HIST1H4A (Ab-88) Antibody

What is HIST1H4A and what is the significance of the Tyr88 phosphorylation site?

HIST1H4A is a histone H4 family member that plays critical roles in chromatin structure and gene regulation. The tyrosine 88 (Tyr88) residue in histone H4 is evolutionarily conserved, suggesting an important physiological function . Phosphorylation at this site (pY88-H4) has been identified in multiple castration-resistant prostate cancer (CRPC) biospecimens, indicating its potential role in disease progression . This post-translational modification appears to be involved in regulating androgen receptor (AR) gene expression, making it particularly relevant for prostate cancer research .

How is the specificity of HIST1H4A (Ab-88) Antibody validated?

The specificity of HIST1H4A (Ab-88) Antibody is validated through multiple complementary approaches:

• Peptide competition assays - The antibody specifically recognizes Tyr88-phosphorylated H4 peptide but not unphosphorylated peptide

• Cross-reactivity screening - Testing against 59 different histone modifications (acetylation, methylation, phosphorylation, and citrullination) using Histone Peptide Array demonstrates no cross-reactivity

• Mutation studies - Point mutant constructs (Y88F-H4 and Y72F-H4) confirm that the antibody specifically recognizes phosphorylation at the Tyr88 site but not at Tyr72

• Western blotting - Direct immunoblotting and immunofluorescence analyses further validate specificity

What sample preparation protocols are recommended for histone H4 phosphorylation detection?

For optimal detection of histone H4 phosphorylation at Tyr88, the following protocols are recommended:

• Histone Purification: Total histones should be purified from cells or tissues using acid extraction methods

• Cell Treatment: For stimulation studies, serum and androgen starvation followed by growth factor treatment (e.g., insulin-like growth factor or platelet-derived growth factor) can induce robust phosphorylation

• Protein Extraction: For immunoblotting applications, standard cell lysis and nuclear extraction protocols are appropriate with the addition of phosphatase inhibitors to preserve phosphorylation status

• ChIP Sample Preparation: For chromatin immunoprecipitation applications, standard crosslinking procedures followed by sonication to generate chromatin fragments of appropriate size (~200-500bp) are recommended

How can HIST1H4A (Ab-88) Antibody be effectively used in ChIP-seq experiments?

HIST1H4A (Ab-88) Antibody has been successfully employed in ChIP-seq experiments to map the genome-wide distribution of pY88-H4 marks. Based on published research methodologies:

• Immunoprecipitation Protocol: The antibody can be used to immunoprecipitate chromatin fragments containing the pY88-H4 mark

• Sequencing Depth: For comprehensive coverage, high-depth sequencing is recommended (~20-30 million uniquely mapped reads)

• Data Analysis: The resulting data can identify distinct genomic locations where pY88-H4 marks are deposited (e.g., upstream of the AR transcription start site)

• Validation: ChIP-qPCR with site-specific primers should be used to validate key findings from ChIP-seq data

Research using this approach has identified approximately 370 distinct genomic locations with pY88-H4 deposition, including specific regions upstream of the AR gene that appear critical for its transcriptional regulation .

What is the relationship between ACK1 kinase and histone H4 Tyr88 phosphorylation?

Evidence from multiple experimental approaches demonstrates that ACK1 (TNK2) is the primary kinase responsible for phosphorylating histone H4 at Tyr88:

• Mass Spectrometry Analysis: Total histones purified from cells expressing activated ACK1 show Tyr88 phosphorylation, while histones from SRC-expressing cells do not

• Protein Interaction: Pull-down studies followed by mass spectrometry reveal that ACK1 (phosphorylated at Tyr827) binds directly to histone H4

• In Vitro Kinase Assay: Purified recombinant human ACK1 directly phosphorylates histone H4 at Tyr88 in cell-free conditions, and this activity is inhibited by ACK1 inhibitor (R)-9bMS

• Specificity Testing: When incubated with all four core histones, ACK1 specifically phosphorylates H4 but not H2A, H2B, or H3

• Knockdown Studies: siRNA-mediated knockdown or CRISPR-Cas9 deletion of ACK1 significantly reduces H4 Tyr88 phosphorylation

How does pY88-H4 contribute to gene regulation and what are the implications for disease?

The phosphorylation of histone H4 at Tyr88 appears to play a critical role in gene regulation, particularly for the androgen receptor (AR) gene:

• Chromatin Marking: ChIP-seq analysis reveals that pY88-H4 marks are deposited at specific locations upstream of the AR transcription start site, termed AR enhancer modules (AREMs)

• Transcriptional Activation: The deposition of pY88-H4 marks is associated with increased AR gene expression

• Mechanism: The pY88-H4 mark appears to promote recruitment of the WDR5/MLL2 complex, which likely contributes to transcriptional activation

• Functional Consequence: Mutation studies using the Y88F mutant of H4 demonstrate that this phosphorylation site is necessary for AR upregulation, as expression of the non-phosphorylatable mutant results in decreased AR and PSA mRNA levels

• Disease Relevance: The pY88-H4 mark has been identified in castration-resistant prostate cancer samples but not in normal prostate tissue, suggesting its potential role in disease progression

What controls should be included when using HIST1H4A (Ab-88) Antibody in experiments?

To ensure robust and reproducible results when using HIST1H4A (Ab-88) Antibody, the following controls should be included:

• Peptide Competition: Pre-incubation of the antibody with phosphopeptide should abolish signal

• Kinase Inhibition: Treatment with ACK1 inhibitor ((R)-9bMS) should reduce pY88-H4 signal

• Genetic Controls: Cells expressing Y88F mutant H4 should show negative results

• Positive Controls: Cells treated with growth factors known to activate ACK1 (e.g., IGF, PDGF) should show increased signal

• Knockdown Controls: ACK1 knockdown or knockout cells should show reduced signal

• Isotype Control: Use of an isotype-matched non-specific antibody to assess background binding

• Technical Replicates: Multiple replicates to ensure reproducibility

How can HIST1H4A (Ab-88) Antibody be integrated with other methodologies to study chromatin structure?

HIST1H4A (Ab-88) Antibody can be effectively combined with other methodologies to provide comprehensive insights into chromatin structure and function:

• HiChIP Applications: The antibody can potentially be used in HiChIP experiments to identify chromatin interactions associated with pY88-H4 marks, similar to approaches used with other histone modifications like H3K27ac

• Multi-omics Integration: Data from pY88-H4 ChIP-seq can be integrated with RNA-seq, ATAC-seq, and other histone modification ChIP-seq datasets to comprehensively map the regulatory landscape

• Allele-Specific Analysis: For studies involving genetic variants, the antibody could be used to assess allele-specific deposition of pY88-H4 marks, potentially contributing to interaction QTL (iQTL) studies

• Time-Course Experiments: The antibody can be used to monitor dynamic changes in pY88-H4 deposition following various stimuli, providing insights into temporal regulation

How should researchers address potential cross-reactivity with other histone modifications?

While the HIST1H4A (Ab-88) Antibody has been shown to be highly specific, researchers should consider the following strategies to address potential cross-reactivity concerns:

• Histone Peptide Array: Comprehensive testing against multiple histone modifications to confirm specificity

• Point Mutation Studies: Expression of mutant histones (e.g., Y88F-H4) as negative controls

• Comparison with Total H4: Parallel analysis with antibodies against unmodified H4 to normalize for total H4 levels

• Multiple Antibody Validation: Confirmation of key findings using alternative antibodies or detection methods when available

• Mass Spectrometry Validation: For critical findings, mass spectrometry-based validation of the modification can provide definitive evidence

What are the best practices for quantifying pY88-H4 levels in experimental settings?

For accurate quantification of pY88-H4 levels:

• Western Blot Quantification: Normalization to total H4 levels is essential for comparative analysis

• ChIP-qPCR: Standard curves should be generated for accurate quantification, with normalization to input DNA

• ChIP-seq Analysis: Quantification of peak heights or areas using appropriate bioinformatic tools, with normalization to sequencing depth and input controls

• Statistical Analysis: Multiple biological replicates (minimum n=3) should be used for statistical comparisons

• Dose-Response Studies: For treatment effects, full dose-response curves rather than single-dose experiments provide more robust quantification

How can researchers distinguish between direct and indirect effects of pY88-H4 on gene regulation?

Distinguishing direct from indirect effects requires multiple complementary approaches:

• Temporal Analysis: Time-course experiments to determine the sequence of events following stimulation

• Proximity Analysis: ChIP-seq combined with RNA-seq to correlate pY88-H4 deposition with changes in nearby gene expression

• Perturbation Studies: Targeted disruption of the modification (e.g., by expressing Y88F-H4 mutant) coupled with gene expression analysis

• Mechanistic Studies: Investigation of protein complexes recruited to pY88-H4 marks using techniques like ChIP-MS or protein-protein interaction studies

• Functional Genomics: CRISPR-based editing of potential pY88-H4 binding sites to assess functional consequences

What are the potential applications of HIST1H4A (Ab-88) Antibody in studying chromatin looping and 3D genome organization?

Recent advances in chromatin interaction studies suggest several potential applications:

• HiChIP Applications: The antibody could be used for HiChIP experiments to identify chromatin loops associated with pY88-H4 marks, similar to approaches using H3K27ac HiChIP

• Interaction QTL Studies: Integration with genotype data could identify genetic variants associated with pY88-H4-mediated chromatin interactions

• Cell Type-Specific Interactions: Comparison of pY88-H4-associated chromatin loops across different cell types could reveal tissue-specific regulatory mechanisms

• Dynamic Reorganization: Analysis of changes in pY88-H4-associated loops in response to stimuli could provide insights into dynamic genome reorganization

How might pY88-H4 interact with other histone modifications in the epigenetic regulation of gene expression?

Understanding the interplay between pY88-H4 and other histone modifications represents an important research direction:

• Co-occurrence Analysis: ChIP-seq for multiple modifications to identify patterns of co-occurrence or mutual exclusivity

• Sequential ChIP: Sequential ChIP experiments (Re-ChIP) to determine if pY88-H4 co-exists with other modifications on the same nucleosomes

• Enzymatic Crosstalk: Investigation of how pY88-H4 might influence the activity of other histone-modifying enzymes

• Modification Readers: Identification of proteins that specifically recognize pY88-H4 and how they might interact with readers of other modifications

• Integrative Analysis: Computational integration of multiple histone modification datasets to identify combinatorial patterns associated with specific gene expression states