Acetyl-HIST1H4A (K77) Antibody

What is Acetyl-HIST1H4A (K77) and why is it significant in epigenetic research?

Acetyl-HIST1H4A (K77) refers to the acetylation of lysine 77 on histone H4, one of the core components of the nucleosome. Histone H4 is essential to chromatin structure, working alongside other core histones (H2A, H2B, and H3) to form the nucleosome octamer around which DNA wraps . The acetylation of this specific lysine residue represents an important post-translational modification that contributes to chromatin remodeling and gene expression regulation.

This modification has gained particular attention in cancer research, where H4K77ac levels correlate with clinical features including tumor size, microvascular invasion, and elevated alpha-fetoprotein levels in hepatocellular carcinoma . Importantly, patients with high acetylation levels of H4K77ac demonstrate significantly shorter disease-free survival than those with low acetylation levels, highlighting its potential as a prognostic biomarker .

Epigenetic researchers focus on H4K77ac because histone acetylation typically promotes a more open chromatin structure that facilitates transcription, making these modifications critical for understanding gene regulation in both normal development and disease states.

How does Acetyl-HIST1H4A (K77) differ from other histone H4 acetylation sites?

While histone H4 contains multiple lysine residues that can be acetylated (including K5, K8, K12, and K16), H4K77ac has distinct characteristics that differentiate it from these more commonly studied sites:

Unlike the N-terminal acetylation sites (K5, K8, K12, K16) that are widely characterized and have established roles in chromatin regulation, K77 is located within the globular domain of histone H4. This positioning suggests it may have different structural impacts on the nucleosome compared to tail modifications. The acetylation at K77 may affect histone-DNA interactions in ways that differ from tail modifications.

H4K77ac appears to have particularly strong clinical correlations in certain cancer contexts. Research demonstrates that high H4K77ac levels correlate specifically with microvascular invasion, larger tumors, and elevated alpha-fetoprotein in hepatocellular carcinoma patients . These correlations differ from patterns observed with other histone acetylation sites.

While histone acetyltransferase 1 (HAT1) is known to primarily target newly synthesized histone H4 at K5 and K12, the specific enzymes responsible for K77 acetylation are less well-established . This suggests distinct regulatory mechanisms compared to tail acetylation sites.

What are the validated applications for Acetyl-HIST1H4A (K77) antibodies?

Acetyl-HIST1H4A (K77) antibodies have been validated for multiple research applications:

• Western Blotting: These antibodies effectively detect H4K77ac in cell and tissue lysates, typically showing bands at approximately 11-12 kDa. Detection can be enhanced in samples treated with HDAC inhibitors like sodium butyrate, which increases global histone acetylation . The antibodies work effectively on PVDF membranes under reducing conditions .

• Immunohistochemistry (IHC): H4K77ac antibodies have been successfully used in tissue microarrays to evaluate acetylation levels in cancer samples. In HCC studies, tissues were scored based on staining intensity (levels 0-3) and percentage of positive cells (0-3) . This application has provided valuable prognostic information in cancer research.

• Immunofluorescence: Antibodies against acetylated histone H4 can detect nuclear localization patterns in fixed cells. For instance, in HeLa cells, acetylated histone H4 is detected primarily in the nucleus using specific monoclonal antibodies followed by fluorophore-conjugated secondary antibodies and DAPI counterstaining .

• Chromatin Immunoprecipitation (ChIP): Though specific data for K77 is more limited than for other sites, anti-acetyl histone H4 antibodies have been validated for ChIP applications to identify genomic regions where these modifications are present .

• Flow Cytometry: Some antibodies against acetylated histones have been validated for flow cytometry, allowing quantification of acetylation levels at the single-cell level.

How should researchers validate the specificity of an Acetyl-HIST1H4A (K77) antibody?

Thorough validation of Acetyl-HIST1H4A (K77) antibodies is essential for reliable research results. A comprehensive validation approach should include:

• Peptide Competition Assays: Pre-incubate the antibody with synthetic peptides containing acetylated K77 versus unmodified or differently modified peptides. Signal should be blocked only by the specific acetylated K77 peptide, confirming specificity for this modification.

• HDAC Inhibitor Treatment: Cells treated with histone deacetylase inhibitors like sodium butyrate (typically 10mM for 24 hours) should show increased H4K77ac signal compared to untreated controls . This confirms the antibody detects a modification responsive to acetylation dynamics.

• Western Blot Analysis: The antibody should detect a specific band at approximately 11-12 kDa in histone extracts, with increased signal intensity in HDAC inhibitor-treated samples . Cross-reactivity with other proteins or histone modifications should be minimal.

• Multiple Antibody Comparison: Use antibodies from different suppliers or clones to verify consistent patterns of detection across different reagents.

• Mass Spectrometry Correlation: Where possible, compare antibody-based detection with mass spectrometry quantification of H4K77ac to confirm specificity.

• Genetic Controls: In advanced validation, CRISPR-Cas9 modification of histone H4 genes or knockout of HATs that target H4K77 could provide definitive confirmation of specificity.

What is the optimal protocol for detecting Acetyl-HIST1H4A (K77) by Western blot?

For optimal Western blot detection of Acetyl-HIST1H4A (K77), researchers should follow this methodological approach:

• Sample Preparation:

  • Extract histones using acid extraction (e.g., 0.2N HCl) to enrich for histone proteins

  • Add HDAC inhibitors (e.g., sodium butyrate, 10mM) to preservation buffers

  • Include both treated (e.g., sodium butyrate-treated) and untreated controls

  • Quantify protein concentration using Bradford or BCA assay

• Gel Electrophoresis:

  • Use 15-18% SDS-PAGE gels for better resolution of small histone proteins

  • Load 10-20 μg of acid-extracted histones per lane

  • Run at 100-120V until the dye front reaches the bottom

• Transfer:

  • Transfer to PVDF membrane (preferred over nitrocellulose for histone proteins)

  • Use cold transfer buffer containing 20% methanol

  • Transfer at 30V overnight at 4°C for efficient transfer of small proteins

• Blocking and Antibody Incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Dilute primary antibody to 0.1-1 μg/mL in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash extensively (4-5 times, 5 minutes each) with TBST

  • Incubate with HRP-conjugated secondary antibody (typically 1:2000-1:5000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Expect a band at approximately 11-12 kDa for histone H4

  • Include a total H4 antibody on a separate blot as loading control

This protocol has been demonstrated to effectively detect changes in H4 acetylation in response to treatments like sodium butyrate .

How should I design immunohistochemistry experiments using Acetyl-HIST1H4A (K77) antibodies?

When designing immunohistochemistry (IHC) experiments with Acetyl-HIST1H4A (K77) antibodies, follow these methodological guidelines:

• Tissue Preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-5 μm thickness)

  • For tissue microarrays (TMAs), use 1.5-mm-diameter cores from representative tumor areas, avoiding necrotic and hemorrhagic regions

  • Include duplicate cores from contrasting areas (e.g., tumor center and adjacent tissue) to ensure reproducibility

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Heat in pressure cooker or microwave until boiling, then maintain at sub-boiling temperature for 10-20 minutes

  • Allow slides to cool to room temperature before proceeding

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Block non-specific binding with serum-free protein block

  • Dilute primary antibody appropriately (typically 1:1000-1:2000) in PBS containing 1% BSA

  • Incubate overnight at 4°C in a humidified chamber

  • Use automated staining platforms when available for consistent results

• Detection and Visualization:

  • Apply appropriate secondary antibody system (e.g., polymer-based detection)

  • Develop with DAB (3,3'-diaminobenzidine) substrate

  • Counterstain nuclei with hematoxylin

  • Dehydrate, clear, and mount with permanent mounting medium

• Scoring System:

  • Evaluate staining intensity on a scale of 0-3 (0=negative, 1=weak, 2=moderate, 3=strong)

  • Assess percentage of positive cells (0=0%, 1=1-33%, 2=34-66%, 3=67-100%)

  • Calculate final score as sum of intensity and percentage scores (0-6)

  • For duplicate cores, use the higher score as the final result

• Controls:

  • Include positive controls (e.g., tissues known to express high H4K77ac)

  • Include negative controls (omission of primary antibody)

  • Use normal adjacent tissue as internal controls when available

This methodological approach has been successfully used to identify correlations between H4K77ac levels and clinical features in hepatocellular carcinoma .

What are the critical parameters for ChIP-Seq experiments targeting Acetyl-HIST1H4A (K77)?

ChIP-Seq experiments targeting Acetyl-HIST1H4A (K77) require careful attention to several critical parameters:

• Cross-linking and Chromatin Preparation:

  • Optimize formaldehyde cross-linking time (typically 10-15 minutes)

  • Ensure proper sonication to generate 200-500 bp fragments

  • Verify sonication efficiency by agarose gel electrophoresis

  • Keep samples cold throughout processing to preserve acetylation marks

• Antibody Selection and Validation:

  • Use ChIP-grade or ChIP-validated antibodies specifically

  • Verify antibody specificity through Western blot and peptide competition assays

  • Perform preliminary ChIP-qPCR on known positive and negative regions

  • Consider using multiple antibodies to confirm results

• Immunoprecipitation Conditions:

  • Determine optimal antibody amount through titration experiments (typically 2-5 μg per reaction)

  • Include appropriate controls (IgG control, input DNA)

  • Allow sufficient incubation time (overnight at 4°C with rotation)

  • Use magnetic beads for more consistent recovery

• Washing and Elution:

  • Use increasingly stringent wash buffers to reduce background

  • Ensure complete removal of wash buffer between steps

  • Elute under gentle conditions to preserve antibody-epitope binding

  • Reverse cross-links thoroughly (65°C overnight)

• Library Preparation and Sequencing:

  • Start with sufficient ChIP DNA (typically 1-10 ng)

  • Use low-input library preparation kits if necessary

  • Include spike-in controls for normalization

  • Sequence to adequate depth (20-30 million reads minimum)

• Data Analysis:

  • Use appropriate peak-calling algorithms optimized for histone modifications

  • Compare with other histone marks to identify patterns

  • Analyze distribution of peaks relative to genomic features (promoters, enhancers, etc.)

  • Integrate with gene expression data to establish functional relevance

• Validation:

  • Confirm key findings with ChIP-qPCR

  • Correlate with other techniques like CUT&Tag or CUT&RUN

  • Perform biological replicates to ensure reproducibility

These parameters have been adapted from established protocols for histone acetylation ChIP-Seq, which should be optimized specifically for H4K77ac in each experimental system.

How does Acetyl-HIST1H4A (K77) correlate with clinical outcomes in cancer research?

Research on Acetyl-HIST1H4A (K77) has revealed significant correlations with clinical outcomes in cancer, particularly in hepatocellular carcinoma (HCC):

These findings suggest that H4K77ac has potential as a prognostic biomarker in HCC and possibly other cancer types, which could inform clinical decision-making and treatment strategies.

What is the relationship between HAT1 activity and Acetyl-HIST1H4A (K77)?

The relationship between histone acetyltransferase 1 (HAT1) and H4K77 acetylation involves complex regulatory mechanisms:

Understanding this relationship provides insights into the complex interplay between histone production, acetylation, cellular metabolism, and disease processes.

How do different histone deacetylase inhibitors affect Acetyl-HIST1H4A (K77) levels?

Different histone deacetylase (HDAC) inhibitors can have varying effects on Acetyl-HIST1H4A (K77) levels, providing valuable research tools:

• Sodium Butyrate:

  • Commonly used at concentrations of approximately 10mM for 24 hours in experimental settings

  • Effectively increases global histone H4 acetylation, including at K77

  • In HeLa cells, sodium butyrate treatment results in detectable increases in acetylated histone H4, as demonstrated by Western blot analysis

  • Often used as a positive control in experiments studying histone acetylation

• Trichostatin A (TSA):

  • More potent than sodium butyrate, typically used at nanomolar concentrations (50-200 nM)

  • Broadly affects multiple HDAC classes and histone acetylation sites

  • May have different kinetics of K77 acetylation compared to other inhibitors

  • Particularly effective for short-term (4-12 hour) experiments

• Suberoylanilide Hydroxamic Acid (SAHA/Vorinostat):

  • FDA-approved HDAC inhibitor used in cancer treatment

  • Affects multiple histone residues, including H4K77

  • Used at micromolar concentrations (1-5 μM) in most experimental settings

  • Clinically relevant inhibitor that allows translation between experimental and therapeutic contexts

• Class-Specific HDAC Inhibitors:

  • Different HDAC classes may preferentially target specific histone residues

  • Class I HDAC inhibitors (e.g., MS-275/Entinostat) may have different effects on H4K77ac compared to pan-HDAC inhibitors

  • Understanding which HDACs primarily deacetylate H4K77 would help predict inhibitor efficacy

• Experimental Considerations:

  • Time-course experiments are crucial as acetylation patterns evolve over time after inhibitor treatment

  • Dose-response relationships should be established for each cell type

  • Combined treatments with different HDAC inhibitors may reveal synergistic effects

  • Cell type-specific responses should be anticipated due to differential expression of HDACs

When studying these effects, it's important to include appropriate controls and consider the broader impact on other histone modifications and cellular processes beyond H4K77ac.

How does Acetyl-HIST1H4A (K77) interact with other histone modifications?

Acetyl-HIST1H4A (K77) participates in complex interactions with other histone modifications, forming part of the "histone code" that collectively regulates chromatin structure and function:

This complex interplay suggests H4K77ac functions within a broader epigenetic landscape that collectively determines chromatin states and gene expression patterns.

What molecular mechanisms underlie the correlation between H4K77ac and cancer progression?

The correlation between H4K77ac and cancer progression likely involves several molecular mechanisms:

Understanding these mechanisms could provide insights for developing targeted therapeutic approaches that disrupt the epigenetic patterns associated with cancer progression.

How do metabolic changes affect Acetyl-HIST1H4A (K77) levels?

Metabolic conditions significantly impact Acetyl-HIST1H4A (K77) levels through various interconnected mechanisms:

This metabolic connection suggests that H4K77ac levels may serve as indicators of cellular metabolic state, particularly in contexts like cancer where metabolism is frequently altered.

What novel technologies are advancing the study of Acetyl-HIST1H4A (K77)?

Several cutting-edge technologies are enhancing our ability to study Acetyl-HIST1H4A (K77) with unprecedented precision:

• Advanced ChIP-Based Technologies:

  • CUT&Tag and CUT&RUN techniques offer higher sensitivity and lower background than traditional ChIP-seq

  • These methods require fewer cells and provide improved signal-to-noise ratios

  • Automated ChIP platforms increase reproducibility and throughput for H4K77ac studies

• Single-Cell Epigenomics:

  • Single-cell ChIP-seq and CUT&Tag approaches reveal H4K77ac heterogeneity within cell populations

  • Integration with single-cell transcriptomics provides insights into acetylation-expression relationships at the individual cell level

  • These approaches are particularly valuable for understanding tumor heterogeneity in cancer studies

• Mass Spectrometry Advancements:

  • Improved sensitivity in mass spectrometry enables more accurate quantification of histone PTMs

  • Middle-down and top-down proteomics approaches allow analysis of combinatorial modification patterns

  • Targeted MS approaches can specifically measure H4K77ac levels in complex biological samples

• CRISPR-Based Epigenome Editing:

  • Catalytically dead Cas9 (dCas9) fused to histone acetyltransferases or deacetylases allows site-specific modulation of H4K77ac

  • These systems enable causal studies of H4K77ac function at specific genomic loci

  • Inducible systems permit temporal control of acetylation changes

• Advanced Imaging Techniques:

  • Super-resolution microscopy provides detailed spatial information about H4K77ac distribution in the nucleus

  • Multiplexed imaging allows simultaneous visualization of multiple histone modifications

  • Live-cell imaging with acetylation-specific sensors enables real-time monitoring of dynamic changes

• Computational Approaches:

  • Machine learning algorithms predict H4K77ac patterns from underlying DNA sequence and other epigenetic marks

  • Network analysis identifies regulatory relationships involving H4K77ac

  • Multi-omics data integration reveals connections between H4K77ac, gene expression, and clinical outcomes

These technological advances are driving more precise, dynamic, and comprehensive understanding of H4K77ac biology in both normal physiology and disease states.

How might targeting enzymes that regulate Acetyl-HIST1H4A (K77) impact cancer treatment strategies?

Targeting enzymes that regulate Acetyl-HIST1H4A (K77) offers promising therapeutic approaches for cancer:

• HDAC Inhibitor Refinement:

  • Current HDAC inhibitors affect multiple acetylation sites and have significant side effects

  • Developing inhibitors with greater specificity for HDACs that deacetylate H4K77 could improve efficacy while reducing toxicity

  • The correlation between H4K77ac and poor prognosis suggests that elevating this mark may not be beneficial in all contexts

• HAT-Targeting Approaches:

  • Inhibitors of HATs responsible for H4K77 acetylation could potentially normalize high acetylation levels seen in certain cancers

  • Understanding the relationship between HAT1 and histone H4 regulation provides potential targets beyond direct H4K77 acetylation

  • Combination approaches targeting both HATs and HDACs could provide more precise control of acetylation patterns

• Metabolic Intervention Strategies:

  • The connection between acetate metabolism, HAT1 activity, and histone acetylation suggests metabolic approaches to modulating H4K77ac levels

  • Targeting acetyl-CoA-producing pathways could affect global acetylation including H4K77ac

  • The challenge remains in achieving specificity to avoid disrupting essential cellular processes

• Biomarker-Based Patient Stratification:

  • H4K77ac levels could serve as biomarkers for patient stratification in clinical trials

  • Patients with high H4K77ac might benefit from different treatment approaches than those with low levels

  • Combined assessment of multiple histone marks (H4K77ac, H2BK120ac, H3.3K18ac) could improve prognostic accuracy

• Targeted Therapy Based on Modification Patterns:

  • The table below summarizes the correlation between histone modifications and clinical parameters in HCC, which could guide therapeutic targeting:

These correlations highlight the potential for developing targeted therapeutic strategies based on specific histone modification patterns .

What key unresolved questions remain about Acetyl-HIST1H4A (K77) function?

Despite growing research, several critical questions about Acetyl-HIST1H4A (K77) remain unanswered:

• Enzymatic Regulation:

  • Which specific histone acetyltransferases (HATs) are primarily responsible for H4K77 acetylation?

  • Which histone deacetylases (HDACs) specifically remove this mark?

  • How is the balance between these enzymes regulated in different cellular contexts?

  • What is the precise role of HAT1 in regulating H4K77ac compared to its established roles in K5/K12 acetylation?

• Genomic Distribution:

  • What is the genome-wide distribution pattern of H4K77ac in normal versus diseased cells?

  • Does H4K77ac mark specific functional elements (promoters, enhancers, etc.)?

  • How does the distribution pattern change during cellular differentiation, stress response, and disease progression?

  • Are there tissue-specific patterns of H4K77ac distribution?

• Reader Proteins:

  • Which specific reader proteins recognize and bind to H4K77ac?

  • How do these interactions translate into downstream functional outcomes?

  • Are there context-specific readers that recognize H4K77ac in combination with other modifications?

• Cancer Mechanisms:

  • How does H4K77ac mechanistically contribute to microvascular invasion, tumor growth, and elevated AFP in HCC?

  • Why is H4K77ac specifically associated with shorter disease-free survival compared to other modifications?

  • Can modulating H4K77ac levels directly affect cancer cell phenotypes?

• Therapeutic Potential:

  • Can specifically targeting H4K77ac regulation provide therapeutic benefits?

  • How would modulation of H4K77ac affect normal cellular functions?

  • What combination therapies might synergize with approaches targeting H4K77ac?

  • Could metabolic interventions that affect acetyl-CoA availability selectively impact cancer cells through changes in H4K77ac?

Addressing these questions will require multidisciplinary approaches combining epigenomic profiling, functional genomics, structural biology, and clinical studies to fully understand the role of H4K77ac in normal biology and disease.