HIST1H3A (Ab-64) Antibody

What is HIST1H3A and what does the Ab-64 antibody recognize?

HIST1H3A is the gene encoding histone H3.1, one of the core histone proteins essential for chromatin packaging. The Ab-64 antibody specifically recognizes modifications at lysine 64 (K64) on histone H3.1 protein. This antibody is designed against a synthesized peptide derived from Human Histone H3.1 protein (amino acids 59-70) . Lysine 64 is positioned at a structurally significant location - it is the first amino acid of the H3 alpha1 helix in the histone fold and is located on the lateral surface of the histone octamer in close proximity to the inner gyre of DNA . This strategic position makes modifications at this site particularly important for chromatin structure regulation.

The antibody is available as a rabbit polyclonal immunoglobulin that can be used for multiple experimental applications, including ELISA and immunohistochemistry, with specific reactivity against human samples . Proper validation of this antibody is critical for experimental success, as demonstrated in published research protocols using similar antibodies targeting H3K64 modifications.

What are the known post-translational modifications at H3K64 and their functional significance?

Research has identified two major modifications at H3K64 with opposing functions:

H3K64 acetylation (H3K64ac) has been demonstrated to regulate nucleosome stability, facilitate nucleosome eviction, and promote gene expression . It is enriched at transcriptional start sites of active genes and defines transcriptionally active chromatin . The p300 co-activator has been identified as an enzyme responsible for acetylating H3K64 .

Conversely, H3K64 trimethylation (H3K64me3) appears to have a repressive function, opposing the activating effect of H3K64ac . In the malaria parasite Plasmodium falciparum, H3K64me3 shows dynamic stage-specific regulation during the parasite's life cycle and is particularly associated with genes encoding exported proteins .

At imprinted control regions (ICRs), a clear demarcation exists between active alleles that are specifically enriched in H3K64ac and inactive alleles that are enriched in H3K64me3, highlighting the functionally opposing nature of these modifications .

Which experimental applications are validated for the HIST1H3A (Ab-64) antibody?

The HIST1H3A (Ab-64) antibody has been validated for several experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of H3K64 modifications in protein samples .

• IHC (Immunohistochemistry): For visualization of H3K64 modifications in tissue sections, with a recommended dilution range of 1:10-1:100 for IHC-P (paraffin-embedded sections) .

• Immunofluorescence: While not explicitly stated for the catalog number orb752886, related antibodies have been validated for immunofluorescence applications .

Additional applications demonstrated in research using similar H3K64 modification-specific antibodies include:

• ChIP (Chromatin Immunoprecipitation): For genome-wide mapping of H3K64 modifications .

• Western blotting: For detecting H3K64 modifications in histone extracts and assessing modification levels across different biological conditions .

When using this antibody, researchers should store it at 2-8°C for short-term use (up to 2 weeks) or at -20°C in small aliquots for long-term storage to prevent freeze-thaw cycles .

How should researchers optimize ChIP protocols when using H3K64 modification-specific antibodies?

Chromatin immunoprecipitation (ChIP) using H3K64 modification-specific antibodies requires special optimization due to the unique location of K64 within the nucleosome core. Unlike histone tail modifications, K64 is positioned on the lateral surface of the histone octamer where it interacts with DNA . This positioning presents challenges for antibody accessibility that should be addressed through protocol optimization:

• Chromatin preparation: Native ChIP (without formaldehyde cross-linking) may provide better results for detecting H3K64 modifications, as it can preserve nucleosome structure while still allowing antibody access. This approach has been successfully employed in studies mapping H3K64ac genomic distribution .

• Sonication parameters: Precise sonication to obtain mono- to tri-nucleosomes (150-500 bp fragments) is critical for exposing the K64 epitope without disrupting antibody recognition sites.

• Antibody validation: Before performing ChIP experiments, researchers should validate antibody specificity using peptide competition assays. Published protocols have shown that recognition of H3 can be efficiently competed by the immunizing peptide but not by other peptides containing acetylated, methylated, or unmodified histone regions .

• Controls: Include positive control regions known to be enriched for H3K64 modifications. For H3K64ac, these include transcriptional start sites of active genes, while for H3K64me3, heterochromatic regions or specific gene families (like exported protein genes in P. falciparum) may serve as positive controls .

• Validation by orthogonal methods: Limited tryptic digestion of native nucleosomes can be used to confirm antibody specificity, as this removes H3 tails while leaving the DNA-protected H3 core region largely intact .

What impact do H3K64 modifications have on nucleosome stability and chromatin dynamics?

H3K64 modifications have direct biochemical effects on nucleosome properties due to their location at the histone-DNA interface:

The acetylation of H3K64 regulates nucleosome stability by neutralizing the positive charge of the lysine residue, which weakens the electrostatic interactions between the histone octamer and the negatively charged DNA phosphate backbone . This destabilization facilitates nucleosome eviction during transcriptional activation, allowing access for the transcriptional machinery.

In contrast, trimethylation at K64 maintains the positive charge while preventing acetylation, potentially stabilizing nucleosome-DNA interactions and promoting more compact chromatin structures . In Plasmodium falciparum, the dynamic regulation of H3K64me3 throughout the parasite's life cycle suggests it plays a role in stage-specific gene expression patterns .

These direct biophysical effects on nucleosome properties make H3K64 modifications particularly interesting for studies of chromatin remodeling mechanisms and transcriptional regulation. Researchers investigating chromatin dynamics should consider incorporating H3K64 modification analysis into their experimental designs.

How are H3K64 modifications distributed across different genomic features and chromatin states?

Genome-wide mapping studies have revealed distinct distribution patterns for H3K64 modifications across genomic features:

• H3K64ac enrichment:

  • Transcriptional start sites (TSS) of active genes

  • Active alleles of imprinted loci

  • Euchromatic regions

  • Pluripotency genes in undifferentiated embryonic stem cells

• H3K64me3 enrichment:

  • Inactive alleles of imprinted loci

  • Heterochromatic regions

  • Genes encoding exported proteins in ring and trophozoite stages of P. falciparum

ChIP-seq analysis of H3K64ac showed a distribution profile similar to other active histone marks, while H3K64me3 exhibited a profile similar to repressive modifications like H3K9me3 . This correlates with their opposing functional roles in gene regulation.

Interestingly, the H3K64me3 mark shows dynamic stage-specific changes in P. falciparum, being enriched in ring and trophozoite stages but drastically reduced in schizont stages . This finding highlights the importance of considering developmental timing or cell cycle stage when analyzing these modifications.

The distinctive genomic distribution patterns of H3K64 modifications make them valuable markers for specific functional chromatin states and potential regulators of stage-specific gene expression programs.

How does the regulation of H3K64-modifying enzymes contribute to chromatin state transitions?

The regulation of enzymes that modify H3K64 plays a crucial role in defining chromatin states:

• Transcriptional activation: The recruitment of p300/CBP to specific genomic loci leads to H3K64 acetylation, which destabilizes nucleosomes and facilitates transcription initiation . This process is often coordinated with other activating histone modifications and the recruitment of transcription factors.

• Developmental transitions: In P. falciparum, the dynamic regulation of H3K64me3 throughout different life cycle stages suggests that the activity of PfSET4 and PfSET5 enzymes is developmentally controlled . The enrichment of H3K64me3 on genes encoding exported proteins in ring and trophozoite stages, followed by its reduction in schizont stages, correlates with the expression patterns of these genes .

• Genomic imprinting: The observed mutually exclusive distribution of H3K64ac and H3K64me3 at imprinted control regions (ICRs) suggests that the enzymes depositing these marks are selectively recruited to specific alleles . This selective recruitment contributes to the establishment and maintenance of allele-specific expression patterns.

Understanding the regulation of these enzymes provides insights into the mechanisms controlling chromatin state transitions during processes such as development, differentiation, and disease progression. Researchers studying these transitions should consider incorporating analysis of H3K64 modifications and their modifying enzymes into their experimental designs.

What cross-talk exists between H3K64 modifications and other histone marks?

H3K64 modifications function within a complex histone code network:

• Cooperative activation marks: H3K64ac co-occurs with other activating histone modifications such as H3K9ac at transcriptionally active regions . This suggests coordinated deposition of these marks, potentially by the same or interacting enzyme complexes.

• Opposing modification patterns: H3K64ac and H3K64me3 show mutually exclusive distribution patterns, particularly evident at imprinted loci where active alleles are enriched for H3K64ac while inactive alleles are enriched for H3K64me3 . This indicates that these modifications form part of broader activating or repressive histone modification landscapes.

• Correlation with specific chromatin states: In P. falciparum, the H3K64me3 mark shows a distribution profile similar to the repressive H3K9me3 modification . This correlation suggests these marks may function together to establish repressive chromatin environments.

• Distribution across histone variants: H3K64ac shows differential distribution among histone H3 variants (H3.1, H3.2, and H3.3), with quantification showing variable levels of this modification across these variants . This adds another layer of complexity to the histone code, where both the underlying histone variant and its modifications contribute to functional outcomes.

This cross-talk between different histone modifications creates a complex regulatory network that fine-tunes chromatin structure and function. Comprehensive analysis of multiple histone marks is therefore essential for understanding the full regulatory landscape of chromatin.

What are common challenges in detecting H3K64 modifications and how can they be addressed?

Studying H3K64 modifications presents several technical challenges due to the location of K64 within the nucleosome core:

Researchers have successfully addressed these challenges through rigorous antibody validation procedures. For example, peptide competition assays have shown that antibody recognition of H3K64ac is efficiently competed by the immunizing peptide but not by other peptides containing acetylated, methylated, or unmodified histone regions . Additionally, limited tryptic digestion of native nucleosomes has been used to confirm antibody specificity for the core region rather than tail modifications .

For mass spectrometry-based detection, optimization of histone extraction and digestion protocols is essential. Acid extraction of histones followed by appropriate enzymatic digestion has been successfully employed to identify H3K64me3 in P. falciparum . The mass shift of 43 Da in modified peptides validates the presence of trimethyl groups .

Proper sample preparation, including careful timing of sample collection for dynamic modifications like H3K64me3 in P. falciparum , is also critical for successful detection and meaningful biological interpretation.

How can researchers validate the specificity of antibodies targeting H3K64 modifications?

Rigorous antibody validation is essential for studies of H3K64 modifications. A comprehensive validation strategy should include:

• Peptide competition assays: Research has demonstrated that specific H3K64ac antibodies can be efficiently competed with the immunizing peptide but not with other peptides containing acetylated, methylated, or unmodified histone regions . This approach confirms binding specificity.

• Limited proteolysis: Tryptic digestion of native nucleosomes removes histone tails while leaving the core region intact. Antibodies specific to H3K64 modifications should still recognize the core fragment, confirming their specificity for these core modifications rather than tail modifications .

• Western blot analysis: Immunoblotting of histone extracts from cells with manipulated levels of modifying enzymes (e.g., p300/CBP knockdown or overexpression for H3K64ac) should show corresponding changes in modification levels .

• Mass spectrometry validation: Mass spectrometry analysis of histone peptides can confirm the presence and identity of specific modifications. For H3K64me3, a mass shift of 43 Da in the modified peptide validates the presence of trimethyl groups .

• Cross-reactivity testing: Testing the antibody against a panel of modified histone peptides can assess potential cross-reactivity with other similar modifications. Published validation has shown that high-quality H3K64 modification antibodies recognize their target with high specificity compared to other histone modifications .

What controls should be included in experiments investigating H3K64 modifications?

Proper experimental controls are crucial for reliable H3K64 modification studies:

• Antibody specificity controls:

  • Peptide competition assays using the immunizing peptide versus irrelevant peptides

  • Immunoblotting with recombinant histones or histone mutants (K64A or K64R) where available

  • Technical replicates to assess reproducibility

• Biological context controls:

  • Positive control regions: For H3K64ac, active gene promoters; for H3K64me3, heterochromatic regions or specific gene families in P. falciparum

  • Negative control regions: Gene deserts or regions known to lack the modification of interest

  • Cell type or developmental stage controls: Given the dynamic nature of these modifications, appropriate stage-matched controls are essential

• Enzyme manipulation controls:

  • Knockdown or overexpression of modifying enzymes: p300/CBP for H3K64ac; PfSET4/PfSET5 for H3K64me3 in P. falciparum

  • Enzyme inhibitor treatments where available

• Parallel analysis controls:

  • Analysis of both H3K64ac and H3K64me3 to understand the complete regulatory landscape

  • Integration with other histone modifications data for contextual interpretation

• Technical controls:

  • Input samples for ChIP experiments

  • Isotype control antibodies for immunoprecipitation

  • Loading controls for Western blotting

Inclusion of these controls helps ensure experimental rigor and facilitates accurate interpretation of results in H3K64 modification studies.

How does analysis of H3K64 modifications contribute to understanding disease mechanisms?

H3K64 modifications play important roles in regulating gene expression and chromatin structure, making them potentially significant in disease contexts:

• Cancer research: Aberrant histone modifications are hallmarks of many cancers. Given that H3K64ac is associated with active transcription and is regulated by p300/CBP , which are frequently dysregulated in cancer, investigating H3K64ac patterns could provide insights into oncogenic transcriptional programs. The HIST1H3A antibody could be valuable for examining these patterns in tumor samples.

• Parasitic diseases: In P. falciparum, H3K64me3 shows stage-specific regulation and association with genes encoding exported proteins . These proteins are critical for host-parasite interactions and virulence. Understanding the role of H3K64me3 in regulating these genes could lead to new therapeutic approaches for malaria.

• Developmental disorders: Imprinted loci show distinct H3K64 modification patterns on active versus inactive alleles . Disruption of these patterns could contribute to imprinting disorders. Studying H3K64 modifications at imprinted regions may provide insights into the molecular basis of these conditions.

• Inflammatory diseases: Given the role of p300/CBP in inflammatory gene regulation and their involvement in H3K64 acetylation , investigating H3K64ac in inflammatory contexts could reveal novel regulatory mechanisms and potential therapeutic targets.

The HIST1H3A antibody provides a valuable tool for such investigations, allowing researchers to map H3K64 modification changes in disease states and potentially identify new biomarkers or therapeutic targets.

What innovative experimental approaches can advance H3K64 modification research?

Several cutting-edge approaches can significantly advance our understanding of H3K64 modifications:

• Single-cell epigenomics: Applying single-cell ChIP-seq or CUT&Tag techniques to analyze H3K64 modifications can reveal cell-to-cell heterogeneity and identify rare cell populations with distinct epigenetic states. This approach is particularly valuable for studying complex tissues or developmental processes.

• Real-time dynamics: Developing tools for live-cell imaging of H3K64 modifications, such as engineered antibody fragments or modification-specific reader domains coupled with fluorescent proteins, could provide unprecedented insights into the dynamic regulation of these modifications during cellular processes.

• Causal manipulation: CRISPR-based epigenome editing tools specifically targeting H3K64 modifications (e.g., dCas9 fused to p300 for site-specific H3K64 acetylation) would allow researchers to test the causal role of these modifications in gene regulation and chromatin function.

• Structural studies: Cryo-EM or X-ray crystallography of nucleosomes with H3K64 modifications could reveal the precise structural impacts of these modifications on nucleosome properties and stability, complementing the functional studies already performed.

• Multimodal analyses: Integrating H3K64 modification mapping with other epigenomic, transcriptomic, and proteomic data can provide comprehensive views of regulatory networks. For example, combining ChIP-seq for H3K64ac/me3 with RNA-seq, ATAC-seq, and Hi-C could reveal how these modifications influence three-dimensional chromatin organization and gene expression programs.

These innovative approaches could significantly expand our understanding of how H3K64 modifications contribute to chromatin regulation and cellular function.

How do findings on H3K64 modifications contribute to our understanding of the histone code?

Research on H3K64 modifications has significantly expanded our understanding of the histone code in several ways:

These contributions significantly enhance our understanding of chromatin regulation beyond the classical histone code focused on tail modifications, revealing a more complex and nuanced regulatory landscape.