HIST1H2BC (Ab-14) Antibody

What is HIST1H2BC and what is its role in chromatin structure?

HIST1H2BC is a core component of the nucleosome, the fundamental unit of chromatin. Nucleosomes wrap and compact DNA, limiting accessibility to cellular machinery that requires DNA as a template. Histones play a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . As part of the histone H2B family, HIST1H2BC is encoded by genes located in histone cluster 1 on chromosome 6p22-p21.3 .

The protein functions by forming an octamer with other core histones (H2A, H3, and H4), around which approximately 147 base pairs of DNA are wrapped. DNA accessibility is regulated through post-translational modifications of histones (the "histone code") and nucleosome remodeling . The positioning and modification state of HIST1H2BC directly affects chromatin architecture and gene expression patterns.

What structural features distinguish HIST1H2BC from other H2B variants?

HIST1H2BC is a canonical H2B histone with a molecular weight of approximately 14 kDa . Human genome studies have revealed considerable variation in encoded histone proteins that exceeds commonly used subtype designations . While canonical H2BC is encoded by multiple genes (including H2BC4, H2BC6, and H2BC7), these genes can exhibit distinct mutation patterns linked to different cancers, suggesting unique functional impacts despite sequence similarity .

H2B variants like H2BK and H2BJ differ from canonical H2BC by only one or two amino acid substitutions, yet they significantly alter nucleosome properties. For example, H2BK has a single substitution (S125A), while H2BJ has two substitutions (V40I and S125A). Despite these minimal differences, these variants decrease DNA accessibility compared to canonical nucleosomes containing H2BC .

What applications is the HIST1H2BC (Ab-14) antibody validated for?

The HIST1H2BC (Ab-14) antibody has been validated for multiple research applications:

The antibody shows reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species . Its specificity for the region around Serine-14 makes it particularly useful for studies involving phosphorylation or other modifications at this residue.

How is HIST1H2BC post-translationally modified?

HIST1H2BC undergoes various post-translational modifications that regulate its function:

• Ubiquitination: Mono-ubiquitination at K120 (ubH2B) is associated with transcriptionally active regions and plays a critical role in gene expression regulation .

• Phosphorylation: Occurs at various residues including Ser14, the target site for the HIST1H2BC (Ab-14) antibody .

• Acetylation: Modifications at sites like K12, K16, and K20 influence chromatin structure and gene activation .

• Crotonylation: Documented at residues K12, K16, K20, and K23, with specific antibodies available for each modification site .

• Butyrylation: Occurs at sites like K5, representing another layer of the histone code .

These modifications form the complex "histone code" that regulates chromatin structure and dynamics, influencing processes like gene transcription, DNA repair, and replication.

How does H2B ubiquitination regulate nucleosome stability and transcription?

H2B mono-ubiquitination (ubH2B) plays crucial roles in transcription and chromatin dynamics:

Nucleosome Stability Effects:

• ubH2B increases nucleosome stability primarily by affecting H2A-H2B dimers

• It enhances H2B levels over both active and repressed genes

• Histone amounts are increased on native chromatin in the presence of high levels of H2B ubiquitination

Trans-histone Crosstalk:

• ubH2B participates in a trans-histone crosstalk pathway where its presence is required for H3K4 and H3K79 methylation

• H3K4me1, H3K4me2, H3K4me3, and H3K79me3 are abolished in the absence of H2B ubiquitination

• Conversely, increased H2Bub1 levels (due to loss of deubiquitinases Ubp8 and Ubp10) cause increased H3K4 and H3K79 methylation

Transcriptional Effects:

• ubH2B impairs the mechanical stability of the nucleosome and helps recruit FACT (FAcilitates Chromatin Transcription) complex by enhancing FACT binding

• It can both activate and repress transcription depending on context

• For repressed/inducible genes (PHO5, GAL1, ADH2), elevated H2Bub1 levels reduce basal expression

These mechanisms highlight how H2B ubiquitination serves as a critical regulator of chromatin accessibility and gene expression.

How do H2B variants alter nucleosome structure and gene expression profiles?

H2B variants significantly impact nucleosome properties and gene expression profiles:

Structural Alterations:

• Nucleosomes containing H2BK and H2BJ had significantly increased S1/2 values relative to canonical (H2BC) nucleosomes (1.6- and 3.5-fold differences, respectively), indicating decreased DNA accessibility

• H2BJ nucleosomes showed ~2-fold higher resistance to Gal4-DNA binding domain access compared to H2BK nucleosomes, despite differing by only one additional amino acid substitution

• These variants appear to alter the trajectory of DNA exiting the nucleosome and potentially change higher-order chromatin organization

Cancer Correlations:

• Specific H2B variants are associated with particular cancers and outcomes

• Low grade glioma patients with high H2BC9 (H2BH) expression have worse prognosis than adenoid cystic carcinoma patients with similar expression

• Low grade glioma patients with low H2BC12 expression show better 5-year survival rates than those with low H2BC11 expression

Molecular Interactions:

• H2B variants exhibit co-expression with known oncogenes and chromatin remodelers like MYC and EZH2

• Different variants show distinct mutational hotspots linked to specific cancer types (e.g., H2BC E76K in bladder cancer versus H2BJ G53S/D in non-small cell lung cancer)

These findings suggest that subtle changes in H2B protein sequence can profoundly affect chromatin structure and gene regulation.

What are the mechanisms of HIST1H2BC degradation and their physiological significance?

Histone H2B degradation represents an important regulatory mechanism with significant physiological impacts:

Degradation Pathway:

• H2B degradation is mediated by K48-linked polyubiquitination

• Lysine 31 (K31) is a key residue mediating H2B polyubiquitination and degradation

• E2 ubiquitin-conjugating enzyme UBC-20 (human UBE2K) and E3 ligase HECT-1 (human HECTD1) are involved in this process

• The degradation can be blocked using proteasome inhibitors like MG-132

Physiological Context:

• Starvation conditions trigger systemic H2B loss in organisms

• Replication-dependent H2B proteins can be replaced by replication-independent variants under starvation conditions

• The mechanism involves both transcriptional regulation and protein degradation

• In C. elegans, HIS-41 (a replication-independent H2B) was the only H2B isoform that maintained its levels during starvation

Signaling Impact:

• H2B degradation affects insulin/IGF signaling pathways

• Maintaining appropriate levels of histone H2B is critical for animal development and survival

• Knockdown of H2B partially suppresses larval lethality and rescues dauer formation in C. elegans insulin/IGF receptor mutants

• This suggests a direct link between histone dynamics and metabolic signaling

These findings establish histone H2B degradation as a key regulatory mechanism linking chromatin structure to cellular metabolic states.

What are the optimal conditions for using HIST1H2BC (Ab-14) antibody in Western blot experiments?

Sample Preparation and Loading:

• Use histone extraction protocols for optimal results (acid extraction with 0.2M H₂SO₄ or 0.4N HCl)

• Run samples on 15-18% gels for better resolution of the 14 kDa HIST1H2BC protein

• Include phosphatase inhibitors if phosphorylation status is important

Antibody Conditions:

• Recommended dilution: 1:100-1:1000 for Western blot

• Optimal concentration: 0.15-1.2μg/ml

• Secondary antibody: Goat polyclonal to rabbit IgG at 1/50000 dilution

Validated Sample Types:

• Cell lysates: HeLa, 293, A549, HepG2, HL60, MCF-7, K562

• Tissue samples: Rat liver, Mouse kidney

Expected Results:

• A single band at approximately 14 kDa

• If multiple bands appear, increase antibody dilution or optimize extraction conditions

• If signal is weak, consider using enhanced chemiluminescence detection systems with longer exposure times

How can HIST1H2BC (Ab-14) antibody be effectively used in ChIP experiments?

For effective ChIP experiments using HIST1H2BC (Ab-14) antibody, follow this optimized protocol based on validated research approaches:

Sample Preparation:

• Start with approximately 4×10^6 cells

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with 0.125M glycine

• Lyse cells and isolate nuclei

• Treat with Micrococcal Nuclease to partially digest chromatin

• Sonicate to create appropriately sized chromatin fragments (200-500bp optimal)

Immunoprecipitation:

• Use 5μg of anti-HIST1H2BC antibody per ChIP reaction

• Include normal rabbit IgG as a negative control

• Incubate chromatin-antibody mixture overnight at 4°C

• Capture complexes using protein A/G magnetic beads

• Wash extensively to remove non-specific binding

• Elute bound chromatin and reverse cross-links

DNA Analysis:

• Purify the ChIP DNA using column-based methods

• Quantify enrichment using real-time PCR

• For genome-wide analysis, proceed with library preparation for ChIP-seq

• Validate findings using primers against regions of known HIST1H2BC occupancy

• The beta-Globin promoter can serve as a control region for quantification

Data Interpretation:

• Calculate fold enrichment relative to IgG control and input samples

• Compare occupancy patterns with known histone modifications

• Correlate with gene expression data for functional insights

• Consider H2B ubiquitination status when interpreting results

What controls should be implemented when studying HIST1H2BC modifications?

Proper experimental controls are essential for accurate interpretation of HIST1H2BC modification studies:

Antibody Controls:

• Specificity Validation:

  • Peptide competition assays using the immunizing peptide

  • Testing against recombinant HIST1H2BC with and without the modification

  • Cross-reactivity assessment with other H2B variants

• Negative Controls:

  • Normal rabbit IgG (same species as the primary antibody)

  • Primary antibody omission

  • Isotype controls

Sample Controls:

• Positive Controls:

  • Cell lines with confirmed HIST1H2BC expression (HeLa, 293, A549, HepG2)

  • Treatment conditions known to induce specific modifications (e.g., HDAC inhibitors for acetylation)

• Negative Controls:

  • HIST1H2BC knockdown or knockout cells

  • Mutation of modification sites (e.g., K to R for preventing ubiquitination)

Modification-Specific Controls:

• For Ubiquitination Studies:

  • Deubiquitinase inhibitors to preserve modifications

  • UBE2K/UBC-20 or HECTD1/HECT-1 knockout cell lines

  • Proteasome inhibitors like MG-132 to block degradation

• For Phosphorylation Studies:

  • Phosphatase inhibitors during sample preparation

  • Phosphatase treatment as negative control

  • Kinase inhibitors specific to the pathway of interest

• For Multiple Modifications:

  • Sequential ChIP (Re-ChIP) to verify co-occurrence of modifications

  • Mass spectrometry validation of modification status

How can different histone modifications on HIST1H2BC be simultaneously analyzed?

Studying multiple modifications simultaneously requires specialized approaches:

Mass Spectrometry-Based Methods:

• Bottom-up Approach:

  • Enzymatic digestion of purified histones

  • LC-MS/MS analysis to identify and quantify modified peptides

  • Advantages: High sensitivity and throughput

  • Limitations: Loss of combinatorial information

• Top-down Approach:

  • Analysis of intact histone proteins

  • Preserves information about co-occurring modifications

  • Requires specialized instrumentation

  • Enables identification of modification "signatures"

• Middle-down Approach:

  • Limited proteolysis to generate larger fragments

  • Captures combinatorial modifications within the same fragment

  • Balance between sensitivity and combinatorial information

Antibody-Based Methods:

• Sequential ChIP (Re-ChIP):

  • First ChIP with one modification-specific antibody

  • Second ChIP on the eluate with another antibody

  • Identifies genomic regions with co-occurring modifications

• Multiplexed Immunofluorescence:

  • Multiple antibodies with distinct fluorophores

  • Useful for tissue or cell imaging applications

  • Can reveal spatial relationships of modifications

Available Antibodies for Combined Studies:
Based on search result , antibodies are available for numerous HIST1H2BC modifications:

• Crotonylation: K12, K16, K20, K23

• Butyrylation: K5

• Phosphorylation: Various sites

• Ubiquitination: K120

What are the technical considerations when analyzing H2B variant mutations in cancer research?

Cancer studies involving H2B variants require specific technical approaches:

Mutation Analysis Strategies:

• Hotspot Identification:

  • Analysis of large cancer genomics datasets (>65,000 samples) reveals variant-specific mutation patterns

  • HIST1H2BC shows E76K as a key hotspot mutation, primarily in bladder cancer

  • Different H2B genes show distinct hotspot mutations linked to different cancers

• Functional Validation:

  • Site-directed mutagenesis to introduce specific mutations

  • CRISPR-Cas9 genome editing for endogenous mutation

  • Nucleosome assembly with mutant histones for biophysical studies

Experimental Approaches:

• Nucleosome Stability Assays:

  • FRET measurements to assess DNA accessibility

  • MNase digestion assays reveal asymmetrical breathing patterns

  • Gal4-DBD binding accessibility as a measure of DNA exposure

• Expression Analysis:

  • Co-expression analysis with oncogenes and chromatin remodelers

  • Cancer-specific expression patterns of H2B variants

  • Survival analysis correlations with expression levels

Data Integration Challenges:

• Heterogeneity Considerations:

  • Distinguish driver from passenger mutations

  • Account for tumor heterogeneity in patient samples

  • Consider tissue-specific effects of identical mutations

• Multiomic Integration:

  • Combine mutation data with expression profiles

  • Correlate chromatin accessibility with mutation status

  • Link patient outcomes to molecular findings

These technical approaches provide a framework for investigating how H2B variants and their mutations contribute to cancer development and progression.