively used in immunofluorescence microscopy?

Successful immunofluorescence detection of H4K5 acetylation requires optimized protocols that preserve nuclear architecture while ensuring epitope accessibility:

• Fixation method: For most cell types, 4% paraformaldehyde for 10-15 minutes at room temperature provides an optimal balance between structure preservation and epitope accessibility .

• Permeabilization: A brief treatment with 0.1-0.5% Triton X-100 enables antibody penetration into the nucleus. Over-permeabilization should be avoided as it can disrupt nuclear integrity .

• Antigen retrieval: For some fixed tissues or over-fixed samples, a heat-induced epitope retrieval step in citrate buffer (pH 6.0) may significantly improve signal intensity for H4K5ac detection .

• Blocking conditions: Use 5% BSA or 10% serum (matching the host of the secondary antibody) in PBS for 1 hour at room temperature to minimize non-specific binding .

• Antibody dilutions: Primary antibody concentrations should be optimized, typically starting with 1:50-1:500 dilutions for most commercial H4K5ac antibodies . For directly conjugated antibodies (e.g., Alexa Fluor® 488 Anti-Histone H4 acetyl K5), dilutions of 1:30-1:200 are recommended .

• Incubation parameters: Overnight incubation at 4°C with primary antibodies often yields the best signal-to-noise ratio for nuclear epitopes like H4K5ac .

• Counterstaining: DAPI (4′,6-diamidino-2-phenylindole) at 0.1-1 μg/ml provides nuclear context for interpreting H4K5ac distribution patterns .

Immunofluorescence studies have revealed distinctive patterns of H4K5 acetylation in different cell types. For example, enhanced immunofluorescence signals for H4K5 acetylation are observed in elongating spermatids during spermatogenesis, similar to patterns seen for H4K8 acetylation . These cell type-specific distribution patterns can provide important insights into the biological roles of H4K5 acetylation in different contexts.

What quantitative approaches can measure global and local changes in H4K5 acetylation levels?

Accurately quantifying H4K5 acetylation changes requires specialized techniques depending on whether global or locus-specific measurements are needed:

Global H4K5ac Quantification:

• Western blotting: Use H4K5ac antibodies with total H4 antibodies as loading controls. Densitometric analysis should be performed within the linear range of detection. Fluorescent secondary antibodies offer superior quantitative performance compared to chemiluminescence .

• Mass spectrometry: Provides absolute quantification of H4K5ac levels and can simultaneously detect multiple modifications. Sample preparation typically involves acid extraction of histones, propionylation of unmodified lysines, tryptic digestion, and LC-MS/MS analysis . Stable isotope labeling (SILAC or TMT) enhances quantitative accuracy.

• ELISA-based methods: Commercial kits are available for measuring global H4K5ac levels in histone extracts, offering a higher throughput alternative to Western blotting .

Locus-specific H4K5ac Quantification:

• ChIP-qPCR: Calculate percent input or fold enrichment over control regions to quantify H4K5ac at specific loci. Include appropriate negative regions and positive control regions for validation .

• ChIP-seq with spike-in controls: Adding a defined amount of chromatin from a different species before immunoprecipitation provides a normalization reference for comparing H4K5ac levels across experimental conditions .

• CUT&RUN or CUT&Tag: These newer techniques offer higher signal-to-noise ratios than conventional ChIP, enabling more sensitive detection of H4K5ac changes, particularly at regions with lower enrichment .

• Single-cell epigenomic approaches: Emerging technologies like single-cell CUT&Tag allow assessment of H4K5ac heterogeneity across cell populations, revealing cell type-specific regulation that might be masked in bulk analyses .

For all quantitative approaches, appropriate normalization is critical. When comparing H4K5ac across conditions, consider normalizing to unmodified H4 or using spike-in controls to account for technical variation in immunoprecipitation efficiency or cell number differences .

How does H4K5 acetylation mechanistically influence cross-talk with H3 tail modifications?

The dynamic interplay between H4K5 acetylation and H3 tail modifications represents a sophisticated mechanism of epigenetic regulation that operates at the nucleosomal level. NMR studies and molecular dynamics simulations have revealed how these modifications influence each other through structural alterations of the nucleosome:

Acetylation of the H4 N-tail (including K5) significantly enhances the acetylation rate of the H3 N-tail, particularly at K14, by the histone acetyltransferase Gcn5 . This enhancement occurs through conformational changes in the nucleosome structure. When the H4 N-tail is tetra-acetylated at K5, K8, K12, and K16, it alters the dynamics and accessibility of the H3 N-tail, making it more available for modification by HAT enzymes .

This observation is consistent with genomic data showing that acetylation of H4K8 and H3K14 is strongly correlated in transcribing regions , suggesting functional coordination between these modifications. Similarly, acetylation of H4K16 enhances H3 N-tail acetylation by bromodomain-defective Gcn5 .

These findings demonstrate that histone tails form a dynamic network for regulating their modification patterns on the nucleosome, with modifications on one tail influencing the modification state of another. This cross-talk mechanism likely extends to other tail interactions, such as between the H3 N-tail and H2A C-tail, which may interact dynamically via DNA .

What is the structural significance of H4K5ac in bromodomain recognition?

The recognition of acetylated H4K5 by bromodomain-containing proteins represents a critical mechanism for translating histone modifications into functional outcomes. Structural studies have revealed unexpected complexity in these interactions:

Crystal structure analysis of the second bromodomain of BRD2 (BRD2-BD2) in complex with di-acetylated histone H4 tail (H4K5ac/K12ac) revealed a surprising interaction pattern: a single K5ac/K12ac peptide can interact with two BRD2-BD2 molecules simultaneously . In this arrangement, the K5ac residue binds to one BRD2-BD2 molecule while the K12ac residue binds to another . This dual recognition mechanism suggests that bromodomain proteins could potentially cross-link different nucleosomes through recognition of multiple acetylation sites.

The bromodomain binding pocket that recognizes H4K5ac contains several highly conserved residues, including D385, Y429, N429, D432, and H433 in BRD2-BD2 . These residues directly interact with the H4 peptide chain and significantly affect binding efficiency for H4K5ac/K12ac peptides in solution . Mutagenesis studies have shown that the N429A mutation reduces binding affinity with acetylated H4 peptides, while L381A and L383A mutations moderately affect binding efficiency .

The functional implications of these structural interactions are significant. BET family proteins like BRD2 recognize acetylated chromatin through their bromodomains, acting as transcriptional activators or tethering viral genomes to mitotic chromosomes . The ability of BRD2-BD2 to recognize different acetylation patterns provides a structural basis for interpreting the histone code and translating it into specific biological outcomes.

How does H4K5 acetylation influence chromatin compaction dynamics?

Acetylation of H4K5 significantly alters chromatin compaction states through complex biophysical mechanisms that have been elucidated through advanced structural studies:

Recent research using 13C direct-detect NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations has revealed that acetylation, including at K5, profoundly impacts the conformational ensemble of the histone H4 tail . Contrary to the traditional view that acetylation simply neutralizes positive charges to reduce histone-DNA interactions, the data shows more complex structural effects.

Acetylation significantly alters the chemical environment of basic patch residues (16–20) in the H4 tail and leads to tail compaction . SAXS experiments determined the radius of gyration (Rg) for differently acetylated H4 tail constructs: unacetylated (unAc), intermediately acetylated primarily at K5, K8, and K12 (3Ac), and uniformly acetylated at all five available lysines (5Ac) . The measurements showed Rg values of 15.75 ± 0.17, 14.86 ± 0.18, and 14.02 ± 0.15 Å respectively, demonstrating progressive compaction with increased acetylation .

This trend was corroborated by molecular dynamics simulations, which revealed that the 5Ac ensemble uniquely favors compact conformers that are mostly absent in unAc and 3Ac ensembles . The probability distribution for 5Ac is distinctly skewed toward lower Rg values, suggesting a conformational shift toward more compact states .

Interestingly, these structural changes are influenced by the protonation state of H18 within the basic patch. The pKa of H18 shifts higher (+0.1 units) upon acetylation, making it slightly less acidic in the 5Ac sample . When H18 is protonated, the conformational ensemble shifts: the Rg increases from 13.10 to 14.31 Å, the probability distribution becomes more uniform, and intramolecular contacts between the N-terminus and the basic patch are mitigated . This suggests that H18 in the center of the basic patch may provide a conformational buffer against entrapment in collapsed conformations .

These structural insights reveal that H4K5 acetylation contributes to a complex conformational landscape that influences histone tail interactions with DNA and other nuclear proteins, ultimately affecting chromatin accessibility and gene regulation.

What are the most common sources of false positives and negatives in H4K5ac detection?

Reliable detection of H4K5 acetylation can be compromised by several technical issues that researchers should actively address:

Sources of False Positives:

• Cross-reactivity with other acetylated lysines: H4 contains multiple acetylation sites (K5, K8, K12, K16) with similar surrounding sequences. Inadequately validated antibodies may detect these other modifications, particularly H4K8ac due to its proximity to K5 .

• Non-specific antibody binding: Insufficient blocking or washing steps can lead to background signal unrelated to H4K5ac. This is particularly problematic in techniques like ChIP where the amount of target protein is small relative to total protein .

• Over-crosslinking in ChIP experiments: Excessive formaldehyde treatment can create protein-protein crosslinks that artifactually bring H4K5ac-containing chromatin to genomic regions where it doesn't normally reside .

• Secondary antibody cross-reactivity: In multi-labeling experiments, secondary antibodies may recognize primary antibodies from different species imperfectly, creating false co-localization signals .

Sources of False Negatives:

• Epitope masking by neighboring modifications: As demonstrated with antibody CMA405, which recognizes K5ac only when K8 is unacetylated, the modification state of neighboring residues can prevent antibody binding even when K5ac is present .

• Insufficient epitope retrieval: Particularly in fixed tissue samples, inadequate antigen retrieval can prevent access to H4K5ac epitopes .

• Protein-protein interactions blocking the epitope: In the native chromatin context, interactions between histones and other nuclear proteins may obscure the H4K5ac site .

• Sample preparation issues: Harsh extraction conditions can lead to loss of histones or deacetylation of samples, particularly if histone deacetylase (HDAC) inhibitors are not included in extraction buffers .

To minimize these issues, researchers should implement rigorous controls including: peptide competition assays to verify specificity, use of validated knockout/knockdown samples when available, testing multiple antibodies targeting the same modification, and including positive and negative control regions or samples in each experiment .

How can researchers ensure reproducibility in H4K5ac antibody-based experiments across different batches?

Ensuring experimental reproducibility when working with H4K5ac antibodies requires systematic approaches to control for technical variables:

• Antibody selection strategies:

  • Opt for recombinant monoclonal antibodies which offer high batch-to-batch consistency and reproducibility compared to polyclonal alternatives

  • Request detailed validation data from manufacturers, including peptide array specificity profiles and lot-specific quality control metrics

  • When possible, purchase larger antibody lots that can support entire research projects rather than switching between batches mid-study

• Lot validation protocols:

  • Implement in-house validation for each new antibody lot using peptide dot blots or ELISA with modified and unmodified H4 peptides

  • Perform side-by-side comparisons with previous lots on reference samples before deploying new lots in experiments

  • Maintain positive control samples with known H4K5ac levels to calibrate new antibody lots

• Standardized experimental procedures:

  • Develop detailed standard operating procedures (SOPs) for each application (ChIP, IF, Western blot)

  • Use automated liquid handling where feasible to minimize technical variation in antibody dilutions and wash steps

  • Implement consistent fixation, blocking, and incubation times across experiments

• Normalization approaches:

  • Include spike-in controls (e.g., chromatin from another species) in ChIP experiments to enable normalization across batches

  • For Western blots, use standard curves with recombinant acetylated histones for quantitative comparisons

  • In microscopy studies, include calibration slides with standardized fluorescent beads to normalize signal intensity

• Data analysis considerations:

  • Develop computational pipelines that account for batch effects in high-throughput data analysis

  • Consider experimental design that distributes controls and treatments across antibody batches rather than processing them separately

  • Document all batch information in laboratory records and publications to support transparency and reproducibility

By implementing these systematic approaches, researchers can significantly improve the reproducibility of H4K5ac detection across different experimental batches, enabling more reliable comparative studies and longitudinal research projects.

What controls are essential for validating experimental findings related to H4K5 acetylation?

Robust experimental design for studying H4K5 acetylation requires incorporating multiple levels of controls to ensure valid and interpretable results:

Antibody Specificity Controls:

• Peptide competition assays: Pre-incubating H4K5ac antibodies with acetylated and unacetylated H4 peptides should selectively block binding to acetylated targets .

• Genetic validation: Where available, using histone H4K5 mutants (K5R or K5Q) provides definitive specificity confirmation, particularly valuable for ChIP studies .

• Deacetylase treatment controls: Treating samples with recombinant histone deacetylases should reduce or eliminate H4K5ac signal, confirming modification-specific detection .

Technical Controls:

• Isotype controls: Including appropriate isotype-matched control antibodies helps establish background signal levels for immunostaining and ChIP experiments .

• Input controls for ChIP: Analyzing pre-immunoprecipitation chromatin is essential for calculating enrichment and controlling for differences in chromatin preparation or DNA amount .

• Loading controls for immunoblotting: Total H4 antibodies or total protein stains must be used to normalize H4K5ac signals when comparing samples with potentially different histone content .

Biological Controls:

• Known positive and negative regions for ChIP: Include genomic regions with established H4K5ac enrichment patterns, such as active transcription start sites (positive) and heterochromatic regions like telomeres (negative) .

• HDAC inhibitor treatment: Cells treated with HDAC inhibitors like trichostatin A (TSA) or sodium butyrate should show increased H4K5ac levels, providing a positive control for detection systems .

• HAT mutant cells: Cells with mutations or knockdowns of HATs known to acetylate H4K5 should show reduced signal, confirming biological specificity .

Validation through orthogonal techniques:

• Multiple detection methods: Confirm key findings using different techniques (e.g., ChIP-seq findings validated by ChIP-qPCR; immunofluorescence results confirmed by immunoblotting) .

• Mass spectrometry validation: For critical observations, mass spectrometry-based confirmation of H4K5ac changes provides an antibody-independent verification method .

• Functional validation: Connect H4K5ac changes to expected downstream effects, such as altered chromatin accessibility (ATAC-seq) or transcriptional changes (RNA-seq) .

By systematically incorporating these controls, researchers can establish the validity of their H4K5ac findings and distinguish genuine biological effects from technical artifacts, leading to more reliable and reproducible epigenetic research.