Phospho-HIST1H1E (T145) Antibody

What is HIST1H1E and what is its biological function?

HIST1H1E (also known as Histone H1.4) is a linker histone protein that binds to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. Histones H1, including HIST1H1E, are necessary for the condensation of nucleosome chains into higher-order structured fibers. HIST1H1E also functions as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . Recent research has revealed that mutations in the HIST1H1E gene are associated with Rahman syndrome, a rare congenital anomaly syndrome characterized by variable somatic overgrowth, macrocephaly, distinctive facial features, intellectual disability, and behavioral problems . These findings highlight the crucial role of HIST1H1E in normal development and cellular function.

What is the significance of threonine 145 (T145) phosphorylation in HIST1H1E?

Threonine 145 (T145) phosphorylation represents a critical post-translational modification in the C-terminal domain (CTD) of HIST1H1E. The CTD of histone H1 proteins is known to interact with DNA and other nuclear proteins to regulate chromatin structure. Phosphorylation at T145 likely alters these interactions, potentially affecting chromatin accessibility and gene expression patterns . Understanding this specific phosphorylation event provides insights into the dynamic regulation of chromatin states during various cellular processes, including the cell cycle, differentiation, and stress responses. Studies using phospho-specific antibodies allow researchers to track when and where this modification occurs, helping to elucidate its precise functions in different biological contexts.

What are the optimal storage conditions for Phospho-HIST1H1E (T145) Antibody?

For long-term preservation of antibody activity, store Phospho-HIST1H1E (T145) Antibody at -20°C or -80°C upon receipt . The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage . For short-term storage and frequent use, the antibody can be kept at 4°C for up to one month . It is critical to avoid repeated freeze-thaw cycles as these can significantly decrease antibody performance and specificity . When working with the antibody, aliquot into smaller volumes before freezing to minimize freeze-thaw cycles.

How can I validate the specificity of Phospho-HIST1H1E (T145) Antibody in my experimental system?

Comprehensive validation of antibody specificity is essential for reliable research outcomes. For Phospho-HIST1H1E (T145) Antibody, implement the following validation approaches:

• Peptide Competition Assay: Pre-incubate the antibody with a synthetic phospho-peptide containing the T145 site. This should block specific binding, resulting in signal reduction in subsequent assays.

• Phosphatase Treatment Control: Treat duplicate samples with lambda phosphatase to remove phosphate groups. The antibody should show reduced or absent signal in treated samples compared to untreated controls.

• CRISPR/Cas9 Knockout or siRNA Knockdown: Generate HIST1H1E-deficient cells to compare antibody reactivity with wild-type cells. Significant signal reduction should be observed in knockout/knockdown cells.

• Mutational Analysis: Express wild-type HIST1H1E alongside a T145A mutant (preventing phosphorylation) and compare antibody reactivity. The antibody should recognize only the wild-type protein when phosphorylated.

• Cross-reactivity Testing: Evaluate reactivity against other phosphorylated histone H1 variants to ensure specificity to phosphorylated HIST1H1E.

These validation steps provide cumulative evidence for antibody specificity, increasing confidence in experimental results.

What are the recommended antibody dilutions and optimal conditions for different experimental applications?

Based on product documentation and standard practices for phospho-specific antibodies, the following table outlines optimal conditions for various applications:

These conditions should be optimized for each specific experimental system, as factors like cell type, fixation method, and protein abundance can influence optimal antibody concentrations.

How should I prepare cell or tissue samples to preserve HIST1H1E phosphorylation status?

Preserving phosphorylation status during sample preparation is critical for accurate analysis:

• Immediate Processing: Process samples immediately after collection or flash-freeze in liquid nitrogen to prevent phosphatase activity.

• Phosphatase Inhibitors: Include multiple phosphatase inhibitors in all buffers (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate, and commercial phosphatase inhibitor cocktails).

• Temperature Control: Maintain samples at 4°C throughout all preparation steps to minimize enzymatic activity.

• Lysis Buffer Composition: Use lysis buffers containing 1-2% SDS for complete protein denaturation and phosphatase inactivation. For non-denaturing applications, use buffers with non-ionic detergents and high concentrations of phosphatase inhibitors.

• Fixation for Microscopy: For immunostaining applications, use rapid fixation with pre-warmed 4% paraformaldehyde (10-15 minutes) to preserve nuclear architecture while maintaining phospho-epitope accessibility.

• Controlled Extraction: For nuclear proteins like HIST1H1E, consider using sequential extraction protocols that preserve nuclear structure during initial steps.

Following these guidelines ensures that observed phosphorylation patterns reflect the true biological state rather than artifacts of sample preparation.

How can Phospho-HIST1H1E (T145) Antibody be used to study chromatin dynamics during cell differentiation?

Investigating chromatin remodeling during cell differentiation using Phospho-HIST1H1E (T145) Antibody requires a multi-faceted approach:

• Temporal Profiling: Track changes in HIST1H1E T145 phosphorylation levels throughout differentiation using western blotting. Create a time course from pluripotent state through lineage commitment, correlating phosphorylation changes with expression of differentiation markers.

• Spatial Distribution Analysis: Employ immunofluorescence microscopy to visualize nuclear distribution patterns of phosphorylated HIST1H1E before, during, and after differentiation. Co-stain with markers of heterochromatin/euchromatin (H3K9me3, H3K4me3) to identify relocalization events that may signify chromatin reorganization.

• Genomic Occupancy Mapping: Perform ChIP-seq at key differentiation stages to identify genomic regions showing dynamic changes in phosphorylated HIST1H1E binding. Integrate with RNA-seq data to correlate with gene expression changes during lineage specification.

• Functional Studies: Use CDK inhibitors to modulate HIST1H1E phosphorylation levels (CDKs often phosphorylate H1 histones). Alternatively, express phospho-mimetic (T145E) or phospho-deficient (T145A) HIST1H1E mutants to assess effects on differentiation efficiency and directional bias.

• Single-Cell Resolution Analysis: Combine with single-cell technologies to detect heterogeneity in differentiating populations, potentially identifying subpopulations with distinct chromatin states that predict cell fate decisions.

This comprehensive approach provides insights into how HIST1H1E phosphorylation contributes to the epigenetic regulation of cell differentiation programs.

What role does HIST1H1E T145 phosphorylation play in DNA damage response, and how can this be investigated?

The role of HIST1H1E T145 phosphorylation in DNA damage response can be systematically investigated through:

• DNA Damage Induction Models:

  • UV irradiation: Primarily induces nucleotide excision repair

  • Ionizing radiation: Causes double-strand breaks

  • Chemical agents: Etoposide (topoisomerase II inhibitor), cisplatin (crosslinking agent)

• Temporal Dynamics Analysis:

  • Time-course western blotting of phosphorylated HIST1H1E after damage induction

  • Correlation with γH2AX foci formation and resolution

  • Monitoring throughout different phases of repair (initial response, repair process, and recovery)

• Spatial Relationship Studies:

  • Co-localization analysis with DNA damage markers (γH2AX, 53BP1, RAD51) using immunofluorescence

  • Evaluation of phosphorylated HIST1H1E recruitment to damage sites using laser microirradiation

  • Chromatin accessibility changes at damage sites (ATAC-seq, DNase-seq)

• Functional Analysis:

  • CRISPR-mediated knockdown of HIST1H1E and complementation with phospho-mutants

  • Assessment of DNA repair efficiency using reporter assays

  • Cell survival and genomic stability measurements following damage

• Kinase Identification:

  • Inhibitor screens to identify kinases responsible for damage-induced T145 phosphorylation

  • In vitro kinase assays with candidate kinases (ATM, ATR, DNA-PK, CDKs)

  • Mass spectrometry analysis of damage-induced phosphorylation sites

Understanding the role of HIST1H1E phosphorylation in DNA damage response could reveal important mechanisms of chromatin regulation during genomic stress.

How do phosphorylation patterns of HIST1H1E change during the cell cycle?

HIST1H1E phosphorylation is dynamically regulated throughout the cell cycle, with specific patterns that can be investigated using the following approaches:

• Cell Synchronization Methods:

  • Double thymidine block for G1/S boundary

  • Nocodazole treatment for M phase arrest

  • Serum starvation followed by readdition for G0/G1 transition

  • Thymidine-nocodazole sequence for G2/M boundary

• Time-Course Analysis: Collect cells at defined intervals after synchronization release and analyze phosphorylation levels using:

  • Western blotting with Phospho-HIST1H1E (T145) Antibody

  • Immunofluorescence microscopy to examine nuclear distribution patterns

  • Flow cytometry for quantitative population analysis (combining with DNA content staining)

• Dual Labeling Approaches: Combine Phospho-HIST1H1E (T145) Antibody with:

  • Cell cycle phase markers (e.g., Cyclin B1 for G2/M, Cyclin E for G1/S)

  • DNA content staining (propidium iodide or DAPI) for precise cell cycle positioning

  • Other histone modifications with known cell cycle dynamics (e.g., H3S10ph)

• Quantitative Mass Spectrometry: Apply targeted proteomics to quantify multiple HIST1H1E phosphorylation sites simultaneously across cell cycle phases, revealing potential hierarchical or cooperative phosphorylation events.

• Functional Manipulation: Inhibit cell cycle-regulated kinases (CDKs, Aurora kinases) to determine their contribution to HIST1H1E phosphorylation during specific phases.

These approaches can reveal how T145 phosphorylation contributes to chromatin reorganization during cell division.

I'm experiencing weak or no signal when using Phospho-HIST1H1E (T145) Antibody in Western blotting. What could be the issue?

Several factors could contribute to suboptimal Western blot results with Phospho-HIST1H1E (T145) Antibody:

• Sample Preparation Issues:

  • Insufficient phosphorylation preservation: Add phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to all buffers from cell lysis through gel loading

  • Protein degradation: Add protease inhibitors and process samples rapidly at 4°C

  • Low protein loading: Increase the amount of nuclear extract loaded (30-50 μg recommended)

  • Inefficient extraction: Use stronger lysis buffers containing 1-2% SDS for complete extraction of nuclear proteins

• Technical Considerations:

  • Blocking agent: Use 5% BSA instead of milk (phospho-epitopes can be masked by casein phosphoproteins in milk)

  • Antibody concentration: Increase to 1:500 dilution for stronger signal

  • Incubation time: Extend primary antibody incubation to overnight at 4°C

  • Transfer efficiency: Verify complete transfer using a stained membrane or ladder

• Biological Variables:

  • Low phosphorylation levels: Include positive controls such as mitotic cells where histone phosphorylation is elevated

  • Cell-type specific expression: Confirm HIST1H1E expression in your cell type

  • Treatment conditions: Manipulate phosphorylation status with phosphatase inhibitors (calyculin A, okadaic acid)

• Detection System Optimization:

  • Enhanced chemiluminescence (ECL) substrate: Use high-sensitivity detection reagents

  • Exposure time: Increase exposure time for digital imaging systems

  • Secondary antibody: Ensure compatibility with primary antibody and consider using signal amplification systems

Methodical troubleshooting of these parameters should identify the source of the problem and lead to improved results.

How can I optimize Phospho-HIST1H1E (T145) Antibody performance in ChIP and ChIP-seq experiments?

Optimizing ChIP and ChIP-seq with Phospho-HIST1H1E (T145) Antibody requires careful attention to several critical parameters:

• Crosslinking Optimization:

  • Test multiple formaldehyde concentrations (0.5-2%) and times (5-15 minutes)

  • Consider dual crosslinking with EGS (ethylene glycol bis(succinimidyl succinate)) before formaldehyde to better preserve protein-protein interactions

  • Quench with 125 mM glycine for exactly 5 minutes to prevent over-crosslinking

• Chromatin Preparation:

  • Sonication: Optimize cycles, amplitude, and duration to achieve 200-500 bp fragments

  • Verify fragment size distribution by agarose gel electrophoresis

  • Ensure consistent chromatin concentration across samples (10-20 μg per ChIP)

• Antibody Parameters:

  • Titrate antibody amounts (2-10 μg per ChIP)

  • Include proper controls: IgG negative control, input sample (5-10% of starting material)

  • Perform pilot ChIP-qPCR on regions known to contain H1 histones before proceeding to sequencing

• Washing Conditions:

  • Optimize salt concentration in wash buffers (150-500 mM NaCl)

  • Include non-ionic detergents (NP-40, Triton X-100) to reduce non-specific interactions

  • Extend washing times for higher stringency (5-10 minutes per wash)

• Library Preparation for ChIP-seq:

  • Use sufficient material (minimum 10 ng ChIP DNA)

  • Minimize PCR cycles to reduce amplification biases

  • Include spike-in controls for normalization

• Data Analysis Considerations:

  • Use peak calling algorithms appropriate for histone modifications (MACS2)

  • Consider histone H1's broader distribution pattern compared to core histone modifications

  • Integrate with other epigenomic datasets (histone modifications, chromatin accessibility)

These optimizations will help ensure robust and reproducible ChIP-seq data for phosphorylated HIST1H1E.

What are the common pitfalls when performing immunofluorescence with Phospho-HIST1H1E (T145) Antibody?

Several technical challenges can affect immunofluorescence results with Phospho-HIST1H1E (T145) Antibody:

• Fixation and Permeabilization Issues:

  • Overfixation: Excessive formaldehyde fixation can mask nuclear epitopes; limit to 10-15 minutes with 4% PFA

  • Inadequate permeabilization: Nuclear proteins require sufficient permeabilization; use 0.2-0.5% Triton X-100 for 10-15 minutes

  • Timing: Perform fixation rapidly after experimental treatment to preserve phosphorylation state

• Antibody-Related Factors:

  • Concentration: Start with manufacturer's recommended dilution (1:50-1:200) and optimize as needed

  • Incubation time: Extended incubation (overnight at 4°C) often yields better results than short incubations

  • Background reduction: Include 0.1-0.2% BSA and 0.05% Tween-20 in antibody dilution buffer

• Signal Detection Challenges:

  • Autofluorescence: Include an unstained control to identify intrinsic cellular fluorescence

  • Photobleaching: Use anti-fade mounting media and minimize exposure to excitation light

  • Detection sensitivity: Use high-quality secondary antibodies and appropriate filter sets

• Controls and Validation:

  • Negative controls: Include secondary antibody-only control and non-specific IgG control

  • Positive controls: Include cells with known high levels of phosphorylation (e.g., mitotic cells)

  • Phosphatase treatment: Treat duplicate samples with lambda phosphatase as a specificity control

• Image Acquisition and Analysis:

  • Exposure settings: Standardize exposure times across all experimental conditions

  • Quantification: Use appropriate software for unbiased quantification of nuclear signal intensity

  • Colocalization: When examining relationship with other nuclear markers, employ proper colocalization analysis

Addressing these potential pitfalls will significantly improve the quality and reliability of immunofluorescence data with Phospho-HIST1H1E (T145) Antibody.

What is the relationship between HIST1H1E mutations and Rahman syndrome?

Recent research has established a causal relationship between HIST1H1E mutations and Rahman syndrome, providing important context for understanding HIST1H1E function:

Rahman syndrome is a rare congenital anomaly syndrome resulting from pathogenic variants in the HIST1H1E gene . The condition is characterized by variable somatic overgrowth, macrocephaly, distinctive facial features, intellectual disability, and behavioral problems . This syndrome has been identified in 48 individuals globally, with 20 pathogenic protein-truncating variants reported in the C-terminal domain (CTD) of HIST1H1E .

A novel heterozygous frameshift variant in the CTD of HIST1H1E (p.Lys169IlefsTer27) was identified in a patient with features consistent with Rahman syndrome . This variant replaces the last 51 amino acids of the HIST1H1E protein with 26 incorrect amino acid residues, of which the last 25 amino acids share exactly the same sequence with those in all 20 CTD pathogenic variants reported with Rahman syndrome .

Expression studies of one pathogenic variant (c.435dup or p.Thr146Hisfs*50) demonstrated reduced protein expression, suggesting that haploinsufficiency of HIST1H1E protein and loss of function could be the underlying mechanism leading to neurodevelopmental phenotypes such as intellectual disability . This connection between HIST1H1E dysfunction and developmental disorders underscores the critical role of this histone protein in normal brain development and function.

How does HIST1H1E phosphorylation contribute to epigenetic regulation?

HIST1H1E phosphorylation plays a multifaceted role in epigenetic regulation through several mechanisms:

• Chromatin Compaction Modulation: Phosphorylation of HIST1H1E, particularly at sites like T145, alters its interaction with DNA, generally decreasing its binding affinity and promoting a more open chromatin structure. This dynamic regulation allows for controlled access of transcription factors and chromatin remodeling complexes to specific genomic regions.

• Interplay with Other Histone Modifications: HIST1H1E phosphorylation functions within the broader histone code, working in concert with modifications on core histones (H3, H4, H2A, H2B). For example, regions with phosphorylated HIST1H1E often correlate with active histone marks like H3K4me3 and H3K27ac.

• Cell Cycle-Dependent Regulation: Phosphorylation of HIST1H1E increases progressively during the cell cycle, reaching maximum levels during mitosis. This pattern suggests a role in chromosome condensation and segregation during cell division, with subsequent dephosphorylation allowing proper chromatin reorganization in daughter cells.

• Transcriptional Control: Differential phosphorylation of HIST1H1E can either facilitate or repress transcription, depending on genomic context and the presence of other regulatory factors. The reduced binding of phosphorylated HIST1H1E to DNA can create accessible regions for RNA polymerase II and associated factors.

• DNA Damage Response: Emerging evidence suggests that HIST1H1E phosphorylation changes in response to DNA damage, potentially facilitating the access of repair machinery to damaged sites while maintaining global chromatin integrity.

Understanding these mechanisms provides a framework for investigating how HIST1H1E phosphorylation contributes to normal development and how its dysregulation may lead to disease states.

What new research directions are emerging for HIST1H1E phosphorylation studies?

Several promising research frontiers are expanding our understanding of HIST1H1E phosphorylation:

• Single-Cell Epigenomics: New technologies enabling single-cell ChIP-seq and CUT&Tag will reveal cell-to-cell variability in HIST1H1E phosphorylation patterns within heterogeneous populations, potentially uncovering subpopulation-specific regulatory mechanisms previously masked in bulk analyses.

• Phase Separation Biology: Emerging research suggests that histone modifications, including phosphorylation, can influence biomolecular condensate formation in the nucleus. Studies examining how HIST1H1E phosphorylation affects liquid-liquid phase separation properties could reveal new mechanisms of nuclear compartmentalization and gene regulation.

• Therapeutic Targeting: As connections between HIST1H1E dysregulation and conditions like Rahman syndrome become established , targeting the kinases and phosphatases that regulate HIST1H1E phosphorylation could provide novel therapeutic approaches for developmental disorders and potentially certain cancers.

• Integrative Multi-Omics: Combined analysis of phosphorylated HIST1H1E genomic distribution with transcriptomics, chromatin accessibility, and 3D genome architecture data will provide a comprehensive understanding of how this modification influences the nuclear environment and gene expression programs.

• Evolutionary Conservation: Comparative studies across species could reveal conserved and divergent aspects of HIST1H1E phosphorylation, illuminating fundamental mechanisms versus species-specific adaptations in chromatin regulation.

• Non-Canonical Functions: Investigation of potential roles for HIST1H1E phosphorylation beyond chromatin, such as involvement in signaling cascades or interactions with non-nuclear cellular components, may uncover unexpected functions.

These emerging directions highlight the continuing importance of phosphorylation-specific antibodies like Phospho-HIST1H1E (T145) Antibody in advancing our understanding of epigenetic regulation.