HIST1H4A (Ab-77) Antibody

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

HIST1H4A is a canonical histone H4 gene that encodes one of the core histone proteins essential for chromatin structure in eukaryotic cells. As a fundamental component of nucleosomes (the basic unit of chromatin consisting of DNA wrapped around histone octamers), histone H4 plays a critical role in genome organization and regulation. This protein undergoes various post-translational modifications (PTMs) including acetylation at lysine residues K5, K8, K12, and K16, and methylation at K20, which collectively regulate gene expression, DNA repair, and chromatin structure . These modifications create what is often termed the "histone code," which influences DNA accessibility and recruitment of regulatory proteins. The study of HIST1H4A and its modifications provides crucial insights into epigenetic mechanisms that control cellular differentiation, disease progression, and genome stability.

What detection methods are compatible with HIST1H4A (Ab-77) antibody?

The HIST1H4A antibody can be utilized across multiple experimental platforms:

• Chromatin Immunoprecipitation (ChIP): For investigating genomic locations of histone H4, often coupled with sequencing (ChIP-seq) for genome-wide analysis .

• Immunofluorescence (IF): To visualize nuclear localization patterns of histone H4 and its relation to chromatin organization.

• Western Blotting: For detecting and quantifying histone H4 protein levels in extracted histones or nuclear proteins.

• ELISA: Enables quantitative measurement of histone H4 in various sample types with detection ranges typically between 37.5-2400 pg/mL for human HIST1H4A .

• Flow Cytometry: For cell-by-cell analysis of histone modifications in heterogeneous populations.

Each application requires specific optimization of antibody concentration, incubation conditions, and sample preparation protocols to achieve optimal signal-to-noise ratios.

What sample types can be analyzed using the HIST1H4A antibody?

The HIST1H4A antibody has been validated for detecting histone H4 in multiple sample types:

For optimal results, samples may require dilution to bring concentrations within the detection range. The linearity assessment data indicates that serum samples can be diluted up to 1:8 while maintaining reliable detection, with recovery percentages ranging from 88% to 110% .

How can I validate the specificity of HIST1H4A (Ab-77) antibody in my experimental system?

Antibody validation is essential for generating reliable research data. For HIST1H4A antibodies, implement these methodological approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific binding should be blocked, resulting in signal reduction.

• Knockout/knockdown controls: Compare signals between wild-type samples and those where HIST1H4A has been knocked out using CRISPR-Cas9 or knocked down using siRNA. An example methodology is described in literature where sgRNA (5′-CACCGTTCGGGGCAAGGCCGGAAA-3′) targeting HIST1H4G was used to establish knockout lines in MCF7 cells .

• Recombinant protein controls: Test reactivity against recombinant histone H4 produced in E. coli (unmodified) versus purified histones from mammalian cells (with modifications) .

• Dot blot analysis: Test antibody specificity against modified and unmodified peptides encompassing various sites of H4 modification, as demonstrated with H4K91 acetylation-specific antibodies .

• Mass spectrometry validation: Perform immunoprecipitation followed by LC-MS to confirm target protein identity, as has been done to detect low amounts of histone variants in cell lines .

What are the optimal conditions for using HIST1H4A antibody in ChIP-seq experiments?

For successful ChIP-seq experiments using HIST1H4A antibody, follow these methodological guidelines:

Sample Preparation:

• Cross-link protein-DNA complexes with 1% formaldehyde (10 minutes, room temperature)

• Quench with 125 mM glycine (5 minutes)

• Isolate nuclei and sonicate chromatin to 200-500 bp fragments

• Verify fragment size distribution by gel electrophoresis

Immunoprecipitation Protocol:

• Pre-clear chromatin with protein A/G beads

• Use 2-5 μg antibody per reaction (optimize through titration)

• Include input DNA control and IgG negative control

• Incubate overnight at 4°C with rotation

Data Analysis:

• Use established peak-calling algorithms (e.g., MACS)

• Analyze differential histone H4 occupancy between conditions using statistical tools like EdgeR

• Compare results with published datasets on histone H4 localization

When interpreting ChIP-seq data, remember that histone H4 modifications show distinct genomic distribution patterns. For example, research has shown that H4K91 acetylation is significantly enriched in active regions of the genome and present at low levels at telomeres and the HMR locus .

How do post-translational modifications of histone H4 affect HIST1H4A antibody recognition?

Post-translational modifications can significantly impact antibody recognition in complex ways:

N-terminal Tail Modifications:

• Histone H4 acetylation at K5, K8, K12, and K16 residues may enhance or impede antibody binding depending on the epitope .

• Some antibodies show exquisite specificity for particular modification patterns. For example, certain H4K5ac-specific antibodies only recognize K5ac when the neighboring K8 is unacetylated , allowing distinction between newly assembled H4 (diacetylated at K5 and K12) and hyperacetylated H4.

Core Domain Modifications:

• Less studied modifications in the globular domain, such as acetylation at K91, may also affect antibody recognition .

• These modifications can alter nucleosome stability and chromatin structure, potentially affecting epitope accessibility.

Combinatorial Effects:

• Multiple simultaneous modifications can create conformational changes that mask or expose antibody epitopes.

• This complexity necessitates careful antibody selection and validation when studying specific histone H4 modification states.

To address these concerns, researchers should review manufacturer's specificity data and consider using multiple antibodies targeting different epitopes or modification states when studying histone H4 dynamics.

What are the challenges in distinguishing between histone H4 variants using antibodies?

Differentiating between histone H4 variants presents several technical challenges:

• High Sequence Conservation: Histone H4 is extremely conserved evolutionarily, with variants sharing extensive sequence similarity. This high conservation complicates generating variant-specific antibodies .

• Limited Variant-Specific Regions: The regions that differ between variants may be small, conformationally hidden, or poorly immunogenic, making them difficult targets for antibody generation.

• Modification Interference: Post-translational modifications can mask variant-specific epitopes or create epitopes that cross-react with antibodies intended for other variants .

To overcome these challenges, researchers should:

• Target regions with maximum sequence divergence between variants

• Extensively validate using recombinant proteins, variant-specific peptides, and knockout systems

• Combine antibody-based detection with mass spectrometry and RNA analysis

• Consider using sequential immunoprecipitation to deplete major variants before detecting minor variants

Research on the histone H4 variant H4G demonstrates the difficulty of detecting low-abundance variants, requiring sensitive detection methods such as LC-MS .

How can HIST1H4A antibody be used to study nucleosome assembly and chromatin remodeling?

HIST1H4A antibodies are valuable tools for investigating chromatin dynamics through several methodological approaches:

Pulse-Chase Experiments:

• Track newly synthesized versus existing histones during replication and repair

• Combine with modification-specific antibodies to differentiate newly assembled H4 (typically diacetylated at K5 and K12) from pre-existing histones

Chromatin Assembly Analysis:

• The acetylation of the NH2-terminal tail of histone H4 by type B histone acetyltransferases (HATs) is involved in chromatin assembly processes

• Co-immunoprecipitation can identify proteins interacting with histone H4 during assembly

DNA Damage Response:

• Mutations that alter histone H4 residues, particularly those in the interface between histone dimers like K91, confer phenotypes consistent with defects in chromatin assembly such as sensitivity to DNA damaging agents

• ChIP-seq using HIST1H4A antibodies before and after DNA damage can map redistribution during repair

Heterochromatin Formation:

• H4 plays important roles in silent chromatin structure

• Loss of specific H4 modifications, such as K91 acetylation, causes substantial alteration of telomeric silent chromatin structure, resulting in upregulation of telomere-proximal genes

The combination of ChIP-seq, immunofluorescence microscopy, and biochemical approaches can provide comprehensive insights into histone H4's role in chromatin biology.

What is the relationship between histone H4 acetylation and behavioral variation in model organisms?

Research reveals intriguing connections between histone H4 acetylation and behavioral phenotypes:

• Behavioral Variability Regulation: Studies in zebrafish demonstrate that histone H4 acetylation levels can influence behavioral inter-individual variability. Treatment with HDAC inhibitors that increase H4 acetylation reduces behavioral variability in population studies .

• Class-Specific HDAC Effects: Different classes of HDAC inhibitors have distinct effects on H4 acetylation and behavior:

  • Inhibitors of class I and II HDACs (NaBu, TSA) increase H4 acetylation and reduce behavioral variability

  • Inhibitors of class III HDACs (cambinol) increase H4 acetylation but do not alter behavioral variability

• Genetic Evidence: Heterozygotic mutants of the class I histone deacetylase hdac1 (hdac1+/-) show reduced behavioral inter-individual variability compared to controls, along with increased histone H4 acetylation, supporting pharmacological findings .

• Epigenomic Profiles: ChIP-seq analysis of H4 acetylation reveals specific genomic regions where acetylation differences correlate with behavioral phenotypes, providing mechanistic insights into how epigenetic modifications influence neural function .

This research highlights the importance of histone H4 modifications beyond their classical role in transcriptional regulation, extending to complex phenotypic outcomes relevant to neuroscience and behavioral research.

What are the common technical issues when working with HIST1H4A antibody in different applications?

Researchers commonly encounter several technical challenges when working with histone H4 antibodies:

When working with histone antibodies, batch-to-batch variation can affect reproducibility. Regular validation using positive and negative controls is essential, especially when starting new projects or using new antibody lots.

How can I quantitatively measure HIST1H4A in different sample types?

For quantitative measurement of HIST1H4A in various biological samples, ELISA is the method of choice. Based on available data :

Detection Parameters:

• Sensitivity: 9.3 pg/mL

• Detection range: 37.5 pg/mL-2400 pg/mL

• Intra-assay precision: CV% <8% (samples tested twenty times on one plate)

• Inter-assay precision: CV% <10% (samples tested in twenty different assays)

Sample Preparation Guidelines:

• Serum samples: No special preparation needed; dilution may be required for high-concentration samples

• Plasma samples: EDTA plasma shows good recovery (94%, range 90-98%)

• Cell lysates: Extraction using acid extraction or commercial histone extraction kits

• Tissue homogenates: Homogenization followed by histone extraction

Dilution Linearity:
Serum samples show good linearity across multiple dilutions :

• 1:1 dilution: 101% recovery (range 95-108%)

• 1:2 dilution: 88% recovery (range 83-99%)

• 1:4 dilution: 89% recovery (range 87-92%)

• 1:8 dilution: 110% recovery (range 106-115%)

For accurate quantification, always generate a standard curve for each experiment, as indicated in the technical guidelines: "These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed" .

How does HIST1H4A expression and modification vary across cancer types?

Studies of histone H4 and its variants in cancer reveal important tissue-specific patterns:

• Expression Level Variations: Histone H4 variant expression differs across cancer types. For example, the histone H4 variant H4G shows tumor-stage dependent overexpression in tissues from breast cancer patients . Expression analysis indicates that h4g expression in breast cancer cell lines (MCF7, LCC1, LCC2) is higher than in non-cancerous breast epithelial cells (MCF10A) or embryonic kidney cells (HEK293T) .

• Functional Significance: Modulating H4G expression affects rRNA expression levels, protein synthesis rates, and cell-cycle progression. H4G expression promotes breast cancer cell growth in mouse xenograft models, suggesting a role in cancer progression .

• Subcellular Localization: Unlike canonical histone H4, H4G localizes primarily to nucleoli, where it interacts with nucleophosmin 1 (NPM1), a nucleolar histone chaperone involved in ribosomal biogenesis and tumor progression .

• Chromatin Modifications: H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues, potentially contributing to the increased protein synthesis required for rapid cancer cell proliferation .

These findings highlight the importance of considering histone variant expression and specific modifications when studying cancer biology and developing potential therapeutic approaches targeting epigenetic mechanisms.

What emerging technologies are enhancing the study of histone H4 variants and modifications?

Several cutting-edge technologies are transforming histone H4 research:

• CUT&RUN and CUT&Tag: These techniques offer advantages over traditional ChIP by providing higher signal-to-noise ratios, requiring fewer cells, and enabling in situ chromatin profiling of histone H4 and its modifications.

• Single-cell epigenomics: New methods allow measurement of histone modifications at single-cell resolution, revealing heterogeneity in H4 modification patterns within seemingly homogeneous populations.

• Multi-omics integration: Combining histone modification data with transcriptomics, proteomics, and 3D chromatin structure provides comprehensive views of how H4 modifications influence gene expression and genome organization.

• Antibody engineering: Development of recombinant antibodies with higher specificity for particular histone H4 variants and modification states enables more precise epigenomic profiling.

• Mass spectrometry advances: Improved sensitivity in mass spectrometry now allows detection of low-abundance histone variants and combinatorial modifications that were previously undetectable .

These technological advances will continue to expand our understanding of histone H4 biology in development, disease, and cellular function.