Acetyl-HIST1H4A (K5) Antibody

What is Acetyl-HIST1H4A (K5) and why is it significant in epigenetic research?

Acetyl-HIST1H4A (K5) refers to the acetylation of lysine 5 on histone H4, one of the core histones in nucleosomes. This post-translational modification is significant in epigenetic research because it plays a critical role in chromatin structure and function. H4K5ac is predominantly associated with euchromatin (transcriptionally active regions) and contributes to chromatin decondensation and transcriptional regulation . Additionally, H4K5ac is associated with newly assembled chromatin since H4 in predeposition complexes is diacetylated at K5 and K12 by histone acetyltransferases . This makes H4K5ac a valuable marker for studying processes like DNA replication, chromatin assembly, and cellular responses to DNA damage.

How do I select the appropriate Acetyl-HIST1H4A (K5) antibody for my experiment?

When selecting an Acetyl-HIST1H4A (K5) antibody, consider these key factors:

• Application compatibility: Confirm the antibody has been validated for your specific application (Western blot, ChIP, immunofluorescence, etc.) .

• Species reactivity: Ensure the antibody recognizes your species of interest. Many antibodies target human H4K5ac, but cross-reactivity with mouse and rat should be verified if working with these models .

• Clonality: Monoclonal antibodies offer higher specificity and reproducibility, while polyclonal antibodies might provide greater sensitivity .

• Modification specificity: Some antibodies recognize H4K5ac only when neighboring residues are in specific states (e.g., CMA405 detects K5ac only when K8 is unacetylated) .

• Validation data: Review the validation data, including specificity tests against other histone modifications and controls like peptide competition assays .

What are the common applications for Acetyl-HIST1H4A (K5) antibodies in basic research?

Acetyl-HIST1H4A (K5) antibodies are utilized in several fundamental research applications:

• Western blotting: To detect and quantify H4K5ac levels in cell or tissue lysates. Typically appears as a band at approximately 11-12 kDa .

• Immunofluorescence/Immunocytochemistry (IF/ICC): To visualize the nuclear localization and distribution patterns of H4K5ac in fixed cells, usually showing specific staining in nuclei .

• Chromatin Immunoprecipitation (ChIP): To identify genomic regions enriched with H4K5ac, often combined with quantitative PCR or sequencing (ChIP-seq) .

• ELISA: To quantitatively measure H4K5ac levels in purified histone preparations .

• Immunohistochemistry (IHC): To examine H4K5ac distribution in tissue sections, providing insights into epigenetic states in different cell types within a tissue context .

What controls should I include when using Acetyl-HIST1H4A (K5) antibodies?

When using Acetyl-HIST1H4A (K5) antibodies, include these essential controls:

• Positive control: Cell lines treated with histone deacetylase inhibitors like sodium butyrate, which increases global histone acetylation levels .

• Negative control: Use of appropriate IgG matching the host species of the primary antibody .

• Specificity control: Peptide competition assays using acetylated and non-acetylated peptides to confirm antibody specificity.

• Biological controls: Cell lines or conditions known to have different H4K5ac levels. For example, comparing proliferating cells (higher H4K5ac) with quiescent cells.

• Loading control: For Western blots, include total H4 antibody or other stable proteins to normalize loading variations.

• Cross-reactivity control: Testing against other acetylated lysines on histone H4 (K8, K12, K16) to confirm specificity for K5 acetylation.

How can I distinguish between newly assembled H4 and hyperacetylated H4 using specific antibodies?

Distinguishing between newly assembled H4 (diacetylated at K5 and K12) and hyperacetylated H4 (acetylated at multiple residues including K5, K8, K12, and K16) requires antibodies with high specificity for particular acetylation patterns:

• Context-dependent antibodies: Some antibodies, like CMA405, recognize H4K5ac only when the neighboring K8 is unacetylated . This unique feature allows researchers to specifically detect newly assembled H4 (diacetylated at K5 and K12) and distinguish it from hyperacetylated H4 (where both K5 and K8 are acetylated).

• Sequential ChIP (ChIP-reChIP): Perform initial ChIP with antibodies against H4K5ac, followed by a second IP with antibodies against other acetylation marks. Newly assembled chromatin would be enriched for H4K5ac and H4K12ac but not H4K8ac or H4K16ac.

• Mass spectrometry approach: Using quantitative mass spectrometry to analyze immunoprecipitated histones can provide precise identification of combinatorial modification patterns, revealing the proportions of differently modified H4 molecules.

• Pulse-chase experiments: Combine metabolic labeling of newly synthesized histones with immunoprecipitation using H4K5ac antibodies to specifically track newly assembled chromatin versus existing modified chromatin.

What are the mechanistic differences between H4K5ac and other H4 acetylation marks in transcriptional regulation?

The mechanistic differences between H4K5ac and other H4 acetylation marks in transcriptional regulation involve distinct functions, readers, and genomic distribution patterns:

• Functional differences:

  • H4K5ac and H4K12ac are primarily associated with newly assembled chromatin and histone deposition during DNA replication .

  • H4K8ac and H4K16ac are more directly involved in active transcription, with enrichment around transcription start sites as revealed by ChIP-seq analyses .

  • H4K16ac uniquely prevents the formation of compact 30-nm chromatin fibers and inhibits ATP-dependent chromatin remodeling, functions not shared by H4K5ac.

• Bromodomain recognition:

  • Different bromodomain-containing proteins show varying affinities for specifically acetylated lysines on H4.

  • BRD4 preferentially binds to H4K5ac and H4K8ac, while BRDT recognizes H4K5ac in combination with other acetylation marks.

• Genomic distribution:

  • ChIP-seq analysis reveals different enrichment patterns, with H4K8ac and H4K16ac showing stronger association with transcription start sites compared to H4K5ac .

  • H4K5ac is more broadly distributed and associated with replication-dependent histone deposition throughout the genome.

• Contextual effects:

  • H4K5ac functions may depend on neighboring modifications, creating a complex "histone code" that influences transcription factor binding and chromatin structure.

How do H4K5ac patterns change during DNA damage response and what implications does this have for repair mechanisms?

H4K5ac undergoes dynamic changes during DNA damage response (DDR), with significant implications for repair mechanisms:

• Initial response to damage:

  • Upon DNA damage, there is often a global decrease in H4K5ac levels as part of chromatin compaction to prevent further damage.

  • This is followed by localized increases of H4K5ac at damage sites to facilitate access for repair proteins.

• Double-strand break (DSB) repair pathway choice:

  • H4K5ac levels influence the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways.

  • Higher levels of H4K5ac create a more open chromatin environment that favors HR, which requires greater chromatin accessibility.

• Cell cycle-dependent regulation:

  • The relationship between H4K5ac and DNA repair varies across the cell cycle.

  • In S phase, when newly assembled chromatin (marked by H4K5ac and H4K12ac) is abundant, H4K5ac patterns contribute to replication-coupled repair mechanisms.

• Interaction with repair machinery:

  • H4K5ac serves as a binding platform for bromodomain-containing proteins involved in the DNA damage response.

  • The acetylation-dependent recruitment of chromatin remodelers facilitates access to damaged DNA for repair proteins.

• Restoration phase:

  • Following successful repair, proper restoration of chromatin structure requires precise regulation of H4K5ac and other histone modifications.

  • Failure to reset proper acetylation patterns may lead to epigenetic instability and increased mutation rates.

What are the optimal conditions for using Acetyl-HIST1H4A (K5) antibodies in ChIP experiments?

Optimizing ChIP experiments with Acetyl-HIST1H4A (K5) antibodies requires careful attention to several parameters:

• Crosslinking conditions:

  • For most H4K5ac ChIP applications, 1% formaldehyde for 10 minutes at room temperature provides adequate crosslinking.

  • Excessive crosslinking may mask epitopes and reduce antibody accessibility.

• Chromatin fragmentation:

  • Micrococcal nuclease digestion is often preferred for histone modification ChIP, as demonstrated in protocols using Acetyl-HIST1H4A (K5) antibodies .

  • Aim for chromatin fragments of 150-500 bp for optimal resolution.

• Antibody amount and incubation:

  • Typically, 5 μg of anti-Acetyl-HIST1H4A (K5) antibody per ChIP reaction is recommended .

  • Overnight incubation at 4°C with rotation ensures adequate antibody-chromatin interaction.

• Washing stringency:

  • Include progressively stringent wash steps to reduce background without compromising specific signal.

  • For H4K5ac, standard RIPA buffer washes followed by LiCl and TE washes are generally effective.

• Controls:

  • Include input chromatin control (typically 5-10% of starting material).

  • Use normal rabbit IgG as a negative control to assess non-specific binding .

  • Consider using cell treatments that alter H4K5ac levels (e.g., HDAC inhibitors) as biological controls.

• Quantification method:

  • For targeted analysis, quantitative PCR with primers for known H4K5ac-enriched regions.

  • For genome-wide analysis, ChIP-seq with appropriate sequencing depth (minimum 20 million uniquely mapped reads).

How can I effectively troubleshoot weak or non-specific signals in Western blots using Acetyl-HIST1H4A (K5) antibodies?

For optimal results with Acetyl-HIST1H4A (K5) antibodies in Western blot:

• Use PVDF membrane for better protein retention

• Run under reducing conditions using Immunoblot Buffer Group 1

• Include both acetylated and non-acetylated control samples

• Ensure adequate transfer of low molecular weight histones (11-12 kDa)

What are the critical parameters for immunofluorescence staining when using Acetyl-HIST1H4A (K5) antibodies?

For optimal immunofluorescence staining with Acetyl-HIST1H4A (K5) antibodies, attention to these critical parameters is essential:

• Fixation method:

  • Paraformaldehyde (4%) for 10-15 minutes is generally recommended for preserving nuclear architecture.

  • Methanol fixation may expose epitopes better in some cell types but can disrupt nuclear morphology.

• Permeabilization:

  • Adequate nuclear permeabilization is crucial for antibody access to nuclear epitopes.

  • Use 0.2-0.5% Triton X-100 for 10 minutes at room temperature.

• Antigen retrieval:

  • Some protocols benefit from antigen retrieval using citrate buffer (pH 6.0) heating.

  • This may be particularly important for detecting H4K5ac in cells with compact chromatin.

• Blocking conditions:

  • Thorough blocking (1 hour) with 5% normal serum from the same species as the secondary antibody.

  • Addition of 0.1-0.3% BSA can reduce non-specific binding.

• Antibody concentration and incubation:

  • Optimal dilution ranges from 1:50 to 1:200 for most Acetyl-HIST1H4A (K5) antibodies .

  • For HeLa cells, 0.1 μg/mL has been validated .

  • Extending primary antibody incubation to 3 hours at room temperature or overnight at 4°C improves signal quality .

• Washing steps:

  • Multiple PBS washes (at least 3×5 minutes) after both primary and secondary antibody incubations.

  • Include 0.05% Tween-20 in wash buffers to reduce background.

• Secondary antibody selection:

  • Use highly cross-adsorbed secondary antibodies to minimize non-specific binding.

  • For Acetyl-HIST1H4A (K5) rabbit antibodies, anti-rabbit IgG conjugated to fluorophores like NorthernLights 557 has been validated .

• Nuclear counterstain:

  • DAPI is recommended for nuclear visualization and colocalization analysis with H4K5ac signals .

  • Ensure DAPI concentration and incubation time are optimized to avoid oversaturation.

How can I quantitatively analyze H4K5ac levels in ChIP-seq data and integrate with other epigenetic marks?

Quantitative analysis of H4K5ac ChIP-seq data and integration with other epigenetic marks requires sophisticated computational approaches:

• Quality control and preprocessing:

  • Assess sequencing quality using FastQC.

  • Filter low-quality reads and trim adapters before alignment.

  • Map reads to reference genome using Bowtie2 or BWA.

  • Remove PCR duplicates to prevent bias in quantification.

• Peak calling and signal quantification:

  • Use MACS2 or SICER for peak calling, with input DNA as control.

  • For H4K5ac, which can show broad domains, consider broad peak calling parameters.

  • Generate normalized coverage tracks (bigWig format) for visualization and quantification.

• Differential binding analysis:

  • Compare H4K5ac enrichment between conditions using DiffBind or MAnorm.

  • Normalize for sequencing depth and potential global changes in acetylation levels.

  • Apply appropriate statistical thresholds (FDR < 0.05) for identifying significant changes.

• Genomic feature association:

  • Analyze distribution of H4K5ac relative to genomic features (promoters, enhancers, gene bodies).

  • Use tools like HOMER or ChIPseeker for annotation of H4K5ac peaks.

  • Create metaplots and heatmaps showing H4K5ac distribution around transcription start sites.

• Integration with other epigenetic marks:

  • Perform correlation analysis between H4K5ac and other histone modifications.

  • Use ChromHMM or EpiSig for chromatin state modeling based on multiple marks.

  • Apply multivariate analysis to identify combinatorial patterns of histone modifications.

  • Compare H4K5ac with H4K8ac and H4K16ac to distinguish newly assembled chromatin from transcriptionally active regions .

• Functional interpretation:

  • Perform gene ontology and pathway enrichment analysis of genes associated with H4K5ac peaks.

  • Integrate with gene expression data to correlate H4K5ac changes with transcriptional outcomes.

  • Analyze motif enrichment within H4K5ac peaks to identify potential regulatory factors.

How should I interpret discrepancies between H4K5ac antibodies from different sources in my experiments?

When faced with discrepancies between H4K5ac antibodies from different sources, consider these analytical approaches:

What is the relationship between H4K5ac and other histone modifications in different cellular contexts?

The relationship between H4K5ac and other histone modifications varies across cellular contexts, creating complex regulatory networks:

• Co-occurrence patterns:

  • H4K5ac frequently co-occurs with H4K12ac in newly assembled chromatin .

  • H4K5ac may be mutually exclusive with H4K20me3, as these marks are associated with opposite chromatin states (open vs. condensed) .

  • In transcriptionally active regions, H4K5ac often coincides with H3K4me3 and H3K27ac.

• Cell cycle-dependent relationships:

  • During S phase, newly deposited histones show enrichment of H4K5ac and H4K12ac, which are subsequently modified as chromatin matures .

  • This pattern differs from the stable H4K8ac and H4K16ac enrichment at transcription start sites throughout the cell cycle .

  • In mitotic cells, global reduction of H4K5ac accompanies chromosome condensation.

• Development and differentiation contexts:

  • Stem cells show distinct patterns of H4K5ac distribution compared to differentiated cells.

  • During differentiation, dynamic changes in H4K5ac correlate with developmental gene regulation.

  • Tissue-specific patterns of H4K5ac reflect specialized gene expression programs.

• Disease states:

  • Cancer cells often exhibit altered patterns of H4K5ac and other histone modifications.

  • Similar to H4K16ac and H4K20me3, H4K5ac levels may have diagnostic and prognostic value in certain cancers .

  • Neurodegenerative disorders show disruptions in the balance between histone acetylation and deacetylation, affecting H4K5ac levels.

• Response to environmental stimuli:

  • Stress conditions can rapidly alter H4K5ac distribution as part of adaptive transcriptional responses.

  • Nutritional status influences global histone acetylation, including H4K5ac, through metabolic regulation of acetyl-CoA availability.

What are the emerging roles of H4K5ac in regulating non-coding RNA expression and three-dimensional chromatin organization?

Emerging research reveals complex roles for H4K5ac in non-coding RNA regulation and chromatin architecture:

• Long non-coding RNA (lncRNA) regulation:

  • H4K5ac enrichment at lncRNA promoters correlates with their expression, similar to protein-coding genes.

  • Dynamic changes in H4K5ac can mediate tissue-specific expression of regulatory lncRNAs.

  • Some lncRNAs themselves affect H4K5ac deposition through interactions with histone acetyltransferases or deacetylases.

• Enhancer RNA (eRNA) production:

  • Enrichment of H4K5ac at active enhancers, often in combination with H3K27ac, correlates with eRNA transcription.

  • eRNAs may subsequently influence broader H4K5ac distribution through feedback mechanisms.

  • The presence of H4K5ac at enhancers facilitates chromatin accessibility for transcription factor binding.

• Topologically associating domains (TADs):

  • H4K5ac distribution patterns often respect TAD boundaries, suggesting coordination with three-dimensional chromatin organization.

  • Changes in H4K5ac levels can precede or accompany alterations in chromatin interaction frequencies.

  • The presence of H4K5ac may influence the flexibility of chromatin fibers, affecting long-range interactions.

• Chromatin accessibility and nucleosome positioning:

  • H4K5ac contributes to local chromatin accessibility by weakening histone-DNA interactions.

  • This modification can alter nucleosome stability and positioning, particularly when combined with other acetylation marks.

  • Advanced techniques combining H4K5ac ChIP with ATAC-seq or DNase-seq provide insights into the relationship between this modification and chromatin accessibility.

• Nuclear compartmentalization:

  • Regions with high H4K5ac often localize to the active A compartment in the nucleus.

  • During cell differentiation or response to stimuli, changes in H4K5ac can accompany repositioning of genomic regions between compartments.

  • The relationship between H4K5ac and nuclear organization reveals higher-order regulation beyond sequence-specific effects.

What emerging technologies will advance our understanding of H4K5ac dynamics and function?

Several cutting-edge technologies are poised to revolutionize our understanding of H4K5ac biology:

• Single-cell epigenomics:

  • Single-cell ChIP-seq and CUT&Tag approaches will reveal cell-to-cell variation in H4K5ac patterns.

  • Integration with single-cell transcriptomics will clarify the relationship between H4K5ac heterogeneity and gene expression variability.

  • These approaches will be particularly valuable for understanding H4K5ac dynamics in heterogeneous tissues and during development.

• Live-cell imaging of H4K5ac:

  • Development of acetylation-specific intrabodies and fluorescent probes for real-time visualization of H4K5ac in living cells.

  • FRET-based sensors to monitor dynamic changes in H4K5ac in response to stimuli with high temporal resolution.

  • These techniques will provide unprecedented insights into the kinetics of H4K5ac establishment and removal.

• Mass spectrometry innovations:

  • Improved sensitivity to detect combinatorial histone modifications including H4K5ac.

  • Development of targeted approaches for quantifying specific histone proteoforms.

  • Integration with crosslinking techniques to identify proteins that specifically interact with H4K5ac-modified chromatin.

• Genomic engineering of acetylation sites:

  • CRISPR-based approaches for site-specific manipulation of H4K5ac levels.

  • Creation of acetylation-mimetic or acetylation-deficient mutants to dissect functional consequences.

  • These interventions will help establish causality between H4K5ac and specific cellular processes.

• Computational modeling:

  • Machine learning algorithms to predict H4K5ac patterns from DNA sequence and other epigenetic features.

  • Structural modeling of how H4K5ac affects nucleosome and chromatin fiber dynamics.

  • Systems biology approaches integrating multiple data types to place H4K5ac in broader regulatory networks.

These emerging technologies will address current knowledge gaps and provide more comprehensive understanding of H4K5ac function in genome regulation, development, and disease.