Acetyl-HIST1H4A (K5) Antibody

What is Acetyl-HIST1H4A (K5) Antibody and what biological significance does it detect?

Acetyl-HIST1H4A (K5) Antibody is a specialized immunological reagent designed to detect histone H4 acetylated specifically at lysine 5 (K5). This antibody is available in both monoclonal and polyclonal formats with different production methods. The monoclonal version is produced through cloning of genes encoding the HIST1H4A antibody (including both heavy and light chains), integration into expression vectors, transfection into host cells, and purification via affinity chromatography . Polyclonal versions are typically raised in rabbits using a peptide sequence around the acetylated K5 site of human Histone H4 as the immunogen .

The biological significance of H4K5 acetylation is substantial. This modification promotes chromatin decondensation and is primarily associated with transcriptional activation. Additionally, H4K5 acetylation plays important roles in DNA repair processes and frequently occurs in conjunction with other histone modifications to form a complex regulatory code that fine-tunes gene expression .

What are the recommended applications and dilutions for Acetyl-HIST1H4A (K5) Antibody?

Acetyl-HIST1H4A (K5) Antibody has been validated for multiple research applications with specific recommended dilutions:

These dilutions should be optimized based on specific experimental conditions and sample types . The antibody has been comprehensively tested across these applications, enabling precise detection of human HIST1H4A protein acetylated at K5.

How is H4K5 acetylation functionally different from other histone H4 acetylation marks?

H4K5 acetylation has distinct functional properties compared to other acetylation sites on histone H4. While acetylation at multiple H4 sites (K5, K8, K12, K16) generally promotes transcriptional activation, each site has unique characteristics:

• H4K5 acetylation specifically promotes chromatin decondensation and is often associated with the early stages of transcriptional activation

• H4K5 acetylation, along with K12, is predominantly found on newly synthesized histones during chromatin assembly, catalyzed by Histone Acetyltransferase 1 (HAT1)

• Unlike H4K16 acetylation (which directly affects nucleosome-nucleosome interactions), H4K5 acetylation works in concert with H4K12 acetylation during DNA replication and repair processes

• H4-acetylation (including K5) leads to increased unwrapping of DNA ends but, interestingly, can counteract H3-acetylation in nucleosome disassembly

These functional differences highlight the importance of studying site-specific acetylation rather than general histone acetylation levels.

How can Acetyl-HIST1H4A (K5) Antibody be used to investigate chromatin assembly dynamics?

Acetyl-HIST1H4A (K5) Antibody is an essential tool for studying chromatin assembly dynamics, particularly in replication-coupled processes. Researchers can employ this antibody in several sophisticated experimental approaches:

Time-course ChIP assays can be designed to track the deposition of newly synthesized histones with H4K5 acetylation during S-phase. This approach reveals the temporal dynamics of histone deposition and subsequent modification changes. By synchronizing cells and performing ChIP-seq with Acetyl-HIST1H4A (K5) Antibody at different time points after release, researchers can map genome-wide patterns of newly assembled chromatin .

The antibody can also be utilized in pulse-chase experiments with SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) to distinguish old versus newly incorporated histones. This technique helps quantify histone acetylation turnover rates at specific genomic loci .

Additionally, researchers have used the antibody to demonstrate that HAT1 is not only responsible for acetylation of newly synthesized histone H4 but is also required to maintain acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly . This finding highlights the complex interplay between different histone modifications during chromatin assembly.

What experimental approaches can quantify the impact of H4K5 acetylation on transcriptional regulation?

Multiple experimental approaches can quantify how H4K5 acetylation influences transcriptional regulation:

One sophisticated approach involves reconstituting nucleosomes with site-specifically acetylated histones to directly measure transcriptional outcomes. For example, researchers have reconstituted di-nucleosomes with site-specifically acetylated or unmodified histone H4 containing two copies of the Xenopus somatic 5S rRNA gene. Using mathematically described kinetic models and fitting analysis, they determined that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation approximately 3-fold compared to unmodified histones .

Another approach uses hybridization-assisted fluorescence correlation spectroscopy to analyze the time course of nascent transcript accumulation. This technique allows for precise quantification of transcriptional output from defined chromatin templates with specific acetylation patterns .

Researchers can also couple ChIP-seq using Acetyl-HIST1H4A (K5) Antibody with RNA-seq or PRO-seq (Precision Run-On sequencing) to correlate H4K5 acetylation patterns with active transcription across the genome. This integrative approach reveals how H4K5 acetylation correlates with transcriptional activity at specific loci.

How does H4K5 acetylation interact with bromodomain-containing proteins in chromatin regulation?

H4K5 acetylation serves as a specific recognition site for bromodomain-containing proteins, which play critical roles in chromatin regulation. The structural basis for this interaction has been extensively studied:

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and certain nucleosome remodeling complexes that specifically recognizes acetylated lysine residues. NMR studies have revealed that when bromodomain proteins interact with H4K5ac, the residues exhibiting changed chemical shifts are concentrated in two regions - loops ZA and BC and the α-helical regions immediately flanking the BC loop .

The specificity of bromodomain binding to H4K5ac versus other acetylated lysines is influenced by the sequence context surrounding the acetylated lysine. For instance, the Gcn5p bromodomain may discriminate between different acetylated lysines depending on the sequence in which they are found. Experiments have shown that residues following the acetylated lysine, particularly arginine residues, can strongly influence binding affinity .

Importantly, these interactions form part of a "histone code" where specific combinations of modifications are recognized by different effector proteins. H4K5 acetylation often works in concert with other acetylation marks like H4K8, H4K12, and H4K16 to create binding platforms for bromodomain proteins that subsequently recruit transcriptional machinery .

What controls should be included when using Acetyl-HIST1H4A (K5) Antibody in experimental applications?

When designing experiments with Acetyl-HIST1H4A (K5) Antibody, implementing proper controls is crucial for result interpretation:

Positive Controls:

• Extracts from cells treated with histone deacetylase inhibitors (e.g., TSA or sodium butyrate) to increase global H4K5 acetylation levels

• Recombinant histone H4 peptides with verified K5 acetylation, such as those used in the tetra-acetylated H4 studies

• HAT1-overexpressing cell lines, as HAT1 is known to acetylate H4K5 during chromatin assembly

Negative Controls:

• Unmodified histone H4 peptides or recombinant proteins

• Extracts from HAT1-knockout cells, which show decreased H4K5 acetylation in newly synthesized histones

• Peptide competition assays where the antibody is pre-incubated with acetylated peptides corresponding to the H4K5ac epitope

Specificity Controls:

• Immunoblotting against peptides containing acetylated lysines at other positions (H4K8ac, H4K12ac, H4K16ac) to confirm absence of cross-reactivity

• Parallel experiments with antibodies against other histone modifications to determine modification-specific effects

Researchers should also perform antibody validation using site-specific histone mutants (K5R or K5Q) when possible, as these provide definitive evidence for antibody specificity.

What methods can distinguish between newly deposited and pre-existing H4K5 acetylation marks?

Distinguishing between newly deposited and pre-existing H4K5 acetylation marks requires specialized techniques:

SILAC-Based Approaches:
SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) can be employed to differentiate old from new histones. By growing cells in media containing heavy isotope-labeled amino acids, newly synthesized histones incorporate the heavy isotopes. After a specified period, immunoprecipitation with Acetyl-HIST1H4A (K5) Antibody followed by mass spectrometry analysis can distinguish between pre-existing and newly deposited H4K5ac marks based on their isotope profiles .

Cell Synchronization and Pulse-Chase:
Cells can be synchronized at the G1/S boundary, then released into S phase in the presence of inhibitors of specific HATs or HDACs. Time-course ChIP experiments using Acetyl-HIST1H4A (K5) Antibody can then track the dynamics of H4K5 acetylation during chromatin assembly. This approach has revealed that HAT1 is responsible for H4K5 acetylation on newly synthesized histones .

EdU Labeling Combined with Proximity Ligation Assay (PLA):
By labeling newly synthesized DNA with EdU (5-ethynyl-2'-deoxyuridine) and performing PLA with antibodies against H4K5ac and biotin-tagged EdU, researchers can visualize H4K5ac specifically at sites of new DNA synthesis, distinguishing replication-coupled deposition from pre-existing marks.

These approaches enable researchers to track the dynamic changes in H4K5 acetylation during cellular processes like DNA replication and transcriptional activation.

How can specificity issues with Acetyl-HIST1H4A (K5) Antibody be addressed in complex experimental systems?

Ensuring specificity of Acetyl-HIST1H4A (K5) Antibody in complex experimental systems requires several strategic approaches:

Peptide Array Validation:
Before using the antibody in complex systems, researchers should validate its specificity using peptide arrays containing various histone modifications. This helps identify potential cross-reactivity with similar acetylation sites (H4K8ac, H4K12ac) or with acetylated lysines on other histones.

Knockout/Knockdown Validation:
Using genetic models where H4K5 acetylation is specifically altered provides compelling validation. For example, HAT1 knockout cell lines show significantly reduced H4K5 acetylation on newly synthesized histones, providing a biological system to test antibody specificity .

Combinatorial Epitope Analysis:
When studying chromatin regions with multiple modifications, researchers should use sequential ChIP (re-ChIP) or mass spectrometry to verify the co-occurrence of modifications detected by multiple antibodies. This is particularly important since H4K5 acetylation often occurs in combination with other modifications like H4K8ac, H4K12ac, and H4K16ac .

Dilution Optimization:
Different applications require different antibody dilutions for optimal specificity. Western blotting typically uses higher dilutions (1:500-1:2000) compared to immunocytochemistry or immunofluorescence (1:30-1:200) . Titration experiments should be performed for each new experimental system.

Blocking Optimization:
Modifications to standard blocking protocols can improve specificity. Using acetylated BSA instead of regular BSA in blocking solutions can reduce non-specific binding to acetylated proteins. Additionally, pre-incubation of the antibody with non-specific acetylated peptides (while avoiding the H4K5ac epitope) can reduce cross-reactivity.

How do the dynamics of H4K5 acetylation differ during replication-coupled versus replication-independent chromatin assembly?

The dynamics of H4K5 acetylation show distinct patterns between replication-coupled and replication-independent chromatin assembly:

Replication-Coupled Assembly:
During DNA replication, newly synthesized histone H4 is predominantly diacetylated at K5 and K12 by Histone Acetyltransferase 1 (HAT1). This pattern is evolutionarily conserved and serves as a signature of newly synthesized histones . Studies with HAT1-deficient cells demonstrate that this enzyme is essential for maintaining proper H4K5 acetylation during replication-coupled assembly. Knockout of HAT1 in mice results in neonatal lethality, indicating the critical importance of this process .

The acetylation at H4K5 during replication is typically transient and is removed shortly after deposition onto DNA. This dynamic acetylation-deacetylation cycle is essential for proper chromatin maturation and genome stability, as demonstrated by the increased sensitivity to DNA damaging agents and genome instability observed in HAT1-deficient cells .

Replication-Independent Assembly:
During replication-independent histone exchange, which occurs primarily at transcriptionally active regions, H4K5 acetylation shows different dynamics. This process typically involves histone variants and different HATs than those involved in replication-coupled assembly. H4K5 acetylation in this context is often part of broader acetylation patterns that include K8, K12, and K16, creating a hyperacetylated state that facilitates transcription factor binding .

The acetylation of H4K5 in replication-independent contexts appears to be more stable than in replication-coupled assembly, often persisting as part of active chromatin domains. This stability difference likely reflects the distinct functional roles of H4K5 acetylation in each process - a transient assembly mark versus a stable activating modification.

What role does H4K5 acetylation play in nucleosome stability and chromatin higher-order structure?

H4K5 acetylation significantly influences nucleosome stability and chromatin higher-order structure through several mechanisms:

Effects on Nucleosome Unwrapping:
Biophysical studies using µpsFRET (micro-photoluminescence single-molecule Förster resonance energy transfer) have demonstrated that histone H4 acetylation leads to increased unwrapping of DNA ends from the nucleosome. Interestingly, when H4 acetylation occurs alone, it enhances the unwrapping of nucleosomal DNA ends but does not promote nucleosome disassembly to the same extent as H3 acetylation .

More surprisingly, H4 acetylation can actually counteract the destabilizing effects of H3 acetylation on nucleosome disassembly. This unexpected interplay suggests that the balance between H3 and H4 acetylation can fine-tune nucleosome stability in a highly regulated manner .

Impact on Higher-Order Chromatin Structure:
H4K5 acetylation, particularly when combined with acetylation at K8, K12, and K16, disrupts interactions between the H4 tail and acidic patches on adjacent nucleosomes. This disruption weakens internucleosomal contacts and promotes a more open, accessible chromatin structure. Quantitative analysis has shown that tetra-acetylation of H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation approximately 3-fold compared to unmodified histones .

Crystal structure studies have revealed that despite its functional importance, H4K5 acetylation does not significantly alter the core structure of the nucleosome itself. Instead, its effects are primarily mediated through changes in the dynamic properties of the nucleosome and its interactions with other nucleosomes and nuclear proteins .

How can researchers integrate H4K5 acetylation data with other epigenetic marks for comprehensive chromatin state analysis?

Integrating H4K5 acetylation data with other epigenetic marks requires sophisticated analytical approaches:

Multivariate Statistical Methods:
Advanced statistical techniques like principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), or uniform manifold approximation and projection (UMAP) can be applied to identify patterns across multiple histone modifications. These methods help identify chromatin states defined by specific combinations of modifications, including H4K5ac.

Chromatin State Hidden Markov Models:
Researchers can employ computational frameworks like ChromHMM or EpiCSeg to integrate H4K5ac ChIP-seq data with other histone mark datasets. These algorithms identify recurring combinatorial patterns and define discrete chromatin states based on the co-occurrence of multiple modifications. This approach has revealed that H4K5ac often co-occurs with H3K27ac and H3K4me3 at active promoters, while showing different patterns at enhancers and gene bodies.

Network Analysis of Histone Modifications:
Network-based approaches can reveal the hierarchical relationships between different histone modifications. For H4K5ac, such analyses have shown that it often serves as an early modification that facilitates the subsequent deposition of other active marks. This follows the biological understanding that H4K5ac is involved in the initial stages of chromatin assembly and transcriptional activation .

Integration with Transcription Factor Binding Data:
Combining H4K5ac ChIP-seq with transcription factor ChIP-seq datasets can identify how this modification influences or is influenced by transcription factor binding. Particularly relevant are factors that contain bromodomains, which can specifically recognize acetylated lysines including H4K5ac . This integration helps construct regulatory networks that connect histone modifications, transcription factor binding, and gene expression.

Three-Dimensional Chromatin Structure Correlation:
Techniques like Hi-C, HiChIP, or Micro-C can be integrated with H4K5ac data to understand how this modification relates to three-dimensional genome organization. Such analyses have shown that regions rich in H4K5ac often correspond to open chromatin compartments and are frequently involved in long-range regulatory interactions.

What emerging technologies are enhancing our ability to study H4K5 acetylation at single-cell resolution?

Recent technological advances have significantly improved our ability to study H4K5 acetylation at single-cell resolution:

CUT&Tag and CUT&RUN for Single Cells:
Cleavage Under Targets and Tagmentation (CUT&Tag) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) have been adapted for single-cell analysis of histone modifications including H4K5ac. These methods offer improved signal-to-noise ratios compared to traditional ChIP-seq approaches and require fewer cells, making them ideal for analyzing H4K5 acetylation heterogeneity within populations.

Single-Cell ATAC-seq with Histone Modification Information:
Newer versions of single-cell ATAC-seq incorporate antibody-targeted approaches to simultaneously capture chromatin accessibility and specific histone modifications like H4K5ac. This provides correlated data on both chromatin structure and histone modification status at the single-cell level.

Mass Cytometry (CyTOF) for Histone Modifications:
Mass cytometry using metal-conjugated antibodies against H4K5ac allows simultaneous quantification of multiple histone modifications in thousands of individual cells. This approach has revealed significant heterogeneity in H4K5 acetylation levels even within seemingly homogeneous cell populations.

In Situ Histone Modification Sequencing:
Emerging spatial genomics techniques can map H4K5 acetylation patterns while preserving tissue architecture and cellular context. These approaches combine immunofluorescence using Acetyl-HIST1H4A (K5) Antibody with in situ sequencing to correlate modification patterns with cellular position within tissues.

Live-Cell Imaging of H4K5 Acetylation:
New approaches using acetylation-sensitive fluorescent probes or FRET-based sensors can monitor H4K5 acetylation dynamics in living cells. These techniques offer unprecedented temporal resolution for studying how this modification changes during cellular processes like DNA replication, transcription activation, and cell division.

These technologies are revealing that H4K5 acetylation patterns show substantial cell-to-cell variability, suggesting this modification may contribute to cellular heterogeneity and differential gene expression within tissues.

How does the functional significance of H4K5 acetylation vary across different tissue and cell types?

The functional significance of H4K5 acetylation shows remarkable tissue and cell-type specificity:

Developmental Contexts:
Studies using HAT1 knockout mice have revealed that H4K5 acetylation is particularly critical during embryonic development. Homozygous deletion of HAT1, which catalyzes H4K5 acetylation, results in neonatal lethality with severe defects in lung development, resulting in less aeration and respiratory distress. Many HAT1-deficient neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw . This suggests that H4K5 acetylation plays crucial tissue-specific roles during development, particularly in lung maturation and craniofacial development.

Proliferating versus Differentiated Cells:
H4K5 acetylation patterns differ substantially between highly proliferative cells and terminally differentiated cells. In rapidly dividing cells, where chromatin assembly occurs frequently during DNA replication, H4K5 acetylation is abundant and dynamic, reflecting its role in the deposition of newly synthesized histones. In contrast, post-mitotic differentiated cells show more stable H4K5 acetylation patterns that correlate with tissue-specific gene expression programs.

Cancer Cells:
Altered patterns of H4K5 acetylation have been observed across various cancer types. Some malignancies show global hypoacetylation at H4K5, while others display hyperacetylation at specific genomic loci. For example, studies in malignant human lymphoblast cell lines have employed SILAC to identify and quantify histone acetylation changes at the amino acid level, revealing distinct H4K5 acetylation profiles compared to normal lymphocytes .

Stem Cells versus Differentiated Progeny:
Pluripotent stem cells maintain a generally hyperacetylated chromatin state, including elevated H4K5 acetylation, which contributes to their open chromatin structure and developmental plasticity. During lineage commitment and differentiation, H4K5 acetylation patterns become more restricted and correlate with lineage-specific gene expression programs.

These tissue and cell-type variations in H4K5 acetylation patterns highlight the importance of context-specific studies when investigating the functional roles of this modification in different biological systems.