HIST1H3A (Ab-45) Antibody

What is HIST1H3A and how does it differ from other H3 variants?

HIST1H3A is a gene encoding histone H3.1, one of the canonical histone H3 variants. Histone H3.1 is a 135-amino acid protein (after cleavage of the first methionine) containing a histone tail, four α-helices, and two loop domains . While H3.1 shares high sequence homology with other H3 variants, it has some unique features:

• H3.1 contains a unique cysteine residue at position 96 that can function as a chromatin-embedded redox sensor

• Unlike H3.3, H3.1 is predominantly synthesized and incorporated into chromatin during DNA replication in the S phase of the cell cycle

• The tails of H3.1 and other H3 histones (amino acids 1-89) are identical except for residue 31, where H3.1 contains alanine while other variants contain serine

The specific molecular weight of HIST1H3A protein is approximately 15,404 Da .

What are the key post-translational modifications of HIST1H3A at position Thr-45?

Threonine 45 of histone H3 (H3T45) has been identified as a site of phosphorylation in vivo . This modification is part of the core histone domain rather than the histone tail, giving it the potential to directly modulate nucleosome structure and consequently DNA accessibility . Key characteristics of this modification include:

• H3T45 phosphorylation (H3T45ph) can be detected at low levels in untreated cells but increases substantially following phosphatase inhibition

• This phosphorylation appears to be linked to apoptotic processes, suggesting its role in cellular stress responses

• The modification occurs within the core nucleosomal structure, potentially influencing DNA-histone interactions more directly than tail modifications

How do antibodies against HIST1H3A Thr-45 contribute to epigenetic research?

Antibodies targeting phosphorylated H3T45 provide essential tools for studying this specific modification in various research contexts. These antibodies enable:

• Visualization and quantification of H3T45ph during different cellular processes

• Mapping genomic locations where this modification occurs through chromatin immunoprecipitation (ChIP) experiments

• Investigation of relationships between H3T45ph and other histone modifications or cellular events

• Assessment of how H3T45ph changes in response to cellular stresses or signaling events

These antibodies are typically validated through multiple methods, including peptide competition assays, ELISA, and peptide dot blots to ensure specificity against the phosphorylated form of the residue .

What are the recommended protocols for validating anti-H3T45ph antibody specificity?

Thorough validation of antibodies targeting H3T45ph is essential for reliable experimental results. Based on established methods, the following protocol is recommended:

• Peptide competition assay: Pre-incubate the antibody with increasing concentrations of either phosphorylated or non-phosphorylated peptides corresponding to the region around H3T45, then perform western blotting or immunostaining to demonstrate specific blocking with the phosphorylated peptide only .

• ELISA validation: Coat wells with H3 peptides (phosphorylated at T45 and non-phosphorylated controls), then test antibody binding using standard ELISA protocols. Quantitative measurement of binding affinity and specificity should show strong preference for the phosphorylated epitope .

• Peptide dot blots: Spot varying concentrations of H3T45ph and control peptides on membranes, then probe with the antibody to visualize binding specificity .

• Mass spectrometry correlation: Treat cells with phosphatase inhibitors like calyculin A for 15 minutes, extract histones, and perform parallel analyses using both the antibody of interest and MS/MS to confirm detection of the same modification .

• Knockout/knockdown controls: When possible, validate antibody specificity using genetic models where the target histone or modifying enzyme has been depleted.

What are the optimal conditions for using anti-HIST1H3A antibodies in western blotting applications?

For optimal western blotting results with anti-HIST1H3A antibodies, the following protocol parameters are recommended:

• Sample preparation:

  • Extract histones using acid extraction methods to enrich for histone proteins

  • For whole cell lysates, use buffer systems containing phosphatase inhibitors to preserve phosphorylation status

  • Load 20-40μg of protein per lane (exact amount may vary by cell type)

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels to achieve good separation of histone proteins

  • Include positive control samples (e.g., A549, C6, AML-12, or HepG2 cell lysates)

• Antibody dilution and incubation:

  • Recommended dilution range: 1:500-1:1000 for primary antibody

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Detection methods:

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence (ECL) detection

  • Expected molecular weight band: approximately 15-17 kDa

Table 1: Recommended Western Blotting Conditions for HIST1H3A Antibodies

What are the recommended protocols for immunohistochemistry using anti-HIST1H3A antibodies?

For immunohistochemistry applications with anti-HIST1H3A antibodies, follow these optimized protocol guidelines:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6μm thickness

  • For phospho-specific epitopes, minimize dephosphorylation by rapid processing

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Alternative: Tris-EDTA buffer (pH 9.0) for some applications

• Antibody dilution and incubation:

  • Recommended dilution range: 1:50-1:200

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Use a polymer-HRP based detection system for optimal sensitivity

  • DAB (3,3'-diaminobenzidine) substrate for visualization

  • Counterstain with hematoxylin

• Controls:

  • Positive control tissue: human breast carcinoma tissue has been validated

  • Negative control: omit primary antibody or use isotype control

  • Peptide competition control: pre-absorb antibody with immunizing peptide

How can anti-H3T45ph antibodies be used to study the relationship between histone phosphorylation and apoptosis?

Investigating the role of H3T45 phosphorylation in apoptosis requires sophisticated experimental approaches:

• Time-course analysis during apoptosis:

  • Induce apoptosis using established triggers (e.g., staurosporine, TNF-α with cycloheximide)

  • Collect cells at regular intervals (0, 1, 2, 4, 8, 12, 24 hours)

  • Perform western blotting with anti-H3T45ph antibody to track phosphorylation dynamics

  • In parallel, measure apoptotic markers (cleaved caspase-3, PARP cleavage) to correlate with H3T45ph timing

• Chromatin immunoprecipitation (ChIP) during apoptosis:

  • Perform ChIP-seq with anti-H3T45ph antibody at key timepoints during apoptosis

  • Analyze genomic distribution of H3T45ph to identify regions affected early in apoptosis

  • Correlate with gene expression changes using RNA-seq

  • Identify transcription factors that co-localize with H3T45ph regions

• Mass spectrometry-based quantification:

  • Isolate histones from control and apoptotic cells

  • Perform nano-LC-MS/MS analysis to quantify H3T45ph levels

  • Compare with other histone modifications to identify potential crosstalk

  • Calculate coefficients of variation to assess distribution patterns

• Inhibitor studies:

  • Test effects of kinase inhibitors on both H3T45ph levels and apoptotic progression

  • Test phosphatase inhibitors (e.g., calyculin A) to enhance H3T45ph and observe effects on apoptotic threshold

This multi-method approach enables comprehensive understanding of how H3T45 phosphorylation contributes to apoptotic processes.

What approaches can be used to identify the kinases and phosphatases that regulate H3T45 phosphorylation?

Identifying the enzymatic regulators of H3T45 phosphorylation requires systematic screening approaches:

• Kinase inhibitor screening:

  • Treat cells with a panel of selective kinase inhibitors

  • Measure H3T45ph levels by western blotting

  • Narrow down candidate kinase families based on inhibitor specificity

  • Validate using genetic approaches (siRNA, CRISPR-Cas9)

• In vitro kinase assays:

  • Express and purify recombinant H3 as substrate

  • Test candidate kinases in vitro with γ-32P-ATP or use antibody-based detection

  • Confirm specificity using H3 variants with T45A mutation

  • Determine kinetic parameters (Km, Vmax) for positive hits

• Phosphatase identification:

  • Treat cells with phosphatase inhibitors of varying specificity

  • Analyze H3T45ph levels to identify phosphatase families involved

  • Perform phosphatase substrate-trapping experiments

  • Validate candidates using phosphatase overexpression and knockdown

• Proximity labeling proteomics:

  • Generate H3.1-BioID or H3.1-TurboID fusion constructs

  • Identify proteins in proximity to H3.1 using streptavidin pulldown and mass spectrometry

  • Focus on kinases/phosphatases among proximity interactors

  • Compare interactomes between wild-type and T45A mutant H3.1

These approaches collectively provide a comprehensive strategy for identifying the enzymatic regulators of H3T45 phosphorylation.

How does H3T45 phosphorylation interact with the redox sensing function of H3.1 through Cys96?

Investigating potential crosstalk between H3T45 phosphorylation and the Cys96-mediated redox sensing function of H3.1 represents an advanced research question that integrates multiple histone functions:

• Simultaneous modification analysis:

  • Treat cells with oxidative stress inducers (H₂O₂, menadione)

  • Analyze both H3T45ph and Cys96 oxidation status using antibodies and mass spectrometry

  • Determine if these modifications co-occur on the same histone molecules

  • Analyze temporal dynamics to establish potential sequential ordering

• Structural impact analysis:

  • Perform molecular dynamics simulations of nucleosomes with various modification states:

    • Unmodified H3.1

    • H3T45ph only

    • Cys96 oxidation only

    • Both modifications simultaneously

  • Analyze impacts on nucleosome stability and DNA accessibility

• Mutational studies:

  • Generate cell lines expressing H3.1 variants:

    • Wild-type

    • T45A (phospho-null)

    • T45E (phospho-mimetic)

    • C96S (redox-null)

    • Combined mutations

  • Analyze cellular responses to oxidative stress and apoptotic stimuli

  • Perform ChIP-seq to map genomic distribution changes

• Chromatin exchange dynamics:

  • Investigate whether H3T45 phosphorylation affects the H3.1-to-H3.3 exchange process that occurs following Cys96 oxidation

  • Use FRAP (Fluorescence Recovery After Photobleaching) with fluorescently tagged histones to measure exchange rates

  • Determine if phosphatase inhibition (to increase H3T45ph) affects oxidation-induced histone variant exchange

This integrated approach would illuminate potential functional connections between these two distinct regulatory modifications in H3.1.

What are the most common causes of non-specific binding with anti-HIST1H3A antibodies and how can they be resolved?

Non-specific binding is a common challenge when working with histone antibodies. Here are the most frequent causes and their solutions:

• Cross-reactivity with other H3 variants:

  • Problem: High sequence homology between H3 variants leads to cross-reactivity

  • Solution: Use peptide competition assays with specific peptides from different H3 variants

  • Validation: Test antibody against recombinant H3.1, H3.2, and H3.3 proteins

• Epitope masking by nearby modifications:

  • Problem: Other PTMs near T45 can interfere with antibody binding

  • Solution: Use MS/MS analysis to identify potential interfering modifications

  • Optimization: Try different antigen retrieval methods for IHC/ICC applications

• Insufficient blocking:

  • Problem: High background due to inadequate blocking

  • Solution: Extend blocking time (2-3 hours) and optimize blocking agent (5% BSA often works better than milk for phospho-epitopes)

  • Alternative: Test synthetic blocking peptides specific to the antibody epitope region

• Sample preparation issues:

  • Problem: Dephosphorylation during sample preparation

  • Solution: Include phosphatase inhibitors in all buffers and keep samples cold

  • Optimization: Minimize time between sample collection and fixation/extraction

Table 2: Troubleshooting Guide for Common HIST1H3A Antibody Issues

How can ChIP-seq experiments with anti-H3T45ph antibodies be optimized for maximum signal-to-noise ratio?

Optimizing ChIP-seq experiments for H3T45ph requires specific adjustments to standard protocols:

• Crosslinking optimization:

  • Test multiple formaldehyde concentrations (0.5-2%) and incubation times (5-20 minutes)

  • For some applications, try dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

  • Ensure rapid quenching with glycine to prevent over-crosslinking

• Chromatin fragmentation:

  • Sonication parameters must be carefully optimized for histone PTM ChIP

  • Target fragment size: 150-300bp (slightly smaller than standard ChIP)

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Consider using micrococcal nuclease (MNase) digestion instead of or in addition to sonication

• Antibody specificity measures:

  • Pre-clear chromatin with protein A/G beads to reduce non-specific binding

  • Include IgG control and input normalization

  • Include spike-in controls with chromatin from another species

  • Perform parallel ChIP with total H3 antibody for normalization

• Washing stringency:

  • Increase salt concentration in wash buffers (up to 500mM NaCl) for more stringent washing

  • Add non-ionic detergents (0.1% NP-40 or Triton X-100) to reduce non-specific binding

  • Increase number of washes (5-6 washes) to improve signal-to-noise ratio

• Library preparation considerations:

  • Start with more immunoprecipitated material than standard ChIP-seq

  • Minimize PCR cycles during library amplification to reduce bias

  • Include library preparation controls to ensure quality

These optimizations collectively enhance the signal-to-noise ratio in H3T45ph ChIP-seq experiments, enabling more accurate mapping of this modification across the genome.

How does H3T45 phosphorylation correlate with other histone modifications during cell cycle progression?

H3T45 phosphorylation exists within a complex landscape of histone modifications that change during the cell cycle. Understanding these correlations requires integrated analysis:

• Cell synchronization approaches:

  • Synchronize cells at different cell cycle phases:

    • G1/S boundary: Double thymidine block

    • S phase: Thymidine-aphidicolin sequential block

    • G2/M: Nocodazole treatment

  • Collect synchronized populations for histone PTM profiling

• Multi-modification analysis:

  • Perform Western blotting with antibodies against multiple PTMs (H3T45ph, H3S10ph, H3K9me3, H3K27me3)

  • Use MS/MS to quantify modification levels on the same histone peptides

  • Calculate correlation coefficients between modification levels

  • Determine if specific modifications tend to co-occur or are mutually exclusive

• Immunofluorescence co-localization:

  • Perform dual immunofluorescence staining for H3T45ph and other modifications

  • Use confocal microscopy to assess spatial co-localization

  • Quantify degree of overlap using Pearson's correlation coefficient

• Sequential ChIP (Re-ChIP):

  • Perform ChIP with anti-H3T45ph antibody followed by a second ChIP with antibodies against other modifications

  • Identify genomic regions containing both modifications

  • Compare with single-modification ChIP profiles

This integrated approach reveals how H3T45 phosphorylation fits into the broader epigenetic landscape across cell cycle phases.

What is the role of H3T45 phosphorylation in cancer development and potential therapeutic targeting?

Cancer cells often exhibit altered histone modification patterns. Understanding H3T45ph in this context has important implications:

• Cancer tissue analysis:

  • Compare H3T45ph levels in tumor vs. matched normal tissues

  • Correlate with clinical parameters (stage, grade, survival)

  • Analyze across different cancer types using tissue microarrays

  • Note: Human breast carcinoma tissue has been validated for H3T45ph antibody reactivity

• Relationship to oncogenic signaling:

  • Investigate H3T45ph changes following activation of common oncogenic pathways (RAS, PI3K/AKT, MYC)

  • Determine if H3T45ph is regulated by cancer-associated kinases

  • Analyze correlation with p53 status, as p53 has been shown to regulate histone H3.1 behavior

• Drug resistance mechanisms:

  • Examine H3T45ph changes in drug-resistant vs. sensitive cell lines

  • Determine if modulating H3T45ph can sensitize resistant cells

  • Investigate connection to H3.1's role as a redox sensor in cancer cells developing adaptive phenotypic plasticity

• Therapeutic targeting strategies:

  • Identify druggable enzymes that regulate H3T45ph

  • Test combination treatments targeting both H3T45ph regulatory pathways and standard chemotherapies

  • Develop phospho-H3T45 levels as potential biomarkers for treatment response

This research direction connects basic epigenetic mechanisms to potential clinical applications in cancer diagnosis and treatment.

What are the most promising emerging technologies for studying HIST1H3A modifications?

Several cutting-edge technologies show particular promise for advancing our understanding of H3T45 phosphorylation and other HIST1H3A modifications:

• Single-cell epigenomics:

  • Single-cell ChIP-seq and CUT&Tag approaches enable analysis of H3T45ph heterogeneity

  • Integration with single-cell transcriptomics reveals functional consequences at individual cell level

  • Spatial epigenomics techniques allow mapping histone modifications within tissue architecture

• Live-cell imaging of histone dynamics:

  • FRET-based biosensors for real-time monitoring of H3T45 phosphorylation

  • Engineered antibody fragments (Fabs) for tracking histone modifications in living cells

  • Correlation with chromatin accessibility measured by live-cell DNA-binding dyes

• Cryo-EM of modified nucleosomes:

  • High-resolution structural analysis of nucleosomes containing H3T45ph

  • Investigation of structural impacts on DNA-histone interactions

  • Comparison with other core modifications to build comprehensive structural models

• CRISPR epigenome editing:

  • Targeted modulation of H3T45 phosphorylation at specific genomic loci

  • Development of CRISPR-based readers and writers for T45 phosphorylation

  • Precise control of modification timing using optogenetic or chemical induction approaches

These emerging technologies will provide unprecedented insights into the dynamic regulation and functional consequences of H3T45 phosphorylation in diverse biological contexts.

What are the key unanswered questions regarding HIST1H3A and its Thr-45 phosphorylation?

Despite significant advances, several fundamental questions about H3T45 phosphorylation remain unanswered:

• Enzymatic regulation:

  • What are the specific kinases and phosphatases that regulate H3T45 phosphorylation?

  • How is their activity coordinated across different cellular contexts?

  • What upstream signaling pathways control these enzymes?

• Functional consequences:

  • How does H3T45 phosphorylation directly affect nucleosome structure and stability?

  • What is the relationship between H3T45ph and DNA accessibility?

  • Does H3T45ph coordinate with other histone modifications to form a "histone code"?

• Evolutionary conservation:

  • Is the regulation and function of H3T45 phosphorylation conserved across species?

  • How does this modification contribute to species-specific chromatin functions?

  • What are the evolutionary drivers for maintaining this site of regulation?

• Disease relevance:

  • How do disease-associated mutations in H3.1 affect T45 phosphorylation?

  • Can H3T45ph serve as a biomarker for specific pathological conditions?

  • Is targeted modulation of this modification a viable therapeutic strategy?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genomics, and cell biology to fully understand the biological significance of H3T45 phosphorylation.