Acetyl-HIST1H4A (K91) Antibody

What is the biological significance of Histone H4 Lysine 91 acetylation?

Histone H4 Lysine 91 acetylation represents a core domain modification with critical roles distinct from the more commonly studied N-terminal tail modifications. This acetylation occurs at a lysine residue positioned at the interface between histone H3/H4 tetramers and H2A/H2B dimers, making it structurally significant for nucleosome stability . Mutation studies replacing lysine 91 with alanine (K91A) have demonstrated that this site plays essential roles in three major nuclear processes: chromatin assembly, DNA repair, and transcriptional silencing . The importance of this modification is highlighted by the phenotypic consequences of K91A mutations, which include increased sensitivity to DNA damaging agents and significant alterations in chromatin structure . Unlike N-terminal modifications that primarily affect DNA-histone interactions, K91 acetylation appears to regulate histone-histone interactions within the nucleosome core, suggesting a fundamentally different mechanism of action in chromatin regulation.

How does Acetyl-HIST1H4A (K91) differ from other histone H4 acetylation sites?

Acetyl-HIST1H4A (K91) is distinctive because, unlike the well-characterized acetylation sites at K5, K8, K12, and K16 that occur on the N-terminal tail, K91 is located within the globular core domain of histone H4 . This positional difference has profound functional implications. While tail modifications primarily affect DNA accessibility by altering histone-DNA interactions, K91 acetylation influences the structural integrity of the nucleosome by affecting histone-histone interactions at the critical interface between the H3/H4 tetramer and H2A/H2B dimers . Experimental evidence demonstrates that K91 mutations destabilize the histone octamer, leading to widespread chromatin structural defects not observed with individual tail modifications . Additionally, analysis of gene expression patterns in K91A mutants reveals a distinct profile compared to tail acetylation mutants, with a striking tendency for upregulation of telomere-proximal genes - approximately half of the open reading frames within 10kb of chromosome ends showed at least 1.5-fold increased expression . This telomeric effect pattern is not typically observed with mutations affecting only tail acetylation sites.

What are the fundamental applications of Acetyl-HIST1H4A (K91) antibodies in epigenetic research?

Acetyl-HIST1H4A (K91) antibodies serve as critical tools in epigenetic research, enabling the investigation of this modification's distribution, dynamics, and functional consequences across the genome. The primary applications include:

• Chromatin Immunoprecipitation (ChIP): These antibodies allow researchers to map the genomic distribution of K91 acetylation, identifying regions where this modification may play regulatory roles in gene expression or chromatin organization .

• Immunocytochemistry (ICC): With recommended dilutions between 1:10-1:100, these antibodies enable visualization of nuclear localization patterns of K91-acetylated histones within cells, potentially revealing spatial organization within the nucleus .

• ELISA-based quantification: This application permits quantitative assessment of global K91 acetylation levels under different experimental conditions, facilitating comparative studies across cell types or treatment conditions .

• Western blotting: Though not specifically mentioned for K91 antibodies in the search results, this technique would enable detection of changes in K91 acetylation levels in response to various treatments or genetic manipulations.

These applications collectively enable researchers to investigate how K91 acetylation contributes to chromatin dynamics and gene regulation in normal development and disease states, complementing studies of the better-characterized N-terminal tail modifications.

How do mutations in Histone H4 K91 interact with DNA damage repair pathways?

Genetic interaction studies between H4 K91 mutations and DNA repair pathway components reveal complex relationships that illuminate the specific functions of K91 acetylation in genome maintenance. When H4 K91A mutations are combined with deletions of either MEC1 or MEC3 kinases (critical components of the DNA damage checkpoint machinery), the resulting strains show increased sensitivity to DNA damaging agents like methyl methanesulfonate (MMS) compared to single mutants . This synergistic effect indicates that K91 acetylation functions in DNA repair through mechanisms distinct from the canonical checkpoint response .

Similarly, H4 K91A mutations exacerbate the DNA damage sensitivity of strains defective in either non-homologous end-joining (NHEJ) repair (Δyku70 mutants) or homologous recombination repair (Δrad52 mutants) . This epistatic relationship suggests that K91 acetylation does not directly participate in these specific repair pathways but rather affects a process that impacts multiple repair mechanisms simultaneously.

Notably, when combined with mutations in the ASF1 histone chaperone, H4 K91A mutations show no additive effect on MMS sensitivity . This non-additive relationship strongly suggests that K91 acetylation functions in the same pathway as ASF1 - specifically, the chromatin assembly process during DNA repair . These genetic interaction studies collectively position K91 acetylation as a critical modification for facilitating efficient nucleosome assembly during DNA repair processes, rather than directly participating in damage detection or specific repair mechanisms.

What is the relationship between H4 K91 acetylation and telomeric gene expression?

Genome-wide expression profiling reveals a striking spatial pattern of gene dysregulation in H4 K91A mutants, with pronounced effects on telomere-proximal genes. Microarray analysis demonstrates that of the 242 genes upregulated at least 2-fold in K91A mutants, a disproportionate 20% are located within 20kb of chromosome ends . This telomeric enrichment is statistically significant, with approximately half of all open reading frames within 10kb and a quarter of those between 20-30kb from telomeres showing at least 1.5-fold upregulation .

These upregulated genes frequently occur in clusters (defined as three upregulated genes within 15kb), with 10 of 14 identified clusters located within 33kb of chromosome ends . Conversely, genes downregulated in K91A mutants show a significant depletion within 30kb of chromosome ends but distribute evenly throughout the remainder of the genome .

This distinct pattern suggests that K91 acetylation plays a specialized role in maintaining proper chromatin structure at telomeres, potentially through interactions with silencing complexes like Sir proteins or through effects on telomeric heterochromatin formation. The spatial concentration of expression changes near telomeres contrasts with the more distributed effects typically seen with mutations in N-terminal tail modifications, highlighting the unique functional role of this core domain acetylation in regulating chromosome end structure and function.

How does H4 K91 acetylation influence cross-talk with other histone modifications?

The interaction between H4 K91 acetylation and other histone modifications reveals complex regulatory networks within the epigenetic landscape. Chromatin immunoprecipitation (ChIP) analyses in yeast demonstrate that K91A mutations alter the distribution pattern of histone H3 lysine 79 (H3K79) methylation . This cross-regulation is significant because H3K79 methylation is associated with transcriptionally active chromatin and plays roles in DNA damage response.

The structural position of K91 at the interface between histone dimers and tetramers provides a potential mechanism for this cross-talk. Acetylation at K91 may alter nucleosome conformation in ways that affect the accessibility of other modification sites to their respective enzymes. Additionally, because K91A mutations destabilize the histone octamer, they likely create widespread changes in chromatin structure that indirectly influence the deposition or maintenance of other modifications.

This cross-talk extends beyond individual nucleosomes, as suggested by the global effects of K91A mutations on Sir2p distribution and histone H4 acetylation patterns at telomeres . Sir2p is a histone deacetylase critical for silencing at telomeres, and its altered distribution in K91A mutants suggests that K91 acetylation may influence the recruitment or activity of histone-modifying enzymes that affect multiple residues simultaneously. These findings position H4 K91 acetylation as an important node in the complex network of histone modification cross-talk, with significant consequences for both local nucleosome structure and broader chromatin domains.

What are the optimal protocols for using Acetyl-HIST1H4A (K91) antibodies in chromatin immunoprecipitation experiments?

For successful chromatin immunoprecipitation (ChIP) experiments using Acetyl-HIST1H4A (K91) antibodies, researchers should optimize several critical parameters. While the search results don't provide specific ChIP protocols for K91 antibodies, we can derive best practices based on information about similar histone modification antibodies and general principles:

• Antibody selection: Use affinity-purified polyclonal antibodies specifically validated for ChIP applications . The antibody should demonstrate high specificity for K91-acetylated H4 with minimal cross-reactivity to other acetylation sites.

• Sample preparation:

  • Crosslink chromatin with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine

  • Isolate nuclei and sonicate to generate DNA fragments of 200-500bp

  • Verify fragmentation efficiency by gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 1-5μg of antibody per immunoprecipitation reaction

  • Incubate overnight at 4°C with rotation

  • Add protein A/G beads and continue incubation for 2-4 hours

  • Perform stringent washing to reduce background

• Controls:

  • Include a no-antibody control to assess non-specific binding

  • Use IgG from the same species as the primary antibody as a negative control

  • Include a positive control antibody targeting a well-characterized modification

  • If possible, include a K91A mutant sample as a specificity control

• Analysis:

  • For targeted analysis, design primers flanking regions of interest

  • For genome-wide analysis, prepare libraries for next-generation sequencing

  • Include input normalization and appropriate statistical analysis

By carefully optimizing these parameters, researchers can maximize both the sensitivity and specificity of ChIP experiments using Acetyl-HIST1H4A (K91) antibodies, enabling reliable mapping of this modification's distribution across the genome.

How can researchers validate the specificity of Acetyl-HIST1H4A (K91) antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results when working with histone modifications. For Acetyl-HIST1H4A (K91) antibodies, a comprehensive validation approach should include:

• Peptide competition assays:

  • Pre-incubate the antibody with excess acetylated K91 peptide (immunogen)

  • In parallel, pre-incubate with unmodified peptide or peptides acetylated at other lysine residues

  • The specific signal should be blocked only by the acetyl-K91 peptide

• Western blot analysis:

  • Compare reactivity against recombinant H4 proteins with defined modifications

  • Test against acid-extracted histones from wild-type cells and K91A mutants

  • Include samples treated with histone deacetylase inhibitors to increase acetylation

• Cross-reactivity testing:

  • Test against a panel of synthetic peptides containing different H4 modifications

  • Examine reactivity to acetylated lysines at positions 5, 8, 12, 16, 20, and 31

  • Verify no cross-reactivity with unmodified K91 peptides

• Dot blot titration:

  • Spot decreasing amounts of modified and unmodified peptides

  • Determine the detection limit and dynamic range

  • Assess linearity of signal with antigen concentration

• Immunofluorescence validation:

  • Compare staining patterns in wild-type cells versus K91A mutants

  • Test staining after treatment with histone deacetylase inhibitors like sodium butyrate

  • Verify nuclear localization and expected distribution patterns

This multi-faceted approach, similar to what has been documented for other histone modification antibodies like H4K8ac , ensures that experimental results genuinely reflect the presence and distribution of K91 acetylation rather than cross-reactivity with other epitopes or non-specific binding.

What technical considerations affect immunocytochemistry experiments with Acetyl-HIST1H4A (K91) antibodies?

Successful immunocytochemistry (ICC) experiments with Acetyl-HIST1H4A (K91) antibodies require attention to several technical parameters to maximize signal specificity and intensity:

• Fixation and permeabilization:

  • Optimal fixation is critical for epitope preservation

  • Standard protocols use 4% paraformaldehyde for 10-15 minutes

  • Permeabilization with 0.1-0.5% Triton X-100 enhances antibody access to nuclear antigens

  • Overfixation can mask epitopes, while underfixation may compromise morphology

• Antibody dilution and incubation:

  • The recommended dilution range for ICC applications is 1:10-1:100

  • Titration experiments should determine optimal concentration for each cell type

  • Incubate primary antibody overnight at 4°C to maximize specific binding

  • Use blocking solution containing 1-5% BSA to reduce background

• Detection systems:

  • Fluorophore-conjugated secondary antibodies offer sensitivity and multiplexing options

  • Include DAPI or similar DNA counterstain to visualize nuclei

  • Consider using tyramide signal amplification for low-abundance modifications

  • Multi-channel imaging allows co-localization with other nuclear markers

• Controls and counterstaining:

  • Include secondary-only controls to assess non-specific binding

  • If available, K91A mutant cells provide ideal negative controls

  • Counterstain with markers for specific nuclear compartments (e.g., nucleoli, heterochromatin)

  • Co-staining with antibodies to other histone marks can reveal spatial relationships

• Signal visualization:

  • Use confocal microscopy for optimal spatial resolution

  • Z-stack acquisition helps resolve nuclear distribution patterns

  • Consistent exposure settings are essential for comparative analyses

  • Quantitative image analysis can extract distribution patterns and intensity measurements

Implementing these technical considerations will enhance the reliability and interpretability of ICC experiments using Acetyl-HIST1H4A (K91) antibodies, allowing researchers to accurately visualize the nuclear distribution and relative abundance of this important histone modification.