Acetyl-HIST1H4A (K31) Antibody

What is Histone H4K31 acetylation and why is it significant?

H4K31 acetylation is a post-translational modification (PTM) occurring at lysine 31 on the histone H4 protein. This modification is particularly significant because the K31 residue is located at the N-terminus of the H4 α1 helix with its side chain extended into the major groove of DNA. Acetylation at this position disrupts water-mediated hydrogen bonding between the lysine side chain and the DNA phosphate backbone, potentially destabilizing the protein-DNA interface near the nucleosome dyad axis. This modification was initially underestimated but has recently gained attention for its role in regulating chromatin structure and gene expression .

How does H4K31 acetylation compare to better-known histone modifications?

Unlike many well-studied histone modifications that occur on histone tails, H4K31 is located within the globular domain of histone H4 on the lateral surface of the nucleosome. This positioning gives H4K31ac unique properties compared to tail modifications. While tail modifications often serve as binding sites for effector proteins, H4K31ac directly affects nucleosome stability by altering histone-DNA interactions. Research indicates that H4K31ac may function similarly to other outer surface modifications like H3K36ac in regulating nucleosome mobility and accessibility .

What methods should be used to verify antibody specificity for H4K31ac?

Proper validation of H4K31ac antibodies requires multiple complementary approaches:

• Dot-blot assays using synthetic peptides: Test the antibody against acetylated H4K31 peptides alongside unmodified peptides and peptides containing other histone modifications

• Western blot analysis: Compare histone extracts from cells treated with and without histone deacetylase inhibitors (HDACi)

• Immunofluorescence: Examine nuclear localization patterns and compare signal intensity with and without HDACi treatment

• ChIP-seq reproducibility: Verify consistent enrichment patterns across technical replicates

Research has shown that specific H4K31ac antibodies should not cross-react with unmodified peptides or with previously described acetyl and methyl marks in histone tails and globular domains .

How can researchers distinguish between genuine H4K31ac signals and non-specific binding?

Distinguishing genuine H4K31ac signals from non-specific binding requires implementing several control measures:

• Use histone deacetylase inhibitors (e.g., FR235222) to increase H4K31ac levels while monitoring signal intensity

• Include control antibodies directed at other histone modifications (e.g., H3K14ac) that should not be affected by the same treatments

• Compare immunoprecipitation results with computational predictions of K31 modification sites

• Employ genetic approaches, such as lysine-to-arginine mutations at position 31, which should abolish specific antibody binding

When validating an H4K31ac antibody, a genuine signal will show increased intensity following HDACi treatment in immunofluorescence and Western blot analyses, while signals for other histone modifications like H3K14ac and H3K27ac should remain relatively unchanged under the same conditions .

What are the technical challenges in developing highly specific antibodies against H4K31ac?

Developing specific antibodies against H4K31ac presents several technical challenges:

• The modification occurs within the globular domain of histone H4, making it less accessible than tail modifications

• The surrounding amino acid sequence context may share similarities with other acetylated lysines in histones

• The antibody must distinguish between H4K31ac and H4K31me1, which occupy the same residue but confer opposite functional outcomes

• The relatively low abundance of H4K31ac in certain cell types may require antibodies with high sensitivity

Researchers have overcome these challenges by using carefully designed synthetic peptides that incorporate the unique sequence context surrounding K31, implementing rigorous cross-reactivity testing, and validating antibody performance across multiple experimental techniques .

What are the optimal techniques for detecting and quantifying H4K31 acetylation in different experimental contexts?

Several techniques can be employed for detecting and quantifying H4K31 acetylation:

• Chromatin Immunoprecipitation (ChIP): For genome-wide distribution analysis

  • Standard ChIP-seq protocols with optimized sonication conditions

  • Verify technical reproducibility between replicates

  • Compare with other histone marks for context

• Immunofluorescence: For cellular localization

  • Optimize fixation conditions to preserve nuclear architecture

  • Use appropriate controls (HDACi treatment increases signal)

  • Co-stain with DAPI for nuclear visualization

• Western blotting: For quantitative analysis

  • Use HDACi-treated samples as positive controls

  • Analyze under reducing conditions with appropriate buffer groups

• Mass spectrometry: For unbiased identification

  • Enables detection without antibody-related biases

  • Can distinguish between different modifications at the K31 position

Each technique provides complementary information, with ChIP-seq revealing genomic distribution, immunofluorescence showing nuclear localization, western blotting providing quantitative data, and mass spectrometry offering unbiased detection .

How should ChIP-seq experiments be optimized for H4K31ac detection?

Optimizing ChIP-seq for H4K31ac detection requires specific considerations:

• Chromatin preparation: Use optimized sonication protocols to generate 200-500bp fragments while preserving epitope accessibility

• Antibody selection: Use highly specific antibodies validated by dot blot and other methods

• Controls: Include input DNA, IgG controls, and positive controls (HDACi-treated samples)

• Data analysis: Compare H4K31ac enrichment patterns with other histone marks (H3K14ac, H3K4me3) to contextualize findings

• Reproducibility: Ensure low variability and high similarity in read coverage between technical replicates

Research indicates that H4K31ac exhibits a distinct pattern of enrichment across chromosomes, with approximately 75% of peaks mapping outside gene bodies, primarily in intergenic regions and promoters .

What are the most effective strategies for visualizing H4K31ac in different cell types using immunofluorescence?

For effective immunofluorescence visualization of H4K31ac:

• Fixation optimization:

  • Use 4% paraformaldehyde for preserving nuclear architecture

  • Include permeabilization steps with detergents like Triton X-100

• Antibody dilution series:

  • Determine optimal concentration (typically 0.1-1 μg/mL)

  • Incubate for sufficient time (3-16 hours) at appropriate temperature

• Signal amplification:

  • Employ fluorophore-conjugated secondary antibodies with minimal background

  • Use mounting media with anti-fade properties to preserve signal

• Experimental treatments:

  • Include HDACi treatment (e.g., FR235222) as a positive control

  • Compare with other histone marks as specificity controls

• Imaging parameters:

  • Use confocal microscopy for better resolution

  • Standardize exposure settings across experimental conditions

Studies have shown that H4K31ac is distributed exclusively and uniformly within nuclei of both parasite and human cells in T. gondii infection models, with signal intensity significantly increasing after HDACi treatment .

What is the functional significance of H4K31 acetylation in chromatin regulation?

H4K31 acetylation plays several crucial roles in chromatin regulation:

• Nucleosome stability: H4K31ac likely destabilizes the protein-DNA interface near the nucleosome dyad axis by disrupting water-mediated interactions between the lysine side chain and DNA

• Chromatin accessibility: While crystallography studies using glutamine substitution (H4Q31) to mimic acetylation did not show large structural changes, H4K31ac may increase DNA unwrapping at nucleosome entry-exit points

• Transcriptional regulation: Genome-wide studies in T. gondii and P. falciparum revealed local enrichment of H4K31ac at active gene promoters, suggesting it contributes to a transcriptionally permissive chromatin state

• Nucleosome mobility: H4K31ac may regulate the equilibrium between mobile and stationary nucleosomes, similar to other outer surface modifications like H3K36ac

This modification appears to relieve nucleosomal repression, facilitating DNA template access for the transcriptional machinery .

How does H4K31 acetylation interact with other histone modifications?

H4K31 acetylation functions within a complex network of histone modifications:

• Mutual exclusivity with methylation: H4K31ac and H4K31me1 show mutually exclusive genome-wide distribution patterns, suggesting a binary regulatory mechanism

• Co-occurrence with active marks: H4K31ac shows genomic distribution patterns similar to other active chromatin marks like H3K14ac and H3K4me3, with enrichment at 5'UTR/promoter regions

• Enzyme interactions: H4K31ac can be removed by histone deacetylases (as evidenced by increased signal after HDACi treatment)

• Regulatory mechanisms: Acetylation at K31 prevents methylation at the same residue, ensuring maximal RNA polymerase progression at highly expressed genes

This interplay between different modifications creates a complex "histone code" that regulates gene expression and chromatin structure in a context-dependent manner .

What is the relationship between H4K31 acetylation and methylation in gene expression regulation?

The relationship between H4K31 acetylation and methylation represents a fascinating regulatory mechanism:

• Genomic distribution: H4K31ac and H4K31me1 exhibit mutually exclusive patterns of enrichment across chromosomes

• Localization differences:

  • H4K31ac: Enriched in distinct peaks at intergenic regions and promoters

  • H4K31me1: Spans from translation start sites through entire gene bodies

• Functional opposition:

  • H4K31ac: Associated with transcriptionally permissive chromatin

  • H4K31me1: Likely locks nucleosomes in a repressed or "poised" state

• Gene expression correlation:

  • H4K31ac: Found at active gene promoters

  • H4K31me1: Enriched in transcribed regions of genes with limited RNA polymerase II activity

This binary switch mechanism suggests that H4K31ac facilitates RNA polymerase progression, while H4K31me1 stabilizes DNA wrapping around histones, potentially slowing RNA polymerase processing and reducing transcription levels .

How can researchers effectively use H4K31ac antibodies in multi-omics approaches?

Integrating H4K31ac antibodies into multi-omics approaches requires strategic experimental design:

• ChIP-seq + RNA-seq integration:

  • Compare H4K31ac peak locations with gene expression data

  • Use differential expression analysis following HDACi treatment

  • Correlate changes in H4K31ac enrichment with transcriptional changes

• Proteomics coupling:

  • Combine ChIP-seq with mass spectrometry to identify proteins associated with H4K31ac-enriched regions

  • Use SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) to quantify changes in the acetylome following interventions

• Single-cell applications:

  • Adapt ChIP protocols for low-input samples

  • Use CUT&RUN or CUT&Tag for improved sensitivity

  • Correlate with single-cell RNA-seq data

• Computational integration:

  • Develop models incorporating H4K31ac data with other epigenetic marks

  • Use machine learning approaches to predict functional outcomes based on modification patterns

This integrated approach can reveal how H4K31ac works within the broader epigenetic landscape to regulate cellular functions across different biological contexts .

What are the species-specific variations in H4K31 modification patterns and their functional implications?

H4K31 modification patterns show important species-specific variations:

• Taxonomic distribution:

  • Initially thought to be restricted to metazoans

  • Now identified across a broader range including apicomplexan parasites

  • Not detected in yeast or the ciliated protozoan Tetrahymena by early studies

• Cell type specificity:

  • In mammals: H4K31me1 decorates mitotic chromosomes but is barely detected in interphase nuclei

  • In T. gondii: H4K31ac distributes uniformly within nuclei of dividing parasites

  • In P. falciparum: Present throughout the intraerythrocytic developmental cycle

• Genomic distribution differences:

  • In T. gondii: H4K31ac enriched at intergenic regions/promoters; H4K31me1 spans gene bodies

  • In human cells: H4K31me1 detected by mass spectrometry but shows different nuclear distribution compared to parasites

• Functional implications:

  • Conservation suggests fundamental roles in chromatin regulation

  • Species-specific patterns may reflect adaptation to different genome organizations and regulatory needs

These variations indicate that while the modification itself is conserved, its regulatory functions may have evolved to suit different biological contexts .

How do experimental conditions affect antibody performance in H4K31ac detection?

Several experimental conditions can significantly impact antibody performance for H4K31ac detection:

• Fixation conditions:

  • Over-fixation can mask epitopes by extensive protein crosslinking

  • Under-fixation risks losing nuclear structure integrity

  • Optimal paraformaldehyde concentration and timing are critical

• Buffer composition:

  • Salt concentration affects antibody-epitope interactions

  • Detergent types and concentrations influence nuclear permeabilization

  • pH conditions may alter epitope accessibility or antibody binding

• Blocking parameters:

  • Insufficient blocking increases non-specific binding

  • Excessive blocking may reduce specific signal detection

  • Selection of appropriate blocking agent (BSA, serum, commercial alternatives)

• Antibody incubation:

  • Time and temperature affect binding equilibrium

  • Concentration determines signal-to-noise ratio

  • Multiple wash steps are essential for removing unbound antibody

• Sample preparation for ChIP:

  • Crosslinking efficiency influences epitope preservation

  • Sonication parameters affect chromatin fragmentation

  • Enzymatic digestion alternatives may preserve epitope integrity

Researchers should systematically optimize these conditions for each experimental system, comparing results with known positive controls such as HDACi-treated samples that show enhanced H4K31ac signals .

What are the best practices for sample preparation when studying H4K31 acetylation?

Optimal sample preparation for H4K31 acetylation studies involves several critical steps:

• Cell/tissue preparation:

  • Harvest cells at consistent density and cell cycle stage

  • Minimize stress during collection to prevent artifactual changes

  • Process samples consistently to reduce experimental variation

• Fixation protocols:

  • For ChIP: Use 1% formaldehyde for precise crosslinking time (8-10 minutes)

  • For immunofluorescence: Use 4% paraformaldehyde with proper permeabilization

  • For protein extraction: Use acid extraction methods optimized for histones

• Chromatin preparation:

  • Sonication parameters: Optimize cycles, amplitude, and duration

  • Target fragment size: 200-500bp for standard ChIP-seq

  • Quality control: Verify fragmentation by gel electrophoresis

• Positive controls:

  • Include HDACi-treated samples (e.g., FR235222, sodium butyrate)

  • Compare with other well-characterized histone modifications

  • Use multiple biological replicates to establish reproducibility

Studies have demonstrated that HDACi treatment significantly increases H4K31ac signal intensity in both immunofluorescence and western blot applications, making it an excellent positive control .

What statistical approaches are most appropriate for analyzing H4K31ac ChIP-seq data?

Analysis of H4K31ac ChIP-seq data requires sophisticated statistical approaches:

• Quality control metrics:

  • Fragment length distribution

  • Library complexity assessment

  • Technical replicate correlation

• Peak calling strategies:

  • MACS2 with appropriate q-value thresholds

  • IDR (Irreproducible Discovery Rate) for replicate consistency

  • Signal-to-noise ratio assessment

• Differential binding analysis:

  • DESeq2 or edgeR for count-based comparisons

  • Consider biological variability through appropriate replication

  • Normalize to input DNA or appropriate controls

• Genomic feature association:

  • Enrichment analysis relative to gene features (promoters, gene bodies)

  • Comparison with transcript levels from RNA-seq

  • Integration with other histone modification datasets

• Visualization approaches:

  • Generate average profiles around transcription start sites

  • Create heatmaps stratified by gene expression levels

  • Use genome browsers for locus-specific examination

Research has shown that H4K31ac enrichment patterns are distinctly different from H4K31me1, with the acetylation mark predominantly localized to intergenic regions and promoters, while methylation spans gene bodies - these distinct patterns require appropriate statistical modeling .

How can genetic approaches be used to validate antibody specificity and biological functions of H4K31 acetylation?

Genetic approaches provide powerful tools for validating both antibody specificity and biological functions:

• CRISPR/Cas9 modification strategies:

  • Generate K31R mutations (cannot be acetylated or methylated)

  • Create K31Q mutations (mimics constitutive acetylation)

  • Develop K31M mutations (prevents modification while maintaining charge)

• Enzyme manipulation:

  • Knock down/out histone acetyltransferases (HATs) that target K31

  • Overexpress or inhibit specific histone deacetylases (HDACs)

  • Engineer inducible systems for temporal control

• Specificity validation:

  • Compare antibody signals between wild-type and K31-mutant cells

  • Use K31R mutants as negative controls in immunoprecipitation

  • Perform peptide competition assays with synthetic modified peptides

• Functional assessment:

  • Analyze transcriptome changes following K31 mutation

  • Examine chromatin accessibility alterations

  • Measure effects on cellular phenotypes (e.g., cell cycle progression)

These approaches can provide definitive evidence for both antibody specificity and the biological significance of H4K31 acetylation, as demonstrated by studies showing that disrupting the water-mediated interactions of K31 with DNA affects nucleosome stability and gene expression .