HIST1H2BC (Ab-79) Antibody

What is HIST1H2BC and what is its significance in epigenetic research?

Histone H2B type 1-C (HIST1H2BC), encoded by the HIST1H2BC gene, is a core component of the nucleosome octamer that forms the primary unit of chromatin packaging in eukaryotic cells. This protein plays a critical role in chromatin structure and gene expression regulation through various post-translational modifications. In epigenetic research, HIST1H2BC and its modifications are particularly significant for understanding chromatin dynamics during transcriptional regulation, DNA replication, and DNA repair processes. The protein has a calculated molecular weight of approximately 13.9 kDa and is highly conserved across species . Antibodies against HIST1H2BC enable researchers to investigate these critical epigenetic mechanisms, particularly when studying how specific modifications to histone H2B impact gene expression patterns and cellular functions.

What are the recommended storage conditions for HIST1H2BC antibodies?

For optimal antibody performance and longevity, HIST1H2BC antibodies should be aliquoted upon receipt and stored at -20°C, avoiding repeated freeze/thaw cycles which can significantly reduce antibody activity . When handling the antibody, it's advisable to keep it on ice during experiments and minimize exposure to room temperature. For short-term storage (less than a week), the antibody can be kept at 4°C, but prolonged storage should be at -20°C. Most HIST1H2BC antibodies are provided in liquid form in PBS buffer containing 0.09% sodium azide as a preservative . When preparing working dilutions, use freshly prepared buffer solutions and consider adding protein stabilizers such as BSA (0.1-1%) if diluting the antibody for extended use.

What applications are HIST1H2BC antibodies validated for?

Commercial HIST1H2BC antibodies are typically validated for several applications, with Western blotting (WB) and ELISA being the most common . For Western blotting applications, the recommended dilution is typically 1:1000, though optimal concentrations should be determined empirically by each researcher for their specific experimental conditions . Some HIST1H2BC antibodies may also be validated for immunoprecipitation, chromatin immunoprecipitation (ChIP), and immunohistochemistry applications, though these may require different optimization parameters. When using HIST1H2BC antibodies for novel applications or in different experimental systems, preliminary validation studies should be conducted to confirm specificity and optimal working conditions.

How should I design ChIP-seq experiments using HIST1H2BC antibodies?

For successful ChIP-seq experiments using HIST1H2BC antibodies, careful consideration of multiple technical factors is essential. Start by validating antibody specificity through Western blotting and pilot ChIP-qPCR experiments on known target regions. During experimental design, include appropriate controls: input DNA (pre-immunoprecipitation), IgG negative control, and if possible, a spike-in control for normalization. Crosslinking conditions are particularly critical - standard formaldehyde fixation (1% for 10 minutes) works for most histone targets, but optimization may be needed for specific modifications.

When performing the immunoprecipitation, use 3-5μg of antibody per ChIP reaction with 25-50μg of chromatin. Consider that different histone H2B modifications may require specific sonication conditions to efficiently fragment chromatin while maintaining epitope integrity. Analysis of H2B modification patterns should account for the genomic distribution patterns observed in previous studies - for instance, H2B N-terminus acetylation (H2BNTac) prominently marks candidate active enhancers and a subset of promoters, distinguishing them from ubiquitously active promoters . This pattern differs from H3K27ac distribution, providing complementary information about regulatory element activity.

How do histone H2B post-translational modifications affect antibody recognition?

Post-translational modifications (PTMs) on histone H2B can significantly impact antibody recognition in ways critical to experimental interpretation. When targeting specific modifications like lysine acetylation (e.g., H2BK5ac, H2BK12ac, H2BK16ac, or H2BK20ac), consider that nearby modifications can create epitope masking or influence antibody binding affinity. For example, research has shown that some commercial antibodies targeting H2BK5ac (such as EP857Y) can cross-react with H3K27ac due to sequence similarities near these sites, while some H2BK20ac antibodies may cross-react with H2BK120ac .

To address these challenges, implement rigorous validation using multiple techniques: peptide competition assays with modified and unmodified peptides, testing on knockout/knockdown samples, and correlating results with orthogonal methods. When analyzing H2B acetylation patterns, consider using multiple antibodies targeting different modification sites to create a comprehensive modification landscape. Be aware that CBP/p300 specifically catalyzes H2BNTac, unlike H3K27ac which can be modified by other acetyltransferases, and H2BNTac is differentially regulated by histone deacetylases 1 and 2 . This differential regulation can impact experimental outcomes when using HDAC inhibitors or studying dynamic acetylation changes.

What is the role of H2B acetylation in enhancer activity and how can it be studied?

H2B N-terminus multisite lysine acetylation (H2BNTac) has emerged as a distinctive signature of active enhancers that can discriminate them from other candidate cis-regulatory elements . Research has revealed that H2BNTac intensity predicts enhancer strength and outperforms current models in predicting CBP/p300 target genes. To effectively study this phenomenon, researchers should employ a multi-faceted approach combining genomic, biochemical, and functional techniques.

ChIP-seq experiments targeting various H2BNTac sites (H2BK5ac, H2BK12ac, H2BK16ac, H2BK20ac) should be compared with established enhancer marks like H3K27ac and H3K4me1. When analyzing genomic distribution, note that H2BNTac is enriched at distal regulatory regions compared to promoters, with a higher H2BNTac:H3K27ac ratio in distal regions than active promoters . This distinguishes H2BNTac from other acetylation marks. To investigate the functional significance of H2BNTac at putative enhancers, researchers can use enhancer activity assays (luciferase reporters, STARR-seq) and correlate H2BNTac levels with transcriptional output of nearby genes.

For mechanistic studies, consider that H2BNTac is specifically catalyzed by CBP/p300 (unlike H3K27ac), and that H2A-H2B dimers undergo rapid exchange through transcription-induced nucleosome remodeling . CBP/p300 inhibitors like A-485 can be used to modulate H2BNTac levels, with differential effects observed between promoters and distal regions, providing insights into regulatory mechanisms.

What are the recommended protocols for Western blotting with HIST1H2BC antibodies?

For optimal Western blotting results with HIST1H2BC antibodies, follow this detailed protocol adapted for the detection of histone proteins:

Sample preparation: Extract histones using acid extraction (0.2N HCl or 0.4N H2SO4) to efficiently isolate basic histone proteins from chromatin. Alternatively, use commercial histone extraction kits that maintain native modifications. When working with whole cell lysates, include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in lysis buffers to preserve acetylation states.

Gel electrophoresis: Use 15-18% SDS-PAGE gels or specialized Triton-Acid-Urea (TAU) gels for better separation of histone variants and modified forms. Load 2-10μg of acid-extracted histones or 20-50μg of whole cell lysate.

Transfer conditions: Transfer proteins to PVDF membranes (preferred over nitrocellulose for histones) using buffer containing 25mM Tris, 192mM glycine, 0.025% SDS, and 15-20% methanol. Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of small histone proteins.

Blocking and antibody incubation: Block membranes with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature. Dilute primary HIST1H2BC antibody 1:1000 in blocking buffer and incubate overnight at 4°C . After washing 3-4 times with TBST, incubate with HRP-conjugated secondary antibody (typically anti-rabbit IgG at 1:3000-1:5000) for 1-2 hours at room temperature.

When analyzing results, note that HIST1H2BC has a calculated molecular weight of 13.9 kDa , but migration patterns may vary depending on post-translational modifications.

How can I troubleshoot cross-reactivity issues with HIST1H2BC antibodies?

Cross-reactivity is a common challenge when working with histone antibodies due to sequence similarities between histone variants and modified sites. For HIST1H2BC antibodies, consider these troubleshooting approaches:

Identify potential cross-reactivity: Review the antibody's immunogen sequence (typically from the N-terminal region of human HIST1H2BC/HIST1H2BF, amino acids 1-30) and compare with other histone sequences using alignment tools. Key regions of concern include sequence similarities between H2BK5 and H3K27, and between H2BK20 and H2BK120 .

Validation experiments: Perform peptide competition assays using both the target peptide and potential cross-reactive peptides. Include appropriate knockout/knockdown controls when available. For suspected cross-reactivity with differently modified sites, test the antibody against samples treated with modification-specific enzymes (e.g., histone deacetylase inhibitors or CBP/p300 inhibitors like A-485) .

Optimization strategies: Increase antibody specificity by using more stringent washing conditions (higher salt concentration in wash buffers) or lower antibody concentrations. Consider pre-absorbing the antibody with peptides containing potential cross-reactive epitopes.

Alternative detection methods: Validate findings using multiple antibodies targeting the same modification from different vendors or raised against different epitopes. Complement antibody-based detection with mass spectrometry to confirm modification-specific findings.

Research has shown that some commercial H2BK5ac antibodies (e.g., EP857Y) can cross-react with H3K27ac, while some H2BK20ac antibodies may cross-react with H2BK120ac . Researchers should be vigilant about these specific cross-reactivities when interpreting experimental results.

What controls should be included in ChIP experiments using HIST1H2BC antibodies?

Comprehensive controls are essential for robust ChIP experiments using HIST1H2BC antibodies:

Technical controls: Include input DNA (pre-immunoprecipitation sample, typically 5-10% of starting material) to correct for differences in starting chromatin amounts and biases in DNA fragmentation or amplification. Use species-matched IgG as a negative control to establish background signal levels. For quantitative comparisons between samples, consider spike-in controls with exogenous chromatin (e.g., Drosophila chromatin with Drosophila-specific antibody).

Biological validation controls: Include ChIP-qPCR validation of known positive and negative genomic regions before proceeding to sequencing. For H2B acetylation studies, CBP/p300-bound regions can serve as positive controls, while transcriptionally inactive regions can serve as negative controls. When studying H2BNTac, regions with high H3K27ac but low H2BNTac (typically constitutively active promoters) provide useful reference regions .

Treatment controls: Consider including samples treated with histone deacetylase inhibitors (enhances acetylation signal) or CBP/p300 inhibitors like A-485 (reduces H2BNTac signal) to validate modification-specific binding . These treatments have been shown to differentially affect H2BNTac at promoters versus distal regulatory regions.

Analysis controls: When analyzing ChIP-seq data, compare H2BNTac patterns with other histone marks like H3K27ac, H3K4me1 (enhancers), and H3K4me3 (promoters) to validate expected genomic distributions . Consider evaluating the H2BNTac:H3K27ac ratio, which is higher at distal regulatory regions than at promoters.

How does H2BNTac compare to other histone marks for identifying regulatory elements?

H2B N-terminus multisite lysine acetylation (H2BNTac) offers distinct advantages for identifying and characterizing regulatory elements compared to traditional histone marks. Research has shown that H2BNTac prominently marks candidate active enhancers and a subset of promoters, discriminating them from ubiquitously active promoters . Unlike H3K27ac, which is present at most active promoters (90-98%), H2BNTac shows more selective enrichment at distal regulatory elements.

The H2BNTac:H3K27ac ratio provides a powerful discriminatory metric - this ratio is significantly higher in distal regions compared to active promoters . This contrasts with the H3K9ac:H3K27ac and H3K4me3:H3K27ac ratios, which show the opposite pattern. When identifying enhancers, H2BNTac intensity more accurately predicts enhancer strength and outperforms current models in identifying CBP/p300 target genes.

H2BNTac also shows stronger association with tissue-specific gene regulation. In mouse embryonic stem cells, 88-97% of H2BNTac+ regions in the top quartile were bound by key transcription factors NANOG or OCT4 . Notably, NANOG/OCT4 binding was similar in both promoter and distal H2BNTac+ regions, whereas their binding was much lower in H3K27ac+ promoters than in distal regions.

For optimal regulatory element identification, researchers should consider combining H2BNTac with established marks like H3K4me1, H3K4me3, and H3K27ac in integrative analyses.

What mechanisms regulate H2BNTac deposition and removal?

H2BNTac is regulated through specific enzymatic pathways that differ from those controlling other histone acetylation marks. Two key mechanisms underlie the distinct specificity of H2BNTac:

First, unlike H3K27ac which can be catalyzed by multiple acetyltransferases, H2BNTac is specifically catalyzed by CBP/p300 . This specificity allows for more precise control of H2BNTac deposition at regulatory elements. Inhibition of CBP/p300 using A-485 demonstrates differential effects on H2BNTac at various genomic locations - H2BNTac is more strongly reduced in promoters and actively transcribed gene body regions than in distal regulatory regions following CBP/p300 inhibition .

Second, H2A-H2B dimers (but not H3-H4 tetramers) undergo rapid exchange through transcription-induced nucleosome remodeling . This dynamic turnover affects the distribution and maintenance of H2BNTac marks across the genome.

Removal of H2BNTac is primarily mediated by histone deacetylases (HDACs) 1 and 2 . Depletion of endogenous HDACs 1 and 2 using degradation tag approaches confirms their role in deacetylating H2BNTac . Class I HDAC inhibitors have been shown to increase H2BNTac levels in vivo, suggesting a dynamic equilibrium between addition and removal of these marks.

Understanding these regulatory mechanisms provides insights into how cells control gene expression through differential histone modification patterns at regulatory elements.

How can I integrate H2BNTac data with other genomic datasets?

Integrating H2BNTac ChIP-seq data with other genomic datasets provides comprehensive insights into gene regulatory mechanisms. A systematic approach should include:

Multi-mark chromatin state analysis: Combine H2BNTac with H3K27ac, H3K4me1 (enhancer-associated), H3K4me3 (promoter-associated), and other relevant histone modifications using computational frameworks like ChromHMM. Research shows that including H2BNTac in chromatin state models can refine the identification of enhancer states that were previously classified as H3K4me1-enriched transcriptional start sites .

Transcription factor binding correlation: Analyze co-occurrence of H2BNTac with transcription factors and cofactors like MED1, NANOG, and OCT4. In mouse embryonic stem cells, NANOG and OCT4 bound a larger fraction of H2BNTac+ regions than H3K27ac+ regions, providing insights into regulatory mechanisms .

Gene expression correlation: Link H2BNTac-marked regions to gene expression data using approaches like correlation analysis or regression models. H2BNTac intensity has been shown to predict enhancer strength and outperform current models in identifying CBP/p300 target genes .

Three-dimensional chromatin interactions: Integrate H2BNTac data with chromosome conformation data (Hi-C, Capture-C, ChIA-PET) to determine physical interactions between H2BNTac-marked enhancers and target promoters.

Drug response analysis: Correlate changes in H2BNTac patterns following treatment with epigenetic modulators (HDAC inhibitors, CBP/p300 inhibitors) with gene expression changes to identify direct regulatory relationships.

For robust integration, normalize datasets appropriately and consider using visualization tools like WashU Epigenome Browser or UCSC Genome Browser to create custom tracks displaying multiple data types.

What is the role of H2B modifications in disease mechanisms?

Histone H2B modifications are increasingly recognized as critical factors in disease pathogenesis, particularly in cancer and neurodegenerative disorders. Aberrant patterns of H2B acetylation can disrupt normal enhancer function and gene expression programs, contributing to disease states. Research indicates that H2BNTac specifically marks active enhancers and can predict enhancer strength , suggesting that dysregulation of this modification may impact disease-associated gene expression networks.

In cancer, altered histone modification patterns, including H2B acetylation, contribute to oncogenic transcriptional programs. The specificity of H2BNTac for CBP/p300-mediated acetylation is particularly relevant, as CBP/p300 mutations and dysregulation occur in multiple cancer types . Since H2BNTac predicts enhancer strength and CBP/p300 target genes better than other marks, it may serve as a valuable biomarker for CBP/p300 activity in cancer tissues.

The dynamic regulation of H2BNTac by histone deacetylases 1 and 2 connects this modification to therapeutic applications, as HDAC inhibitors are used clinically for various cancers . Understanding how these drugs specifically affect H2BNTac patterns could provide insights into their mechanism of action and help predict treatment responses.

Future research should investigate H2BNTac patterns in patient samples, correlate these patterns with disease progression and treatment outcomes, and explore how disease-associated genetic variants affect H2BNTac deposition at regulatory elements.

How can HIST1H2BC antibodies be applied in single-cell epigenomic studies?

Applying HIST1H2BC antibodies in single-cell epigenomic studies represents an emerging frontier with significant technical challenges and potential rewards. To effectively implement these approaches:

Antibody selection and validation: Choose antibodies with exceptionally high specificity and sensitivity, as single-cell techniques require robust signal detection from limited material. Validate antibodies using spike-in controls with known quantities of the target protein or modification.

CUT&Tag and CUT&RUN adaptations: These methods require less starting material than traditional ChIP and can be adapted for single-cell analysis. When using HIST1H2BC antibodies in these techniques, optimization of antibody concentration and incubation conditions is critical. Starting with 1:100 dilution and testing a range of concentrations can help determine optimal conditions.

Single-cell ChIP-seq approaches: When adapting HIST1H2BC antibodies for scChIP-seq, consider using microfluidic platforms or combinatorial indexing strategies. The relatively low abundance of H2B compared to H3 may necessitate modified protocols with increased cell lysis efficiency and improved antibody capture.

Data analysis considerations: Single-cell H2BNTac data analysis should account for the known genomic distribution patterns - enrichment at enhancers and specific promoters rather than constitutively active promoters . Integration with single-cell RNA-seq data can be particularly informative, as H2BNTac intensity predicts enhancer strength and target gene expression .

Alternative approaches: Consider alternative methods like CoBATCH (Concurrent Barcoding and Tagmentation of Chromatin) or single-cell ATAC-seq paired with targeted H2B modification analysis using mass spectrometry.

What are the latest developments in multiplexed detection of histone modifications?

Multiplexed detection of histone modifications represents a cutting-edge approach to comprehensively profile chromatin states. Recent developments offer new opportunities for using HIST1H2BC antibodies alongside other histone modification antibodies:

Mass cytometry (CyTOF) adaptations: Metal-tagged antibodies against multiple histone modifications, including H2B marks, enable simultaneous detection of 40+ epitopes at single-cell resolution. When incorporating HIST1H2BC antibodies into CyTOF panels, careful validation of antibody specificity is essential due to the high homology between histone proteins and modification sites, particularly between H2BK5 and H3K27, and between H2BK20 and H2BK120 .

Co-detection by indexing (CODEX): This method allows for iterative antibody staining and imaging cycles, enabling detection of numerous targets in the same sample. For optimal results with HIST1H2BC antibodies, controlling epitope masking through appropriate antibody sequencing and optimizing signal amplification are crucial.

Sequential ChIP (re-ChIP): This technique allows for the identification of genomic regions containing multiple histone modifications. When performing re-ChIP with HIST1H2BC antibodies, consider their position in the sequential immunoprecipitation series - performing H2B modification ChIP first may improve results due to the dynamic exchange of H2A-H2B dimers .

Barcoded antibody approaches: DNA-barcoded antibodies enable multiplexed detection of histone modifications followed by sequencing readout. For H2BNTac studies, consider the differential genomic distribution patterns compared to other histone marks - H2BNTac shows higher enrichment at distal regulatory elements compared to promoters .

Integration with other genomic methods: Combined assays like Paired-Tag, which couples histone modification profiling with chromatin accessibility or transcription factor binding, can provide multidimensional insights into regulatory mechanisms involving H2B modifications.