Recombinant Mouse Histone H2B type 3-B (Hist3h2bb)

What is the fundamental role of Histone H2B type 3-B in mouse cellular biology?

Histone H2B type 3-B functions as a core component of nucleosomes, the fundamental units of chromatin. It works alongside other histone proteins to wrap and compact DNA, thereby regulating DNA accessibility to cellular machinery that requires DNA as a template. This regulation is critical for transcription, DNA repair, DNA replication, and maintaining chromosomal stability . The specific function of H2B involves participating in the formation of the histone octamer that forms the protein core of the nucleosome, around which approximately 146 base pairs of DNA are wrapped.

In mouse models, H2B type 3-B (gene symbol H2BU1, UniProt entry Q8CGP0) has been identified as a key component in maintaining proper chromatin architecture . The structural integrity of nucleosomes containing H2B type 3-B directly impacts how genes are expressed, silenced, or regulated under various cellular conditions.

How does post-translational modification of Histone H2B type 3-B affect its function?

Histone H2B type 3-B undergoes various post-translational modifications that significantly alter its functionality. One of the most studied modifications is mono-ubiquitination at lysine 120 (ubH2B), which plays a crucial role in regulating transcriptional elongation . This modification impairs the mechanical stability of the nucleosome, creating a more dynamic chromatin environment conducive to transcription.

Additionally, phosphorylation of H2B at serine residues, such as Ser32, by kinases like Aurora-B, has been shown to be essential for proper cellular division. When H2B phosphorylation is disrupted, as in HIPK2-defective cells, cytokinesis cannot be completed successfully, leading to binucleated cells, chromosomal instability, and potentially increased tumorigenicity .

These modifications collectively constitute part of the "histone code" that regulates DNA accessibility and chromatin dynamics. The patterns of modification vary across different genomic regions and cellular states, allowing for precise regulation of gene expression and other DNA-dependent processes .

What technical considerations should be addressed when working with recombinant Mouse Histone H2B type 3-B?

When working with recombinant Mouse Histone H2B type 3-B, researchers should consider several technical aspects:

First, recombinant proteins may have different tertiary structures or post-translational modifications compared to native histones, potentially affecting experimental outcomes. Detection methods optimized for native samples may show reduced sensitivity with recombinant proteins . Therefore, validation of antibody specificity and detection methods specifically for recombinant H2B type 3-B is essential.

Second, storage conditions significantly impact protein stability. Recombinant histones should typically be stored at -80°C for long-term storage, with aliquoting recommended to avoid freeze-thaw cycles. Before use in experiments, proper refolding may be necessary to ensure the protein adopts its native conformation .

Third, for quantitative measurements, careful concentration standardization is critical. ELISA-based methods can detect H2B type 3-B in the range of 0.156-10 ng/ml, but sample concentrations must be diluted to the mid-range of detection assays for accurate results .

How does Aurora-B kinase regulate Histone H2B type 3-B localization during cytokinesis?

Aurora-B kinase plays a crucial role in regulating H2B type 3-B localization during cell division through a direct mechanism. Research has demonstrated that Aurora-B directly binds and phosphorylates H2B at Ser32, which is essential for recruiting H2B to the midbody during cytokinesis . This recruitment is independent of nucleic acids and occurs through protein-protein interactions.

Methodologically, this interaction can be verified through in vitro binding assays with purified proteins. When recombinant GST-Aurora-B and His-H2B are combined, direct binding occurs that can be detected via immunoblotting . The phosphorylation of H2B at Ser32 by Aurora-B can be demonstrated through cold kinase assays followed by immunoblot analysis using phospho-specific antibodies.

For researchers investigating this phenomenon, temporally controlled inhibition of Aurora-B using specific inhibitors like Hesperadin can provide valuable insights. When Aurora-B is inhibited during late stages of mitosis, H2B fails to localize at the midbody, confirming Aurora-B's role in H2B recruitment . Immunofluorescence microscopy with antibodies against phosphorylated H2B-Ser32 and midbody markers like β-tubulin provides spatial resolution of this regulatory process.

What is the mechanistic relationship between Histone H2B mono-ubiquitination and FACT in transcriptional regulation?

The collaboration between mono-ubiquitinated H2B (ubH2B) and the histone chaperone FACT (Facilitates Chromatin Transcription) represents a sophisticated mechanism for regulating transcriptional elongation. Research has revealed that ubH2B impairs the mechanical stability of nucleosomes, which paradoxically helps recruit FACT by enhancing its binding to the nucleosome .

The mechanism involves several steps: First, ubH2B destabilizes the nucleosome structure, making it more accessible. Second, FACT preferentially binds to nucleosomes containing ubH2B. Third, FACT specifically recognizes and deposits H2A-ubH2B dimers to form intact nucleosomes. Finally, this preferential binding of FACT to ubH2B-nucleosomes enhances nucleosome stability in an altered state and maintains its integrity during transcription .

To study this interaction experimentally, researchers can employ:

• Single-molecule biophysics approaches to measure nucleosome stability

• Chromatin immunoprecipitation (ChIP) with antibodies against ubH2B and FACT components

• Time-course ChIP-qPCR analyses to track the dynamics of ubH2B and FACT localization during transcription

• In vitro reconstitution of nucleosomes with recombinant H2B versus ubH2B to directly compare FACT binding affinities

These methods have revealed that the stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as confirmed by genome-wide analyses .

How can researchers effectively differentiate between the functions of Histone H2B type 3-B and other H2B variants in experimental systems?

Differentiating between the functions of Histone H2B type 3-B and other H2B variants requires sophisticated experimental approaches that address the high sequence homology among histone variants while capturing their distinct functional roles.

One effective approach is to use CRISPR/Cas9-mediated gene editing to create specific knockout or knock-in models of H2B type 3-B. By targeting unique regions of the H2BU1 gene, researchers can selectively modify H2B type 3-B without affecting other variants. Subsequently, phenotypic analyses of cellular processes like DNA replication, transcription, and chromosome segregation can reveal variant-specific functions.

Antibody-based detection must be carefully validated for specificity. Commercial antibodies like the Mouse mAb #2934 are produced against specific peptide regions of H2B , but cross-reactivity testing is essential. For H2B type 3-B (UniProt Primary AC: Q8CGP0), researchers should verify antibody specificity using knockout cell lines or peptide competition assays .

Mass spectrometry-based proteomics provides another powerful approach for distinguishing between histone variants. Despite high sequence homology, variant-specific peptides can be identified through careful proteolytic digestion and targeted mass spectrometry. This allows for quantitative comparison of variant abundance and modification states across different experimental conditions.

Functional studies should incorporate variant-specific readouts. For instance, the unique role of H2B type 3-B in cytokinesis can be assessed by measuring the frequency of binucleated cells following targeted depletion of this specific variant .

What are the optimal conditions for detecting phosphorylated Histone H2B type 3-B in experimental systems?

Detecting phosphorylated Histone H2B type 3-B requires careful consideration of both sample preparation and detection methods. The optimal conditions include:

Sample Preparation:

• For cell lysates: Extraction in the presence of phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) is crucial to preserve phosphorylation states.

• For tissue samples: Flash-freezing followed by extraction in buffers containing phosphatase inhibitors and HDAC inhibitors (such as sodium butyrate) helps maintain histone modifications.

• Acid extraction methods (using sulfuric acid or hydrochloric acid) are preferable for isolating histones while maintaining their post-translational modifications.

Detection Methods:

• Immunoblotting: Use phospho-specific antibodies targeting H2B-Ser32P with validated specificity. Western blotting should be performed using 15-18% SDS-PAGE gels to adequately resolve the low molecular weight (14 kDa) histone H2B .

• Immunofluorescence microscopy: For detecting phosphorylated H2B at specific cellular locations (like the midbody during cytokinesis), cells should be fixed with paraformaldehyde, permeabilized, and stained with validated phospho-specific antibodies .

• Mass spectrometry: Offers the highest specificity for detecting and quantifying site-specific phosphorylation. Sample preparation should include enrichment steps for phosphopeptides, such as titanium dioxide or immobilized metal affinity chromatography.

For validating phospho-specific detection, appropriate controls are essential:

• Positive controls: Cells treated with okadaic acid to inhibit phosphatases

• Negative controls: Samples treated with phosphatases or cells treated with kinase inhibitors like Hesperadin (for Aurora-B mediated phosphorylation)

• Specificity controls: Phosphopeptide competition assays

How can researchers effectively study the dynamics of Histone H2B type 3-B ubiquitination in transcriptional regulation?

Studying the dynamics of H2B type 3-B ubiquitination in transcriptional regulation requires methods that capture both temporal and spatial aspects of this modification. Researchers can employ these approaches:

Real-time Monitoring:

• Live-cell imaging with fluorescently tagged ubiquitin and H2B type 3-B allows visualization of ubiquitination dynamics during transcription.

• FRET (Förster Resonance Energy Transfer) sensors designed to detect the proximity between H2B and ubiquitin can provide quantitative measurements of ubiquitination states with high temporal resolution.

Genome-wide Mapping:

• ChIP-seq using antibodies specific to ubiquitinated H2B can map the genomic distribution of this modification.

• Sequential ChIP (ChIP-reChIP) with antibodies against ubH2B followed by FACT components can identify genomic regions where both factors co-localize .

• Time-course ChIP-qPCR following transcriptional activation can track the dynamics of ubH2B levels at specific gene loci .

Functional Correlation:

• Nascent RNA-seq (NET-seq or GRO-seq) performed in parallel with ubH2B ChIP-seq can correlate ubiquitination events with active transcription.

• Single-molecule biophysics approaches measuring nucleosome stability in the presence or absence of ubiquitination provide mechanistic insights into how this modification affects chromatin structure .

Manipulation of Ubiquitination:

• Inducible expression systems for wildtype H2B versus ubiquitination-resistant mutants (K120R) allow direct comparison of transcriptional outcomes.

• Small molecule inhibitors of E3 ligases (RNF20/40) or deubiquitinating enzymes (USPs) can be used to modulate ubH2B levels with temporal precision.

• Reconstituted in vitro transcription systems with chemically defined ubiquitinated nucleosomes enable mechanistic dissection of how ubH2B affects transcriptional machinery.

What are the key considerations for experimental design when investigating the role of Histone H2B type 3-B in cytokinesis?

Investigating the role of Histone H2B type 3-B in cytokinesis requires a multifaceted experimental approach addressing both spatial-temporal dynamics and molecular interactions. Key considerations include:

Cellular Models and Manipulation:

• Cell line selection: Choose models with well-characterized cell division dynamics (HeLa, HCT116) and consider species-specific differences in H2B variants.

• Genetic manipulation: CRISPR/Cas9-mediated knockout or knock-in of tagged H2B type 3-B (with minimal disruption of function) enables tracking of endogenous protein.

• Expression systems: For rescue experiments, use inducible expression of wild-type or mutant H2B (phospho-mimetic S32D or non-phosphorylatable S32A) in knockout backgrounds .

Visualization and Quantification:

• Time-lapse microscopy: Track labeled H2B type 3-B during cell division to monitor recruitment to the midbody.

• Quantitative immunofluorescence: Co-staining with midbody markers (β-tubulin) and phospho-specific antibodies (H2B-Ser32P) provides spatial information .

• Cytokinesis failure quantification: Measure binucleation rates, micronuclei formation, and chromosomal instability markers.

Molecular Mechanism Elucidation:

• Temporal inhibition: Use kinase inhibitors (Hesperadin for Aurora-B) at precisely timed intervals during mitosis to dissect stage-specific requirements .

• Protein-protein interaction mapping: IP-MS (immunoprecipitation followed by mass spectrometry) to identify H2B type 3-B interactors at the midbody.

• In vitro reconstitution: Binding assays with purified components (Aurora-B, H2B, MgcRacGAP, PRC1) to establish direct interactions and requirements .

Controls and Validation:

• Specificity controls: Include DICER-defective cells to rule out microRNA involvement in H2B recruitment .

• Rescue experiments: Introduce phosphomimetic H2B-S32D to verify this modification is sufficient to restore proper cytokinesis in Aurora-B inhibited cells.

• Long-term consequences: Monitor genomic stability over multiple cell divisions following perturbation of H2B function.

How should researchers interpret conflicting results between in vitro and in vivo studies of Histone H2B type 3-B function?

Conflicting results between in vitro and in vivo studies of Histone H2B type 3-B are common and require careful interpretation. Several factors should be considered when reconciling such discrepancies:

Complexity Differences:
In vitro systems often lack the full complement of regulatory factors present in cellular environments. For example, while Aurora-B directly binds and phosphorylates H2B at Ser32 in vitro , the in vivo recruitment process may involve additional factors that modify this interaction. Researchers should systematically add complexity to in vitro systems to identify missing components.

Post-translational Modification States:
Recombinant H2B used in vitro typically lacks the complex pattern of modifications present on endogenous histones. Native H2B may have multiple modifications (phosphorylation, ubiquitination, methylation) that collectively influence its behavior. Detection methods optimized for native samples may show reduced efficacy with recombinant proteins lacking these modifications .

Concentration Effects:
In vitro studies often use protein concentrations that differ from physiological levels. Researchers should perform dose-response experiments spanning physiological concentration ranges and consider local concentration effects that may occur in cellular compartments.

Contextual Environment:
The nuclear environment provides a unique biophysical context (molecular crowding, ion concentrations, etc.) that is difficult to recapitulate in vitro. Experiments comparing different in vitro conditions (varying salt concentrations, crowding agents, etc.) can help identify contextual factors influencing H2B function.

When confronted with conflicting results, researchers should:

• Validate antibody specificity in both contexts

• Use complementary methodologies to confirm observations

• Design hybrid experiments that bridge in vitro and in vivo approaches

• Consider cell-type specific regulatory mechanisms that may explain differences

What are the most common technical challenges in studying Histone H2B type 3-B post-translational modifications and how can they be overcome?

Studying post-translational modifications (PTMs) of Histone H2B type 3-B presents several technical challenges that researchers must address:

Challenge 1: Antibody Specificity
Histone antibodies often cross-react with similar epitopes across variants or may recognize the modification but not the specific variant.

Solution: Validate antibody specificity using multiple approaches:

• Peptide competition assays with modified and unmodified peptides

• Western blots comparing wildtype and modification-site mutants

• Use knockout/knockdown models as negative controls

• For phospho-specific antibodies, treat samples with phosphatases as controls

Challenge 2: Transient Nature of Modifications
Many H2B modifications are dynamically regulated and may be present only briefly or in specific cellular compartments.

Solution: Employ strategies to capture transient modifications:

• Use inhibitors of enzymes that remove modifications (phosphatase inhibitors, DUB inhibitors)

• Synchronize cells to enrich for cell-cycle-specific modifications

• Employ rapid fixation methods to "freeze" modification states

• Use targeted mass spectrometry with heavy-labeled internal standards for absolute quantification

Challenge 3: Low Abundance of Modified Forms
Modified histones often represent a small fraction of the total histone pool.

Solution: Implement enrichment strategies:

• Use PTM-specific antibodies for immunoprecipitation before analysis

• Apply chromatographic enrichment methods (IMAC for phosphorylation, affinity resins for ubiquitination)

• Employ targeted mass spectrometry with increased sensitivity for low-abundance peptides

• For ubiquitinated histones, use tandem ubiquitin binding entities (TUBEs) for enrichment

Challenge 4: Preserving Modifications During Sample Preparation
Modifications can be lost during extraction and processing.

Solution: Optimize extraction protocols:

• Add deacetylase inhibitors (sodium butyrate), phosphatase inhibitors, and DUB inhibitors to extraction buffers

• Use acid extraction methods that rapidly denature enzymes that remove modifications

• Minimize time between sample collection and analysis

• Consider snap-freezing samples to halt enzymatic activity

How can researchers distinguish between the direct and indirect effects of Histone H2B type 3-B modifications on cellular processes?

Distinguishing between direct and indirect effects of Histone H2B type 3-B modifications requires experimental designs that establish causality and mechanistic links:

Rapid Induction Systems:
Employing systems that allow rapid and specific induction of modifications can help separate immediate (likely direct) from delayed (potentially indirect) effects.

• Chemically inducible dimerization systems to recruit modifying enzymes to specific genomic loci

• Optogenetic approaches allowing light-controlled activation of enzymes that modify H2B

• Auxin-inducible degron systems for rapid depletion of regulatory proteins

Temporal Resolution Studies:
Capturing the sequence of events following H2B modification helps establish causality.

• Time-course experiments with high temporal resolution after induction of modifications

• Pulse-chase experiments to track modified histones and associated factors

• Sequential ChIP experiments to determine the order of recruitment of different factors

Site-Specific Mutation Analysis:
Creating targeted mutations at modification sites can definitively link specific modifications to phenotypes.

• Compare wildtype, non-modifiable mutants (e.g., S32A), and modification-mimetic mutants (e.g., S32D for phosphorylation)

• Assess whether mutation phenotypes match those observed with inhibition of modifying enzymes

• Use rescue experiments with mutant histones in cells depleted of endogenous H2B

Reconstituted Systems:
In vitro systems with defined components can establish direct biochemical consequences.

• In vitro transcription systems with reconstituted chromatin containing modified or unmodified H2B

• Single-molecule biophysics approaches to directly measure the effect of modifications on nucleosome stability and dynamics

• FRET-based assays to monitor conformational changes induced by modifications

Specific Example: H2B Ubiquitination and Transcription
Research has shown that ubH2B directly affects nucleosome stability and FACT recruitment . To distinguish this direct effect from potential indirect effects:

• Compare transcriptional output immediately after inducing or removing ubiquitination

• Assess whether FACT recruitment occurs in the absence of other transcription-related changes

• Use reconstituted transcription systems with chemically defined ubiquitinated nucleosomes

• Perform nucleosome stability measurements in the presence and absence of ubiquitination to directly measure biophysical effects

What are the emerging areas of research regarding Histone H2B type 3-B function in disease models?

Histone H2B type 3-B is increasingly recognized as a critical factor in disease pathogenesis, particularly in cancer and developmental disorders. Several emerging research areas warrant investigation:

Cancer Biology:
Dysregulation of H2B modifications, particularly ubiquitination and phosphorylation, has been linked to genomic instability and cancer progression. HIPK2-defective cells, which fail to properly phosphorylate H2B, demonstrate chromosomal instability and increased tumorigenicity . Future research should focus on:

• The diagnostic potential of H2B modification patterns as cancer biomarkers

• Therapeutic targeting of enzymes regulating H2B modifications

• The role of H2B variants in cancer stem cell maintenance and therapy resistance

Neurodevelopmental Disorders:
Given the importance of chromatin regulation in neuronal differentiation and function, alterations in H2B type 3-B may contribute to neurodevelopmental disorders. Research directions include:

• Assessing H2B variant expression and modification patterns in neuronal development

• Investigating the consequences of H2B dysregulation in animal models of neurodevelopmental disorders

• Exploring the role of H2B in activity-dependent transcription in neurons

Regenerative Medicine:
Cellular reprogramming and differentiation involve extensive chromatin remodeling, with histone variants playing crucial roles. Emerging research should examine:

• The dynamics of H2B type 3-B incorporation during cell fate transitions

• The potential manipulation of H2B modifications to enhance reprogramming efficiency

• The development of small molecules targeting H2B-modifying enzymes for regenerative applications

Aging and Senescence:
Chromatin organization undergoes significant changes during aging. New research avenues include:

• Characterizing age-associated alterations in H2B modification patterns

• Investigating the role of H2B in senescence-associated heterochromatin formation

• Exploring interventions targeting H2B modifications to delay age-related chromatin deterioration

What novel methodologies are being developed to study Histone H2B type 3-B function with greater precision?

The field of histone biology is witnessing rapid methodological advances that promise to revolutionize our understanding of H2B type 3-B functions:

Spatially Resolved Proteomics:
New methods allow visualization and quantification of histone modifications with unprecedented spatial resolution:

• Imaging mass spectrometry for in situ detection of histone modifications in tissue sections

• Proximity labeling methods (BioID, APEX) to identify proteins interacting with H2B in specific cellular compartments

• Super-resolution microscopy combined with specific antibodies to visualize the nanoscale distribution of modified histones

Single-Cell Epigenomics:
Technologies enabling single-cell analysis of histone modifications reveal cell-to-cell heterogeneity:

• Single-cell ChIP-seq adaptations for H2B modifications

• Mass cytometry (CyTOF) with histone modification-specific antibodies

• Single-cell proteomics methods capable of detecting histone variants and their modifications

Synthetic Biology Approaches:
Designer systems for precise manipulation of histone function:

• Engineered histone genes with orthogonal chemistry for site-specific modification

• Optogenetic tools for spatiotemporal control of histone-modifying enzymes

• CRISPR-based systems for locus-specific recruitment of histone readers and writers

Computational Methods:
Advanced computational approaches enhance interpretation of complex datasets:

• Machine learning algorithms to predict functional consequences of histone modification patterns

• Molecular dynamics simulations to model how modifications affect nucleosome structure and dynamics

• Network analysis tools to integrate histone modification data with other -omics datasets

In Situ Structural Biology:
Methods to visualize histone structure and interactions within cells:

• Cryo-electron tomography to visualize nucleosome organization in situ

• Live-cell FRET sensors to detect conformational changes in H2B upon modification

• Cross-linking mass spectrometry to map protein-protein interactions involving H2B in intact cells

How might understanding Histone H2B type 3-B function contribute to therapeutic strategies in the future?

The growing understanding of Histone H2B type 3-B function opens several promising avenues for therapeutic development:

Targeting Cytokinesis in Cancer Therapy:
The role of H2B in cytokinesis, particularly its phosphorylation by Aurora-B kinase, presents a potential therapeutic target . Cancer cells often exhibit dysregulated cell division, and compounds that modulate H2B phosphorylation could selectively affect rapidly dividing cancer cells. Strategies might include:

• Small molecule inhibitors targeting the H2B-Aurora-B interaction

• Peptide mimetics that compete with H2B for Aurora-B binding

• Targeted degradation of modified H2B in cancer cells using PROTACs (Proteolysis Targeting Chimeras)

Modulating Transcriptional Programs:
H2B ubiquitination influences transcriptional elongation through FACT recruitment . This mechanism could be exploited to alter gene expression patterns in disease states:

• Small molecules that stabilize or disrupt the ubH2B-FACT interaction

• Compounds targeting DUBs (deubiquitinating enzymes) that remove ubiquitin from H2B

• Locus-specific targeting of H2B-modifying enzymes using CRISPR-based approaches

Epigenetic Reprogramming:
Understanding how H2B modifications contribute to cell fate decisions could inform regenerative medicine approaches:

• Chemical cocktails including modulators of H2B-modifying enzymes to enhance cellular reprogramming

• Engineered histone variants with modification-mimetic properties to direct cell differentiation

• Targeted epigenetic editing to restore normal H2B modification patterns in disease states

Biomarker Development:
The patterns of H2B modifications could serve as diagnostic or prognostic biomarkers:

• Antibody-based assays for specific H2B modifications associated with disease states

• Mass spectrometry profiles of H2B variants and modifications as part of liquid biopsy approaches

• Imaging agents targeting specific H2B modifications for non-invasive diagnostics

Combination Therapies:
Targeting H2B modifications could sensitize cells to existing therapies:

• Inhibitors of H2B ubiquitination to enhance sensitivity to transcription inhibitors

• Modulators of H2B phosphorylation to sensitize cells to anti-mitotic agents

• Compounds affecting H2B-dependent DNA damage responses to potentiate radiotherapy or chemotherapy