Phospho-HIST1H1E (T17) Antibody

What is HIST1H1E and what is the significance of its T17 phosphorylation?

HIST1H1E, also known as Histone H1.4, is a member of the H1 histone family that binds to linker DNA between nucleosomes to form the macromolecular structure known as the chromatin fiber. The phosphorylation at threonine 17 (T17) represents a specific post-translational modification that regulates chromatin structure and function. Histone H1.4 is essential for condensing nucleosome chains into higher-order structured fibers and acts as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation. This specific phosphorylation site may play roles in cell cycle progression, transcriptional regulation, and chromatin dynamics during various cellular processes .

What are the common experimental applications for Phospho-HIST1H1E (T17) antibodies?

Phospho-HIST1H1E (T17) antibodies are versatile tools employed in multiple experimental applications:

These applications enable researchers to study expression patterns, localization changes, and functional implications of T17 phosphorylation in various experimental contexts .

What is the difference between monoclonal and polyclonal Phospho-HIST1H1E (T17) antibodies?

When selecting antibodies for HIST1H1E phosphorylation research, understanding the differences between monoclonal and polyclonal options is critical:

Monoclonal antibodies (like clone 3A4) are produced from identical B-cell clones and recognize a single epitope, offering high specificity but potentially limited sensitivity. These antibodies provide consistent results across experiments and are typically purified using Protein A methods. For instance, the bsm-52267R antibody is a rabbit monoclonal that specifically targets the phosphorylated T17 site .

Polyclonal antibodies recognize multiple epitopes on the antigen, offering higher sensitivity but potentially more cross-reactivity. They are often affinity-purified and may provide stronger signals in applications where the epitope might be partially masked or denatured. For example, the CSB-PA010380PA17phHU antibody is a rabbit polyclonal raised against a synthetic peptide derived from the 12-23aa region of Histone H1.4 .

The choice between these types depends on the specific experimental requirements, with monoclonals preferred for quantitative applications requiring high reproducibility, and polyclonals sometimes favored for detection of low-abundance targets.

How can I optimize immunoprecipitation protocols for studying HIST1H1E T17 phosphorylation dynamics?

Optimizing immunoprecipitation (IP) protocols for HIST1H1E phosphorylation studies requires careful consideration of several factors:

• Nuclear extraction efficiency: Since HIST1H1E is a nuclear protein tightly associated with chromatin, conventional cell lysis buffers may be insufficient. Employ specialized nuclear extraction methods that include high salt concentrations (300-400mM NaCl) and chromatin shearing steps.

• Phosphatase inhibitor cocktails: T17 phosphorylation is labile and susceptible to dephosphorylation by endogenous phosphatases. Always supplement lysis buffers with comprehensive phosphatase inhibitor cocktails containing sodium fluoride, sodium orthovanadate, and β-glycerophosphate.

• Antibody-to-lysate ratio optimization: Start with 2-5μg of antibody per 500μg of nuclear extract, then optimize based on preliminary results. For the polyclonal Phospho-HIST1H1E antibodies, pre-clearing lysates with protein A/G beads can reduce background .

• Incubation conditions: For optimal antibody-antigen interaction, perform immunoprecipitation at 4°C overnight with gentle rotation to maintain phospho-epitope integrity while ensuring sufficient binding.

• Elution strategy: To preserve phosphorylation for downstream analysis, consider elution with phosphopeptide competitors rather than harsh denaturing conditions.

When troubleshooting, include non-phosphorylated controls and total HIST1H1E antibodies to validate specificity and estimate phosphorylation enrichment efficiency.

What are the recommended strategies for multiplexing Phospho-HIST1H1E (T17) detection with other histone modifications?

Multiplexing strategies for simultaneous detection of Phospho-HIST1H1E (T17) and other histone modifications require careful antibody selection and protocol optimization:

• Primary antibody compatibility assessment: Select primary antibodies from different host species (e.g., rabbit-derived Phospho-HIST1H1E antibodies can be paired with mouse antibodies targeting other modifications). Both monoclonal and polyclonal options exist for Phospho-HIST1H1E (T17), providing flexibility in experimental design .

• Sequential immunostaining: For challenging multiplexing, implement sequential staining protocols with mild stripping between rounds. Begin with the most labile modification detection (often phosphorylation sites).

• Fluorophore selection: Choose spectrally distinct fluorophores with minimal overlap when designing multiplexed IF experiments. Consider the excitation/emission profiles of your microscopy system when selecting secondary antibodies.

• Signal amplification techniques: For low-abundance histone modifications, incorporate tyramide signal amplification or similar methods while maintaining quantitative capacity.

• Validation controls: Always include single-stained controls and blocking peptide competitions to confirm the specificity of multiplexed signals.

This approach enables comprehensive epigenetic profiling to understand the relationship between T17 phosphorylation and other histone modifications in chromatin regulation contexts.

How can ChIP-seq be optimized for Phospho-HIST1H1E (T17) to study genome-wide distribution patterns?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for Phospho-HIST1H1E (T17) requires special considerations due to linker histone dynamics:

• Chromatin preparation optimization: Standard formaldehyde crosslinking may be insufficient for capturing transient H1 interactions. Consider dual crosslinking approaches using disuccinimidyl glutarate (DSG) followed by formaldehyde to stabilize protein-protein interactions before DNA interactions.

• Sonication parameters: H1 histones associate with linker regions between nucleosomes, requiring careful optimization of sonication conditions. Aim for fragments between 150-300bp rather than standard 200-500bp fragments used in core histone ChIP-seq.

• Antibody selection: The monoclonal Phospho-Histone H1.3/H1.4 (T17) antibody (clone 3A4) demonstrates high specificity suitable for ChIP-seq applications, minimizing off-target signals .

• Input normalization: Due to the dynamic binding nature of linker histones, robust input normalization is essential. Process input samples with identical conditions to IP samples to account for chromatin accessibility biases.

• Bioinformatic analysis adaptations: Employ specialized peak-calling algorithms that account for the broader distribution patterns of linker histones compared to core histones. Integrate with nucleosome positioning data to identify relationships between phosphorylated H1 and nucleosome organization.

These optimizations enable mapping of T17 phosphorylation across the genome, providing insights into the regulatory roles of this modification in chromatin structure and transcriptional control.

What is the role of HIST1H1E T17 phosphorylation in neurodevelopmental disorders?

The relationship between HIST1H1E T17 phosphorylation and neurodevelopmental disorders represents an emerging research frontier with important clinical implications:

Recent research has revealed that mutations in HIST1H1E are associated with a spectrum of neurodevelopmental disorders, including Rahman syndrome. These conditions present with intellectual disability, hypotonia, distinctive craniofacial features, and behavioral problems . While the specific frameshift mutations identified (such as c.416_419dupAGAA) don't directly affect the T17 phosphorylation site, they significantly alter the C-terminal domain of the protein, which could impact how phosphorylation at T17 regulates chromatin architecture.

Phosphorylation of linker histones, including at T17, plays crucial roles in neuronal differentiation and brain development by modulating chromatin accessibility at developmentally regulated genes. In neurodevelopmental disorder contexts, disruptions to normal HIST1H1E phosphorylation patterns may contribute to aberrant gene expression during critical developmental windows.

Researchers investigating this connection should consider employing Phospho-HIST1H1E (T17) antibodies in comparative analyses of patient-derived cells versus controls, potentially revealing whether phosphorylation at this site is dysregulated in disease states . Such studies may provide mechanistic insights into how HIST1H1E mutations lead to the observed clinical phenotypes and identify potential therapeutic targets.

How does cell cycle progression affect HIST1H1E T17 phosphorylation patterns?

HIST1H1E T17 phosphorylation demonstrates dynamic relationships with cell cycle phases that can be studied using phase-specific experimental designs:

During interphase, basal levels of T17 phosphorylation contribute to maintaining chromatin architecture, with localized increases at actively transcribed regions. As cells enter mitosis, this phosphorylation dramatically increases alongside other histone modifications to facilitate chromosome condensation.

To effectively study these dynamics:

• Synchronization methods: Employ double thymidine block or nocodazole treatment followed by release to capture cells at specific cycle phases.

• Co-staining strategies: Combine Phospho-HIST1H1E (T17) antibodies with cell cycle markers (e.g., phospho-Histone H3 (Ser10) for mitosis) in immunofluorescence applications, using the recommended dilutions (1:50-200) .

• Quantitative analysis: Apply flow cytometry with co-staining for DNA content (propidium iodide) and Phospho-HIST1H1E (T17) to quantify modification levels across cell populations.

• Chromatin fractionation: Separate soluble from chromatin-bound histone fractions to track phosphorylation-dependent mobility changes throughout the cell cycle.

These approaches reveal how T17 phosphorylation contributes to chromatin plasticity during cell division and may inform research on cell cycle dysregulation in disease contexts.

What is the interplay between HIST1H1E T17 phosphorylation and other histone modifications in genome stability?

The functional relationship between HIST1H1E T17 phosphorylation and other epigenetic marks creates a complex regulatory network affecting genome stability:

Histone H1.4, encoded by HIST1H1E, plays crucial roles in maintaining genome stability, DNA replication, and repair processes . The phosphorylation at T17 exists within a broader context of histone modifications that collectively regulate chromatin accessibility during these processes.

Key interactions include:

• Coordination with core histone modifications: T17 phosphorylation may work synergistically with H3K9me3 to promote heterochromatin formation at repetitive elements, protecting genome stability. Conversely, it may antagonize active marks like H3K4me3 at specific genomic loci.

• DNA damage response signaling: During DNA damage response, T17 phosphorylation increases at damage sites, potentially facilitating the recruitment of repair factors. This correlates with γH2AX formation but follows distinct temporal dynamics.

• Replication timing regulation: Differential T17 phosphorylation patterns across the genome contribute to replication timing decisions, with hypophosphorylated regions typically replicating earlier than hyperphosphorylated regions.

Researchers can investigate these relationships using sequential ChIP (re-ChIP) approaches with Phospho-HIST1H1E (T17) antibodies followed by antibodies against other modifications or DNA repair factors . Mass spectrometry-based approaches can also quantify co-occurrence of multiple modifications on the same histone molecules.

What are the common sources of non-specific binding when using Phospho-HIST1H1E (T17) antibodies, and how can they be mitigated?

Non-specific binding can compromise experimental results when working with Phospho-HIST1H1E (T17) antibodies. Understanding and addressing these issues is essential:

Common sources of non-specific binding:

• Cross-reactivity with similar phosphorylation sites: The T17 site resides in a region with sequence similarity to other histone proteins. The monoclonal antibody (clone 3A4) recognizes both Phospho-Histone H1.3 (T17) and Histone H1.4 (T17), which may be advantageous or problematic depending on your experimental goals .

• Dephosphorylation during sample preparation: Inadequate phosphatase inhibition can lead to epitope loss and variable results.

• Fixation artifacts: Overfixation in IHC/ICC applications can mask epitopes or create artificial binding sites.

Mitigation strategies:

• Blocking optimization: For Western blotting, use 5% BSA rather than milk-based blocking buffers, as milk contains phosphoproteins that can compete with detection. For IHC/IF applications, include phosphopeptide competitors in blocking steps.

• Validation controls: Always include phosphatase-treated samples as negative controls to confirm signal specificity. Lambda phosphatase treatment of parallel samples can verify phospho-specificity.

• Dilution optimization: Start with the recommended dilution ranges (WB: 1:300-5000; IHC-P: 1:200-400; IF: 1:50-200) but perform titration experiments to determine optimal concentration for your specific sample type .

• Storage and handling: Store antibodies at -20°C and avoid repeated freeze-thaw cycles to maintain specificity. The appropriate storage buffer (containing 1xTBS, pH7.4, 1%BSA, 40%Glycerol, and 0.05% Sodium Azide) helps preserve antibody performance .

Implementing these strategies will significantly improve signal-to-noise ratio and experimental reproducibility.

How can phosphorylation-specific signals be distinguished from total HIST1H1E in experimental analyses?

Differentiating phosphorylation-specific signals from total protein abundance requires careful experimental design:

• Parallel detection strategy: Run duplicate blots/samples using both phospho-specific and total HIST1H1E antibodies. The ratio between phosphorylated and total protein provides a normalized measure of phosphorylation status independent of expression changes.

• Sequential probing approach: For Western blotting, consider mild stripping and reprobing of the same membrane with total HIST1H1E antibodies after phospho-detection. This approach ensures identical protein loading but may compromise sensitivity.

• Phosphatase controls: Treat duplicate samples with lambda phosphatase before analysis. The signal that disappears after treatment represents the phosphorylation-specific component.

• Quantification methods: For immunofluorescence, employ ratiometric imaging using spectrally distinct fluorophores for phospho and total signals, enabling pixel-by-pixel phosphorylation analysis across subcellular compartments.

• Phosphomimetic constructs: When studying functional consequences, compare wild-type HIST1H1E with T17A (phospho-null) and T17E (phosphomimetic) mutants to distinguish effects specific to phosphorylation from those related to total protein levels.

This systematic approach allows researchers to confidently attribute observed phenomena to phosphorylation events rather than changes in HIST1H1E expression or localization.

What are the best practices for preserving phospho-epitopes during tissue preparation for immunohistochemistry?

Preserving labile phosphorylation marks like T17 on HIST1H1E during tissue processing requires specialized approaches:

• Rapid fixation protocols: Minimize time between tissue collection and fixation. For optimal phospho-epitope preservation, fix tissues in 4% paraformaldehyde for no more than 24 hours at 4°C.

• Phosphatase inhibitor implementation: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers used during tissue harvest and processing, including perfusion solutions for animal studies.

• Optimized antigen retrieval: For IHC-P applications with Phospho-HIST1H1E (T17) antibodies, heat-mediated antigen retrieval using citrate buffer (pH 6.0) generally provides better phospho-epitope recovery than EDTA-based methods.

• Section thickness considerations: Thinner sections (4-5μm) generally provide better antibody penetration and signal-to-noise ratio when working with phospho-specific antibodies, including the recommended 1:200-400 dilution for IHC-P applications .

• Signal amplification systems: Consider tyramide signal amplification or similar methods for enhancing detection sensitivity without increasing background, particularly important for low-abundance phosphorylation sites.

• Control slide inclusion: Always process phosphatase-treated serial sections alongside test sections as negative controls to verify phospho-specific staining.

These practices maximize the likelihood of detecting authentic phosphorylation signals while minimizing artifacts in tissue-based experiments.

How are Phospho-HIST1H1E (T17) antibodies being used in single-cell epigenomic profiling?

Recent advances in single-cell technologies have created new opportunities for studying HIST1H1E phosphorylation at unprecedented resolution:

Single-cell epigenomic profiling with Phospho-HIST1H1E (T17) antibodies enables researchers to investigate heterogeneity in histone modifications across individual cells within complex tissues. These approaches reveal cell-type-specific regulatory mechanisms that may be masked in bulk analyses.

Current methodological advances include:

• CUT&Tag adaptations: Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) approaches combined with Phospho-HIST1H1E (T17) antibodies allow simultaneous profiling of this modification and cell surface markers or other epigenetic features at single-cell resolution.

• Microfluidic platforms: Droplet-based systems can now be adapted for chromatin immunoprecipitation with phospho-specific antibodies, enabling high-throughput analysis of thousands of individual cells.

• Mass cytometry applications: Integration of phospho-specific antibodies into CyTOF workflows allows simultaneous detection of multiple phosphorylation events, including HIST1H1E T17, alongside other cellular parameters.

• Spatial epigenomics: Emerging techniques combine immunofluorescence using antibodies at recommended dilutions (1:50-200 for IF) with in situ sequencing to map phosphorylation patterns within tissue architecture.

These approaches are particularly valuable for studying neurodevelopmental disorders linked to HIST1H1E mutations, as they can reveal how phosphorylation patterns vary across different neural cell types and developmental stages .

What are the emerging connections between HIST1H1E T17 phosphorylation and neurodevelopmental disease mechanisms?

Research into connections between HIST1H1E T17 phosphorylation and neurodevelopmental disorders represents an emerging frontier:

Recent studies have identified multiple HIST1H1E variants associated with Rahman syndrome and other neurodevelopmental disorders. These frameshifts affecting the C-terminal domain could potentially disrupt normal phosphorylation patterns, including at T17 .

Key emerging research directions include:

• Genotype-phenotype correlations: Studies are investigating whether different HIST1H1E variants produce distinct patterns of T17 phosphorylation dysregulation that correlate with specific clinical manifestations. Current evidence suggests that all 23 identified frameshift variants create nearly identical shorter proteins with altered C-terminal tails, yet produce heterogeneous clinical presentations .

• Developmental timing effects: Phosphorylation at T17 may have stage-specific roles during neurodevelopment. Research using patient-derived iPSCs differentiated into neural lineages is beginning to map how phosphorylation patterns change during development and how disease-associated variants disrupt these patterns.

• Circuit-specific impacts: Evidence suggests that phosphorylation of HIST1H1E regulates chromatin accessibility differentially across neural circuits, potentially explaining why symptoms like hypotonia, intellectual disability, and autism spectrum features co-occur in patients with HIST1H1E variants .

• Therapeutic targeting potential: Emerging research is exploring whether modulating kinases or phosphatases that regulate T17 phosphorylation could offer therapeutic approaches for patients with Rahman syndrome and related disorders.

These connections highlight the importance of phosphorylation-specific antibodies as tools for understanding complex neurodevelopmental disease mechanisms.

How do different fixation and extraction methods affect the detection of Phospho-HIST1H1E (T17) in ChIP-seq and CUT&Tag experiments?

The choice of fixation and extraction methods significantly impacts the quality of Phospho-HIST1H1E (T17) detection in genome-wide mapping experiments:

Fixation methods comparison:

• Formaldehyde-only fixation (standard 1% for 10 minutes): Provides adequate crosslinking for abundant proteins but may insufficiently capture transient H1-chromatin interactions. This approach typically yields lower enrichment of phosphorylated H1 variants at dynamic regulatory elements.

• Dual crosslinking (DSG followed by formaldehyde): Significantly improves detection of phosphorylated linker histones by stabilizing protein-protein interactions before DNA crosslinking. This method reveals association patterns at enhancers and other regulatory elements that may be missed with standard fixation.

• Native ChIP approaches: Omitting crosslinking altogether can preserve phospho-epitopes better but risks redistribution of H1 during extraction. This method is generally not recommended for Phospho-HIST1H1E (T17) detection due to the dynamic nature of linker histone binding.

Extraction method considerations:

• Sonication-based fragmentation: Requires careful optimization to prevent epitope destruction. Shorter sonication cycles at lower power settings preserve phosphorylation sites better than aggressive protocols.

• Enzymatic digestion: MNase-based chromatin preparation often better preserves phospho-epitopes but can introduce biases based on chromatin accessibility. This approach may be preferred when studying relationships between nucleosome positioning and H1 phosphorylation.

• CUT&Tag adaptations: This newer method often provides superior signal-to-noise ratio for phosphorylated histones compared to traditional ChIP-seq. The gentler cell permeabilization and in situ antibody binding better preserve labile modifications like T17 phosphorylation.

When designing experiments, researchers should conduct pilot studies comparing different preparation methods using the same antibody lot and concentration to determine optimal conditions for their specific research questions.

How can multi-omics approaches integrate Phospho-HIST1H1E (T17) data with other epigenetic and transcriptomic datasets?

Modern multi-omics integration strategies enable comprehensive understanding of HIST1H1E T17 phosphorylation in broader cellular contexts:

• Sequential ChIP-seq and RNA-seq: Performing ChIP-seq with Phospho-HIST1H1E (T17) antibodies followed by RNA-seq on the same samples allows direct correlation between phosphorylation patterns and transcriptional outcomes. This approach has revealed that T17 phosphorylation often marks boundaries between active and repressed chromatin domains.

• HiChIP adaptations: Combining 3D genome organization data with Phospho-HIST1H1E (T17) mapping through HiChIP protocols can reveal how this modification influences higher-order chromatin architecture and enhancer-promoter interactions.

• Integrated epigenomic visualization: Computational platforms now enable simultaneous visualization of Phospho-HIST1H1E (T17) ChIP-seq data alongside other histone modifications, DNA methylation, accessibility, and gene expression data. These tools reveal coordinative and antagonistic relationships between different epigenetic layers.

• Machine learning approaches: Supervised and unsupervised learning algorithms can identify patterns in multi-omics datasets that predict functional outcomes of T17 phosphorylation in different genomic contexts. These computational approaches have identified unexpected relationships between HIST1H1E phosphorylation and neuronal gene expression programs relevant to neurodevelopmental disorders .

• Time-series integration: Measuring multiple epigenetic features, including T17 phosphorylation, across developmental timelines provides insights into the temporal dynamics of regulatory networks and how their disruption may contribute to disorders like Rahman syndrome .

These integrative approaches provide a systems-level understanding of how HIST1H1E phosphorylation contributes to genome regulation in both normal development and disease states.

What are the recommended controls and validation approaches for Phospho-HIST1H1E (T17) antibody specificity in different experimental contexts?

Rigorous validation is essential for confident interpretation of results with Phospho-HIST1H1E (T17) antibodies:

Western blot validation:

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase to demonstrate phospho-specificity.

• Peptide competition: Pre-incubate antibody with phospho-peptide and non-phospho-peptide competitors to confirm epitope specificity.

• Knockout/knockdown control: When possible, include HIST1H1E knockout or knockdown samples to confirm antibody specificity.

• Molecular weight verification: Confirm signal at the expected molecular weight for HIST1H1E (approximately 21.9 kDa).

Immunostaining validation:

• Blocking peptide controls: Include phospho-peptide blocked antibody controls alongside primary experiments.

• Signal pattern assessment: Phospho-HIST1H1E (T17) should show nuclear localization with possible enrichment at specific nuclear domains depending on cell type and condition.

• Co-localization studies: Validate co-localization with other nuclear markers but distinct distribution from cytoplasmic proteins.

ChIP-seq validation:

• Input normalization: Carefully normalize against input samples processed identically to IP samples.

• Technical replicates: Include at least two technical replicates to assess reproducibility.

• Peak distribution analysis: Compare genomic distribution of peaks (promoters, enhancers, gene bodies) with published datasets and expected biology.

• Motif enrichment: Verify enrichment of expected transcription factor binding motifs at Phospho-HIST1H1E (T17) peaks.

These validation approaches ensure that experimental findings truly reflect the biology of HIST1H1E phosphorylation rather than technical artifacts or antibody cross-reactivity.

How does understanding HIST1H1E T17 phosphorylation contribute to therapeutic development for related neurodevelopmental disorders?

The increasing understanding of HIST1H1E T17 phosphorylation mechanisms provides new avenues for therapeutic development in several ways:

• Biomarker potential: Phosphorylation status of HIST1H1E at T17 may serve as a biomarker for neurodevelopmental disorder progression or treatment response. Patient-derived cell models can be assessed using established antibody-based methods at recommended dilutions to monitor changes in phosphorylation patterns .

• Targeted kinase modulation: Identifying the specific kinases responsible for T17 phosphorylation provides potential therapeutic targets. While direct targeting of histone modifications remains challenging, modulating the responsible kinases may normalize phosphorylation patterns in disorders caused by HIST1H1E mutations.

• Epigenetic editing approaches: CRISPR-based epigenetic editing tools are being developed to modulate histone modifications at specific genomic loci. Understanding the genomic distribution of T17 phosphorylation through ChIP-seq studies helps identify critical regulatory regions that might benefit from such targeted interventions.

• Pharmacological screening platforms: Cell-based assays using Phospho-HIST1H1E (T17) antibodies in high-content imaging or ELISA formats enable screening of compound libraries for molecules that normalize phosphorylation patterns disrupted by disease-associated mutations.

• Precision medicine strategies: The comprehensive characterization of genotype-phenotype relationships in HIST1H1E-related disorders suggests that patients with different variants may benefit from tailored therapeutic approaches based on their specific molecular pathology .