HIST1H4A (Ab-31) Antibody

What is HIST1H4A and why is it significant in epigenetic research?

HIST1H4A is one of several genes encoding the histone H4 protein, belonging to the histone cluster 1 H4 family. Histone H4 is one of the core histones that form the nucleosome, the basic unit of chromatin packaging. The significance of HIST1H4A in epigenetic research stems from its critical role in chromatin structure and gene regulation.

HIST1H4A is classified as a replication-dependent histone isoform, which means its expression is tightly coupled with DNA replication during the S phase of the cell cycle. Unlike variant histones, replication-dependent histones like HIST1H4A constitute the bulk of histone proteins and are incorporated into newly synthesized DNA during replication . The post-translational modifications on these histones, such as acetylation at lysine 31 (targeted by Ab-31 antibody), play crucial roles in regulating chromatin accessibility, DNA repair, transcription, and other DNA-dependent processes.

How does HIST1H4A differ from other H4 histone isoforms?

HIST1H4A is one of several H4 histone isoforms that differ in subtle yet potentially significant ways:

HIST1H4A differs from HIST1H4I by a single amino acid substitution at position 70 (V70A). This residue is located in the α2 helix of histone H4, which is critical for interaction with specific chaperones including DAXX and Scm3. This position is important in generating a hydrophobic pocket that can adopt different conformations based on physiological changes, thus regulating interactions with other histones, ubiquitin-binding proteins, and DNA repair machinery . Even these subtle differences can confer unique functions and regulation to the different isoforms.

What is the significance of acetylation at lysine 31 (detected by Ab-31 antibody) compared to other H4 modifications?

Acetylation at lysine 31 (K31) of histone H4 is one of several important post-translational modifications that regulate chromatin structure and function. The Ab-31 antibody specifically recognizes this modification. Compared to better-studied H4 acetylation sites like K5, K8, K12, and K16, K31 acetylation has distinct functional implications:

• Location: K31 is positioned in the histone fold domain rather than the N-terminal tail, potentially affecting nucleosome stability and higher-order chromatin structure

• Accessibility: Unlike N-terminal modifications that extend outward from the nucleosome, K31 modifications may be more relevant during nucleosome assembly or disassembly

• Cellular function: K31 acetylation has been implicated in DNA damage response pathways and cellular differentiation processes

While N-terminal acetylation sites (K5, K8, K12, K16) are predominantly associated with active transcription, the internal K31 acetylation may have more specialized roles in chromatin dynamics and nucleosome stability. The Ab-31 antibody enables researchers to specifically study this modification and its biological significance .

What are the validated applications for HIST1H4A (Ab-31) antibody in research?

The HIST1H4A (Ab-31) antibody has been validated for multiple research applications, each with specific optimized protocols:

When designing experiments with this antibody, researchers should consider that optimization might be required depending on cell or tissue type. The antibody works most reliably with human samples as its reactivity is confirmed for human targets . For applications beyond those listed above, preliminary validation experiments are recommended.

How should I design ChIP experiments using HIST1H4A (Ab-31) antibody?

Chromatin Immunoprecipitation (ChIP) with HIST1H4A (Ab-31) antibody requires careful experimental design to obtain reliable results:

• Sample preparation: Crosslink proteins to DNA using 1% formaldehyde for 10 minutes at room temperature. Quench with 125 mM glycine.

• Chromatin fragmentation: Sonicate to generate 200-1000 bp fragments. Verify fragmentation efficiency via gel electrophoresis before proceeding.

• Immunoprecipitation optimization:

  • Use 2-5 μg of HIST1H4A (Ab-31) antibody per ChIP reaction

  • Include appropriate controls: IgG negative control and a positive control antibody (e.g., RNA Polymerase II)

  • Perform preliminary titration experiments to determine optimal antibody:chromatin ratios

• Washing and elution: Use stringent washing conditions to minimize non-specific binding, followed by elution of chromatin complexes.

• Data analysis: For ChIP-seq applications, compare K31ac enrichment patterns with other histone marks to identify functional correlations. For ChIP-qPCR, design primers for regions of interest and normalize to input DNA.

When interpreting ChIP data with HIST1H4A (Ab-31) antibody, consider that K31 acetylation may show distinct genomic distribution patterns compared to more well-characterized marks like H3K27ac or H3K4me3. The specific patterns may vary based on cell type, differentiation state, and experimental conditions .

What cell fixation and permeabilization methods work best for HIST1H4A (Ab-31) antibody in immunofluorescence?

For optimal immunofluorescence results with HIST1H4A (Ab-31) antibody, the fixation and permeabilization protocols significantly impact epitope accessibility and signal-to-noise ratio:

• Recommended fixation methods:

  • 4% paraformaldehyde (15 minutes at room temperature) provides good structural preservation while maintaining epitope recognition

  • Methanol fixation (-20°C for 10 minutes) may enhance nuclear epitope accessibility but can affect certain cellular structures

• Permeabilization optimization:

  • 0.1-0.5% Triton X-100 (10 minutes at room temperature) for paraformaldehyde-fixed cells

  • Additional permeabilization is typically unnecessary for methanol-fixed samples

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) may enhance signal intensity for certain sample types

  • For tissues, test both HIER and enzymatic retrieval to determine optimal protocol

• Blocking and antibody incubation:

  • Block with 5% normal serum from the same species as the secondary antibody

  • Incubate with primary antibody at 1:50-1:200 dilution overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies at manufacturer-recommended dilutions

Since the HIST1H4A (Ab-31) antibody targets an acetylated lysine residue, maintaining this modification during sample preparation is critical. Inclusion of histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in buffers can help preserve acetylation levels during experimentation .

How can I validate the specificity of HIST1H4A (Ab-31) antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For HIST1H4A (Ab-31) antibody, implement these complementary validation approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with increasing concentrations of the immunizing peptide (containing acetylated K31)

  • Compare with parallel experiments using unmodified peptide

  • Specific binding should be competitively inhibited by the acetylated peptide but not by unmodified peptide

• Genetic validation:

  • Use CRISPR/Cas9 to generate K31R mutants (prevents acetylation)

  • Compare antibody reactivity between wild-type and mutant samples

  • Signal should be significantly reduced or absent in K31R mutants

• Treatment with HDAC inhibitors and HAT inhibitors:

  • Treat cells with HDAC inhibitors to increase acetylation levels

  • Treat parallel samples with HAT inhibitors to decrease acetylation

  • Analyze changes in antibody reactivity corresponding to expected acetylation changes

• Western blot molecular weight verification:

  • Histone H4 has a predicted molecular weight of approximately 11 kDa

  • The HIST1H4A protein should appear at this position in Western blots

  • Verify single band specificity or document and characterize any additional bands

• Cross-reactivity assessment:

  • Test against recombinant histones with different modifications (e.g., K31me, K31ub)

  • Verify specificity against other acetylated lysine residues in H4 (K5ac, K8ac, K12ac, K16ac)

Document all validation steps thoroughly, as antibody specificity can vary between experimental systems and applications .

How do HIST1H4A K31 acetylation patterns change in cancer progression, and what research controls should I include?

Research has revealed that histone H4 acetylation patterns, including K31ac, undergo significant alterations during cancer progression. When studying these changes:

• Cancer-specific alterations in H4K31ac:

  • Several histone H4 isoforms including HIST1H4I show altered expression or modification in various cancers including ovarian cancer, colon cancer, and myelogenous leukemia

  • K31ac may show tissue-specific and cancer-subtype-specific patterns different from better-characterized marks like H4K16ac

• Essential experimental controls:

  • Tissue-matched controls: Compare tumor samples with adjacent normal tissue from the same patient

  • Developmental stage controls: Match tumor grade/stage with appropriate normal tissue developmental stage

  • Treatment response controls: For treatment studies, include appropriate vehicle controls

  • Technical controls: Include isotype controls and secondary-only controls to assess background signal

• Methodological considerations:

  • Analyze K31ac in conjunction with other histone marks (e.g., H3K27me3, H3K9me3) to develop a comprehensive epigenetic signature

  • Consider cell heterogeneity within tumor samples when interpreting bulk ChIP or immunoblotting data

  • When possible, combine with single-cell approaches to resolve population heterogeneity

• Data normalization approaches:

  • Normalize K31ac signals to total H4 levels to account for changes in histone expression

  • For ChIP-seq experiments, use appropriate spike-in controls (e.g., Drosophila chromatin) to enable quantitative comparisons across samples

This comprehensive approach helps distinguish between cancer-specific K31ac changes and those resulting from altered cell cycle dynamics or proliferation rates that commonly occur in cancer .

What is the relationship between HIST1H4A K31 acetylation and other histone modifications in different chromatin states?

The interplay between different histone modifications creates a complex "histone code" that regulates chromatin structure and function. For HIST1H4A K31 acetylation:

• Co-occurrence patterns with other modifications:

  • K31ac often co-occurs with other activating marks like H3K4me3 and H3K27ac at active regulatory elements

  • Anti-correlation is frequently observed with repressive marks like H3K9me3 and H3K27me3

  • More complex patterns emerge at bivalent domains and enhancer regions

• Functional relationships in chromatin regulation:

  • K31ac may act as a binding site for specific reader proteins distinct from those recognizing N-terminal acetylation sites

  • The internal position of K31 within the histone fold domain suggests potential roles in nucleosome stability or higher-order chromatin structure

• Methodology for studying modification relationships:

  • Sequential ChIP (re-ChIP) can determine co-occurrence of K31ac with other modifications on the same nucleosomes

  • Mass spectrometry approaches can quantify combinatorial modification patterns on individual histone molecules

  • Imaging approaches using multiple antibodies can reveal spatial relationships between different modified nucleosomes

The table below summarizes the relationship between K31ac and other key histone modifications:

Understanding these relationships helps interpret the biological significance of K31ac patterns observed in experimental data .

How can HIST1H4A (Ab-31) antibody be used in combination with other techniques to study chromatin dynamics?

Integrating HIST1H4A (Ab-31) antibody with complementary methods creates powerful approaches to investigate chromatin dynamics:

• ChIP-seq integration:

  • Combine K31ac ChIP-seq with ATAC-seq to correlate acetylation with chromatin accessibility

  • Integrate with Hi-C or micro-C data to examine relationships between K31ac and three-dimensional chromatin organization

  • Analyze alongside transcriptome data (RNA-seq) to assess functional impacts on gene expression

• Mass spectrometry approaches:

  • Use K31ac antibody for immunoaffinity enrichment prior to mass spectrometry

  • Identify proteins that specifically interact with K31-acetylated nucleosomes

  • Quantify combinatorial modification patterns on the same histone molecule

• Live-cell imaging applications:

  • Combine with FRAP (Fluorescence Recovery After Photobleaching) to study dynamics of K31ac-enriched chromatin regions

  • Use in proximity ligation assays (PLA) to detect interactions between K31ac and specific chromatin proteins

  • Implement for super-resolution microscopy to visualize K31ac distribution at nanoscale resolution

• Single-cell approaches:

  • Apply in CUT&Tag or CUT&RUN protocols for single-cell epigenomic profiling

  • Integrate with single-cell RNA-seq in multi-omic approaches

  • Use for flow cytometry or mass cytometry (CyTOF) to quantify K31ac across heterogeneous cell populations

• CRISPR-based functional genomics:

  • Combine with CRISPR screens targeting histone acetyltransferases and deacetylases

  • Use with CRISPR activation/inhibition systems to study cause-effect relationships

  • Implement with epigenome editing approaches to manipulate K31ac at specific genomic loci

These integrated approaches provide mechanistic insights beyond what can be achieved with the antibody alone, enabling researchers to address complex questions about K31ac function in chromatin biology .

What are the technical challenges in detecting HIST1H4A K31 acetylation in different sample types, and how can they be overcome?

Detecting HIST1H4A K31 acetylation presents several technical challenges that vary by sample type and application:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Challenge: Formaldehyde crosslinking can mask the K31ac epitope

  • Solution: Optimize antigen retrieval using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0)

  • Validation: Compare staining patterns with frozen sections where epitope preservation is superior

• Frozen tissue sections:

  • Challenge: Acetylation marks can be lost during storage and processing

  • Solution: Include HDAC inhibitors in fixation solutions

  • Optimization: Test multiple fixation protocols (e.g., acetone, methanol, paraformaldehyde)

• ChIP applications:

  • Challenge: Low abundance of K31ac compared to N-terminal acetylation marks

  • Solution: Increase antibody concentration and chromatin amount

  • Control: Include spike-in chromatin for normalization across samples

• Flow cytometry detection:

  • Challenge: Achieving sufficient permeabilization for nuclear epitope access

  • Solution: Test stronger permeabilization agents (e.g., saponin, Triton X-100)

  • Validation: Use positive control samples treated with HDAC inhibitors

• Western blotting:

  • Challenge: Multiple H4 isoforms can create complex banding patterns

  • Solution: Use acid extraction methods for enriching histones

  • Specificity: Include peptide competition controls to confirm band identity

Sample-specific optimization protocols:

Optimization experiments should be documented thoroughly to ensure reproducibility across different batches of samples .

How can I analyze the genome-wide distribution of HIST1H4A K31 acetylation in relation to chromatin domains and gene regulation?

Analyzing genome-wide distribution of K31ac requires sophisticated computational approaches to connect this modification with chromatin architecture and gene regulation:

• ChIP-seq data generation and processing:

  • Aim for >20 million uniquely mapped reads for sufficient coverage

  • Use spike-in normalization (e.g., Drosophila chromatin) for quantitative comparisons

  • Process with appropriate peak callers (e.g., MACS2 with broad peak settings)

• Integrative genomic analysis approaches:

  • Profile analysis: Generate metagene plots and heatmaps around transcription start sites, enhancers, and other genomic features

  • Chromatin state correlation: Compare K31ac distribution with published chromatin state models (e.g., ChromHMM)

  • Motif analysis: Identify transcription factor binding motifs enriched in K31ac peaks

• Three-dimensional chromatin context:

  • Analyze K31ac distribution in relation to TAD (Topologically Associated Domain) boundaries

  • Examine enrichment patterns at chromatin loop anchors

  • Correlate with A/B compartments from Hi-C data

• Gene regulation analysis:

  • Expression correlation: Calculate correlation between K31ac signal and gene expression levels

  • Enhancer activity: Analyze K31ac at candidate enhancers and correlate with target gene expression

  • Response elements: Examine K31ac dynamics at stimulus-responsive genomic regions

• Data visualization and interpretation:

  • Use genome browsers with multiple track visualization

  • Implement dimensionality reduction approaches (e.g., t-SNE, UMAP) to identify patterns

  • Generate correlation heatmaps between K31ac and other epigenetic marks

• Statistical considerations:

  • Apply appropriate multiple testing correction for genome-wide analyses

  • Use permutation tests to assess significance of spatial correlations

  • Implement bootstrapping approaches to estimate confidence intervals

Example findings from such analyses might reveal that K31ac is enriched at specific subsets of regulatory elements with distinct functional properties or that it marks transitions between different chromatin domains, providing insights into its biological roles .

What is the current understanding of the enzymes that regulate HIST1H4A K31 acetylation and deacetylation?

Research into the enzymatic regulation of H4K31 acetylation is still emerging, with several key findings:

• Histone acetyltransferases (HATs):

  • The MYST family HAT KAT8 (MOF) has been implicated in K31 acetylation in some cellular contexts

  • CBP/p300 may contribute to K31 acetylation, particularly during DNA damage response

  • The precise specificity of different HATs for K31 versus other acetylation sites remains under investigation

• Histone deacetylases (HDACs):

  • Class I HDACs (particularly HDAC1 and HDAC2) appear to deacetylate H4K31 based on inhibitor studies

  • Sirtuin family deacetylases (specifically SIRT1) may also target K31ac in certain contexts

  • The substrate specificity of these enzymes is still being characterized

• Regulation in disease contexts:

  • Altered expression of these enzymes in cancer correlates with changes in K31ac patterns

  • Mutations in HATs and HDACs affecting K31ac have been identified in patient samples

  • HDAC inhibitors used in cancer therapy impact K31ac levels alongside other acetylation marks

• Current research limitations:

  • Most studies have not specifically focused on K31ac but rather examined it alongside other marks

  • The development of K31-specific enzymatic assays is needed for definitive characterization

  • Research is complicated by potential redundancy among multiple enzymes

Future research directions include developing specific inhibitors or activators of K31 acetylation/deacetylation and characterizing the structural basis for enzyme specificity toward this site compared to other acetylation sites in histone H4 .

Several cutting-edge technologies are poised to revolutionize our understanding of HIST1H4A modifications, including K31 acetylation:

• Advanced sequencing technologies:

  • CUT&Tag and CUT&RUN: Higher signal-to-noise ratio than traditional ChIP-seq for detecting K31ac genome-wide

  • Single-cell epigenomics: Revealing cell-to-cell variation in K31ac patterns within heterogeneous populations

  • Long-read sequencing: Enabling detection of K31ac in repetitive regions and providing haplotype-specific information

• Mass spectrometry innovations:

  • Top-down proteomics: Analyzing intact histone proteins to capture combinatorial modification patterns

  • Targeted MS methods: Increasing sensitivity for detecting low-abundance modifications like K31ac

  • Crosslinking MS: Identifying proteins that specifically interact with K31-acetylated nucleosomes

• Imaging advances:

  • Super-resolution microscopy: Visualizing K31ac distribution at nanoscale resolution

  • Live-cell imaging with modification-specific probes: Monitoring K31ac dynamics in real-time

  • Multi-modal imaging: Correlating K31ac patterns with chromatin compaction states

• Synthetic biology approaches:

  • Semi-synthetic histones: Incorporating acetyl-lysine analogs at position 31 for functional studies

  • Optogenetic control of K31 acetylation: Enabling spatiotemporal manipulation of this modification

  • Gene editing technologies: Creating precise mutations at K31 or in modifying enzymes

• Computational methods:

  • Deep learning models: Predicting K31ac patterns from DNA sequence and other epigenetic features

  • Multi-omics integration: Combining K31ac data with transcriptomics, proteomics, and metabolomics

  • 4D nucleome modeling: Incorporating K31ac into models of dynamic chromatin organization

These emerging technologies promise to overcome current limitations in studying K31ac, including sensitivity issues, inability to track dynamic changes, and challenges in determining causal relationships between K31ac and biological outcomes .