Acetyl-HIST1H4A (K79) Antibody

What is Acetyl-HIST1H4A (K79) Antibody and what does it detect?

Acetyl-HIST1H4A (K79) Antibody is a polyclonal antibody that specifically recognizes histone H4 acetylated at lysine 79 (K79). It is produced in rabbits using a peptide sequence around the acetylated K79 site derived from human histone H4 as the immunogen . This antibody detects a post-translational modification that plays crucial roles in chromatin structure regulation, DNA damage response, and gene silencing pathways. The antibody is specifically designed to recognize the acetylated form of H4K79 and not the unmodified protein, making it valuable for studying epigenetic regulation mechanisms .

What are the recommended applications for Acetyl-HIST1H4A (K79) Antibody?

Based on validated testing, Acetyl-HIST1H4A (K79) Antibody is suitable for multiple experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of H4K79 acetylation levels

• ICC (Immunocytochemistry): For cellular localization studies

• IF (Immunofluorescence): For visualizing H4K79 acetylation patterns within cells

• ChIP (Chromatin Immunoprecipitation): For identifying genomic regions associated with H4K79 acetylation

For optimal results in each application, laboratory-specific dilution optimization is recommended, as antibody performance can vary depending on experimental conditions and sample types.

How does H4K79 acetylation differ from other histone H4 modifications?

H4K79 acetylation represents a core domain modification rather than the more commonly studied N-terminal tail acetylations. While N-terminal histone acetylations (such as H4K5) are primarily associated with chromatin assembly during DNA replication , H4K79 acetylation appears to have specialized functions:

This core domain acetylation at K79 functions in concert with, but distinctly from, other histone modifications like H3K79 methylation to regulate chromatin states .

What are the optimal protocols for using Acetyl-HIST1H4A (K79) Antibody in ChIP experiments?

When performing ChIP with Acetyl-HIST1H4A (K79) Antibody, researchers should consider these methodological optimizations:

• Cross-linking: Use 1% formaldehyde for 10 minutes at room temperature for optimal chromatin preparation

• Sonication: Adjust sonication conditions to obtain chromatin fragments between 200-500bp

• Antibody specificity control: Perform parallel immunoprecipitation with pre-immune serum as a negative control

• Epitope masking prevention: Pre-incubate the antibody with lysate from H4K79A mutant cells to block potential cross-reactivity with other histone modifications

• Validation strategy: Compare enrichment patterns between acetylated (active) and non-acetylated (silenced) genomic regions using qPCR before proceeding to genome-wide analysis

Research shows that H4K79 acetylation is significantly enriched in transcriptionally active regions of the genome and present at lower levels at telomeres and silenced loci like the HMR locus . This distribution pattern can serve as an internal validation of ChIP efficacy.

How does H4K79 acetylation functionally interact with the DNA damage response pathway?

H4K79 acetylation appears to play critical roles in DNA damage response that are independent of but complementary to other histone modifications:

• Checkpoint response independence: Studies with H4K79A mutants reveal sensitivity to DNA-damaging agents that is distinct from defects in MEC1 or MEC3 kinase-dependent checkpoint responses

• Repair pathway interactions: H4K79A mutations increase DNA damage sensitivity in both non-homologous end-joining (NHEJ) pathway (Δyku70) and recombinational repair (Δrad52) mutants, suggesting it does not function directly in either repair mechanism

• Chromatin assembly connection: H4K79A mutants show epistatic interactions with mutations in chromatin assembly factors like ASF1, suggesting a role in post-damage chromatin restoration

• Synergistic effects: Double mutants of H3K79A and H4K91A display increased sensitivity to DNA double-strand breaks compared to either single mutant, indicating that these modifications function in separate pathways during DNA damage repair

These findings suggest that researchers should consider H4K79 acetylation as a critical component when studying chromatin dynamics during DNA damage and repair processes.

What is the relationship between H4K79 acetylation and H3K79 methylation in epigenetic regulation?

The relationship between H4K79 acetylation and H3K79 methylation reveals complex crosstalk in chromatin regulation:

• Silent chromatin effects: Both H4K79A and H3K79A mutations cause similar defects in silencing at telomeres and the HMR locus, suggesting they operate in a common pathway for maintaining silent chromatin structure

• Reciprocal regulation: H4K79A mutations alter the distribution of H3K79 methylation at telomeres, while H3K79A mutations increase H4K79 acetylation at silenced loci, demonstrating bidirectional influence

• Locus-specific dominance: At the HML locus, H3K79A has a more pronounced effect on silencing than H4K79A, but in double mutants, the H4K91A phenotype is dominant

• Functional independence in DNA repair: Unlike their cooperative roles in silent chromatin, H3K79 methylation and H4K79 acetylation function independently in DNA damage repair, as evidenced by the increased sensitivity of double mutants

This interplay has significant implications for experimental design when studying either modification, as manipulating one can affect the other's distribution and function.

How can non-specific binding be minimized when using Acetyl-HIST1H4A (K79) Antibody?

To maximize specificity and reduce background when working with Acetyl-HIST1H4A (K79) Antibody:

• Antibody pre-adsorption: Pre-incubate the antibody with lysate from cells containing the H4K79A allele to block potential cross-reactivity with other histone modifications

• Blocking optimization: Use 5% BSA rather than milk-based blocking solutions, as milk contains bioactive compounds that may interfere with histone antibody binding

• Validation controls: Always run parallel experiments with:

  • Unmodified recombinant H4 (negative control)

  • Acetylated H4 from HeLa cells (positive control)

• Peptide competition: Use synthetic peptides with acetylated K79 as competitive binding controls to demonstrate specificity

• Dot blot verification: Perform dot blot analysis with modified and unmodified peptides encompassing several sites of H4 acetylation to confirm antibody specificity before proceeding to complex samples

These approaches significantly enhance detection specificity, particularly in complex chromatin environments where multiple histone modifications coexist.

What techniques can distinguish newly deposited H4K79 acetylation from preexisting modifications?

Differentiating newly deposited H4K79 acetylation from established modifications requires temporal resolution techniques:

• Pulse-chase labeling: Use methods similar to those employed for H4 acetylation studies, where newly replicated chromatin is labeled with [³H]thymidine in the presence of deacetylase inhibitors like sodium butyrate

• Immunoprecipitation enrichment: Compare bound and unbound fractions after antibody immunoprecipitation to assess enrichment of newly synthesized DNA and associated histones

• Deacetylase inhibition control: Include parallel experiments with and without deacetylase inhibitors to distinguish between newly deposited (acetylated) and mature (potentially deacetylated) chromatin

• Replication inhibition studies: Analyze histone deposition during DNA replication inhibition to identify acetylation patterns associated with replication-independent histone exchange

• Cytosolic extract analysis: Examine preassembly complexes in cytosolic extracts to detect newly synthesized H4 with K79 acetylation prior to chromatin incorporation

These approaches allow researchers to track the dynamics of H4K79 acetylation during chromatin assembly and remodeling processes.

How does the distribution of H4K79 acetylation compare with other histone modifications across the genome?

Genome-wide distribution of H4K79 acetylation follows distinct patterns that can be compared with other histone modifications:

• Active vs. silenced regions: H4K79 acetylation is significantly enriched in transcriptionally active genomic regions while present at low levels at telomeres and silenced loci such as the HMR locus

• Correlation with H3K79 methylation: H4K79 acetylation and H3K79 methylation show inverse correlation at silenced loci, with mutations in either causing redistribution of the other

• Relationship to H4 N-terminal acetylation: H4K79 acetylation patterns differ from N-terminal tail acetylation, with tail acetylation increasing at telomeres and HMR in H4K79A mutants

• Complementary analysis techniques:

  • ChIP-seq for genome-wide profiling

  • ChIP-qPCR for targeted locus validation

  • Western blotting for global level assessment

When designing experiments to map H4K79 acetylation, researchers should consider these distribution patterns and include appropriate controls for other modifications to accurately interpret results.

What are the implications of H4K79 acetylation for understanding disease mechanisms?

H4K79 acetylation research has potential implications for understanding disease mechanisms, particularly in:

• Cancer epigenetics: Altered histone modification patterns, including core domain acetylations, are frequent in cancer cells and may contribute to oncogenic gene expression patterns

• DNA repair disorders: Given the role of H4K79 acetylation in DNA damage responses, alterations may contribute to genomic instability in conditions like Fanconi anemia or ataxia telangiectasia

• Silencing-related disorders: Since H4K79 acetylation affects telomeric silencing and heterochromatin maintenance, its dysregulation might contribute to diseases involving inappropriate gene silencing

• Experimental approaches for disease research:

  • Compare H4K79 acetylation patterns between normal and disease tissues

  • Assess correlations between H4K79 acetylation alterations and disease progression

  • Investigate pharmacological agents that modulate H4K79 acetylation as potential therapeutic approaches

Research into these connections requires careful experimental design that accounts for the complex interplay between multiple histone modifications.

What emerging technologies might enhance the study of H4K79 acetylation?

Several cutting-edge technologies show promise for advancing H4K79 acetylation research:

• CUT&RUN and CUT&Tag: These techniques offer higher resolution and lower background than traditional ChIP, potentially improving detection of H4K79 acetylation at specific genomic loci

• Single-cell epigenomics: Emerging methods for single-cell histone modification profiling could reveal cell-to-cell variability in H4K79 acetylation patterns

• Live-cell imaging: Development of acetylation-specific intrabodies or FRET-based sensors could enable real-time visualization of H4K79 acetylation dynamics

• Mass spectrometry advances: Improved sensitivity in MS-based approaches may allow quantitative assessment of H4K79 acetylation co-occurrence with other modifications on the same histone tail

• CRISPR-based epigenome editing: Targeted modification of H4K79 acetylation at specific loci using catalytically active or inactive histone acetyltransferases fused to Cas proteins

These technologies will likely permit more precise spatial and temporal resolution of H4K79 acetylation dynamics in diverse biological contexts.

What are the key unanswered questions regarding the enzymes that regulate H4K79 acetylation?

Critical gaps remain in our understanding of the enzymatic regulation of H4K79 acetylation:

• Acetyltransferase identification: Unlike N-terminal tail acetylation by HAT1 and other known HATs, the specific enzyme(s) responsible for H4K79 acetylation remain elusive

• Deacetylase specificity: Which histone deacetylases (HDACs) specifically target H4K79 acetylation, and how is their activity regulated?

• Crosstalk regulation: How do enzymes that modify H4K79 and H3K79 communicate to maintain proper chromatin states?

• Experimental approaches:

  • Systematic CRISPR screening of HATs and HDACs

  • In vitro acetylation assays with candidate enzymes

  • Proteomic identification of proteins specifically binding to acetylated H4K79

  • Structure-function analysis of putative H4K79-modifying enzymes

Addressing these questions will provide crucial insights into the regulatory mechanisms controlling this important histone modification.