HIST1H1E (Ab-145) Antibody

What is the HIST1H1E (Ab-145) Antibody and what epitope does it recognize?

The HIST1H1E (Ab-145) Antibody is a rabbit polyclonal antibody developed using a peptide sequence surrounding the threonine 145 (Thr145) phosphorylation site derived from human histone H1.4, which is encoded by the HIST1H1E gene. This antibody specifically recognizes the phosphorylated form of Thr145 in the C-terminal domain of histone H1.4 . The antibody is primarily used in epigenetic and nuclear signaling research to detect this specific post-translational modification, which plays an important role in chromatin structure regulation . The antibody is available in liquid form, preserved in 0.03% Proclin 300 and supplied in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 .

What validated applications can researchers use the HIST1H1E (Ab-145) Antibody for?

The HIST1H1E (Ab-145) Antibody has been validated for several experimental applications that are crucial for chromatin biology research. It has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Chromatin Immunoprecipitation (ChIP) according to its technical specifications . For ChIP applications, the antibody has demonstrated effectiveness in precipitating histone H1.4-bound chromatin from HeLa cells (4×10^6) treated with Micrococcal Nuclease . Subsequent real-time PCR analysis with primers against the β-Globin promoter has confirmed its specificity and utility in this application . Additionally, researchers have successfully employed this antibody for Western Blotting (WB) to detect a ~22 kDa band corresponding to histone H1.4 in human cell lysates, and for Immunohistochemistry (IHC) to visualize nuclear localization with emphasis on heterochromatin regions.

How does phosphorylation at Thr145 affect HIST1H1E function in chromatin regulation?

Phosphorylation at threonine 145 (Thr145) significantly impacts HIST1H1E (histone H1.4) function in chromatin organization. This post-translational modification occurs in the C-terminal domain of H1.4, which is crucial for DNA binding and chromatin compaction . When Thr145 becomes phosphorylated, the positive charge of the C-terminal domain is reduced, weakening the electrostatic interactions with negatively charged DNA. This modification modulates H1.4's ability to neutralize linker DNA, resulting in altered chromatin accessibility and compaction states.

Research has demonstrated that Thr145 phosphorylation correlates with more open chromatin configurations, characterized by increased accessibility to nucleases and changes in nucleosome repeat length. Functionally, this phosphorylation has been shown to influence methylation patterns, particularly associated with a loss of the repressive H3K27me3 mark. The phosphorylation status of H1.4 changes throughout the cell cycle, increasing during S-phase and peaking during mitosis, suggesting its role in cell cycle-dependent chromatin reorganization . These changes in phosphorylation state help orchestrate the dynamic chromatin remodeling necessary for proper DNA replication, gene expression, and cell division.

What are the optimal ChIP protocol modifications for studying phosphorylated HIST1H1E in different cell types?

Optimizing ChIP protocols for phosphorylated HIST1H1E requires careful consideration of cell type-specific factors to ensure maximum sensitivity and specificity. For standard cell lines like HeLa cells, researchers should follow a protocol that involves treating approximately 4×10^6 cells with Micrococcal Nuclease to fragment chromatin, followed by immunoprecipitation with 5μg of the HIST1H1E (Ab-145) antibody . This baseline protocol has been validated for detecting binding to genomic regions such as the β-Globin promoter .

For rapidly dividing cell lines, researchers should implement a dual fragmentation approach: enzymatic digestion with Micrococcal Nuclease followed by mild sonication to achieve optimal fragment sizes of 200-500 bp. When working with primary cells or tissue samples, increasing the antibody amount to 7-10 μg per sample and extending incubation times (overnight at 4°C) can improve yield .

Critical modifications for phosphorylation-specific ChIP include adding phosphatase inhibitors (such as sodium fluoride, sodium orthovanadate, and β-glycerophosphate) to all buffers to prevent dephosphorylation during processing . Additionally, reducing the time between sample collection and processing is essential for preserving phosphorylation status. For comparative studies, performing parallel ChIP with a total H1.4 antibody allows calculation of phosphorylation enrichment ratios, providing more meaningful quantitative data about the phosphorylation state across different genomic regions.

How can researchers integrate HIST1H1E phosphorylation data with other epigenetic marks to understand chromatin state dynamics?

Integrating HIST1H1E phosphorylation data with other epigenetic marks requires a multi-dimensional approach to capture the complexity of chromatin regulation. Researchers should begin by performing sequential ChIP (Re-ChIP) experiments, first immunoprecipitating with the HIST1H1E (Ab-145) antibody followed by antibodies against other histone modifications of interest, such as H3K27me3 (repressive) or H3K4me3 (active marks) . This reveals co-occurrence patterns of phosphorylated H1.4 with other modifications at specific genomic loci.

Computational integration methods are essential for analyzing the relationship between phosphorylated H1.4 and other epigenetic marks. Correlation analyses between ChIP-seq datasets for phosphorylated H1.4 and various histone modifications can identify genomic regions where these marks show coordinated or antagonistic patterns. For example, research has shown that Thr145 phosphorylation often correlates with loss of the repressive H3K27me3 mark, suggesting a functional relationship in chromatin decompaction and gene activation.

To understand the temporal dynamics of these relationships, time-course experiments following stimulus application or cell cycle progression can reveal the sequential ordering of modifications and their interdependencies. This approach has shown that phosphorylation of H1.4 increases during S-phase and peaks during mitosis, correlating with specific changes in other histone modifications during cell cycle progression . Integrating these datasets with chromosome conformation capture techniques (such as Hi-C) further illuminates how phosphorylated H1.4 distribution relates to three-dimensional chromatin architecture and topologically associating domain (TAD) boundaries.

What advanced immunofluorescence techniques can reveal the nuclear distribution of phosphorylated HIST1H1E?

Advanced immunofluorescence techniques provide critical insights into the nuclear distribution and dynamics of phosphorylated HIST1H1E. The cytoskeletal (CSK) buffer extraction assay represents a sophisticated approach that differentiates between tightly chromatin-bound and loosely associated nuclear proteins . After CSK buffer extraction, cells are fixed and immunostained with the HIST1H1E (Ab-145) antibody to assess how phosphorylation affects chromatin binding stability and distribution patterns . This technique has been successfully applied to both fibroblasts and HeLa cells expressing Xpress-tagged HIST1H1E .

Super-resolution microscopy techniques such as Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), or Stochastic Optical Reconstruction Microscopy (STORM) overcome the diffraction limit of conventional microscopy, enabling visualization of phosphorylated H1.4 distribution at nanoscale resolution. These approaches can reveal the precise localization of phosphorylated H1.4 within heterochromatin versus euchromatin regions and in relation to nuclear compartments.

Multiplexed immunofluorescence combining the HIST1H1E (Ab-145) antibody with antibodies against other nuclear landmarks (such as nuclear lamina proteins, nucleolar markers, or other histone modifications) provides contextual information about phosphorylated H1.4 distribution. Fluorescence Recovery After Photobleaching (FRAP) using fluorescently tagged H1.4 combined with immunostaining for the phosphorylated form can reveal how phosphorylation affects the mobility and binding dynamics of H1.4 in living cells. For quantitative analysis, automated image analysis pipelines can segment nuclei based on DAPI staining and quantify the intensity, distribution patterns, and colocalization coefficients of phosphorylated H1.4 with other nuclear markers across large cell populations.

How can the HIST1H1E (Ab-145) Antibody be used to study Rahman Syndrome mechanisms?

Rahman Syndrome is a neurodevelopmental disorder characterized by intellectual disability, distinctive facial features, and growth abnormalities caused by heterozygous frameshift mutations in the HIST1H1E gene . The HIST1H1E (Ab-145) Antibody provides a valuable tool for investigating the molecular mechanisms underlying this condition. Research has revealed that a specific class of dominantly acting frameshift mutations affecting the C-terminal tail of HIST1H1E disrupts chromatin structure and contributes to cellular senescence and premature aging .

Using the antibody, researchers can compare phosphorylation patterns at Thr145 between patient-derived cells and controls to determine how these mutations affect post-translational modifications. Studies have shown that mutant HIST1H1E proteins resulting from frameshift mutations retain their ability to bind chromatin but compromise proper DNA compaction . Immunofluorescence microscopy using the antibody reveals aberrant nuclear distribution patterns of phosphorylated H1.4 in patient cells .

ChIP-seq approaches with the antibody can map genome-wide changes in phosphorylated H1.4 distribution in patient-derived cells, identifying dysregulated genomic regions that may contribute to the intellectual disability phenotype. Patients with HIST1H1E mutations commonly exhibit accelerated skeletal maturation, overgrowth, amblyopia, and camptodactyly, all observed in over 80% of cases . Correlating these clinical features with molecular findings using the antibody helps establish genotype-phenotype relationships and potential therapeutic targets. Functional studies have demonstrated that cells expressing mutant HIST1H1E exhibit dramatically reduced proliferation rates, rarely enter S-phase, and undergo accelerated senescence, providing a potential explanation for the premature aging observed in patients .

What role does HIST1H1E phosphorylation play in lymphoma development?

Recent studies have identified mutations in linker histone H1 genes, including HIST1H1E, as drivers of peripheral lymphoid malignancies . The HIST1H1E (Ab-145) Antibody enables investigation of how altered phosphorylation contributes to lymphomagenesis. Research has revealed diverse missense mutations in HIST1H1E in lymphomas that result in loss of nucleosome association and/or reduced capacity for chromatin compaction .

Integration of phosphorylated H1.4 binding patterns with other epigenetic marks has shown that HIST1H1E mutations in diffuse large B-cell lymphoma (DLBCL) correlate with altered H3K36me2 levels and activation of stem-cell gene programs . This suggests a mechanism where disrupted H1.4 phosphorylation contributes to malignant transformation through epigenetic reprogramming. Using lymphoma cell lines with engineered HIST1H1E mutations, researchers can investigate how specific mutations affect phosphorylation at Thr145 and subsequent chromatin remodeling, providing insights into potential therapeutic vulnerabilities.

How does aberrant HIST1H1E function contribute to cellular senescence and premature aging?

Research using the HIST1H1E (Ab-145) Antibody has revealed critical connections between altered HIST1H1E function, cellular senescence, and premature aging. Studies have demonstrated that a specific class of frameshift mutations affecting the C-terminal tail of HIST1H1E results in stable proteins that bind to chromatin but disrupt proper DNA compaction, leading to accelerated cellular senescence . This premature cellular aging has been identified as a previously unrecognized feature of HIST1H1E-related disorders .

Immunoblotting and immunofluorescence analysis using the antibody have shown altered phosphorylation patterns in cells expressing mutant HIST1H1E proteins . Functional characterization has revealed that these cells demonstrate dramatically reduced proliferation rates, hardly enter into S phase, and undergo accelerated senescence . To study this mechanism, the antibody can be used to track changes in phosphorylated H1.4 distribution during senescence progression in patient-derived fibroblasts compared to controls .

The CSK (cytoskeletal) buffer extraction assay, combined with immunostaining using the antibody, has been particularly valuable for assessing how mutations affect the chromatin binding stability of phosphorylated H1.4 . ChIP-seq approaches using the antibody can map genome-wide changes in phosphorylated H1.4 distribution during senescence, identifying critical regulatory regions where altered binding contributes to senescence-associated gene expression patterns. Clinical assessment of individuals with HIST1H1E mutations has established a direct link between aberrant chromatin remodeling (potentially involving altered phosphorylation patterns), cellular senescence, and accelerated aging , providing important insights into fundamental aging mechanisms and potential therapeutic targets for age-related conditions.

What controls should be included when using HIST1H1E (Ab-145) Antibody for phospho-specific detection?

When using the HIST1H1E (Ab-145) Antibody for phospho-specific detection, proper controls are essential to ensure data reliability and specificity. First, researchers should include a phosphatase treatment control, where parallel samples are treated with lambda phosphatase to remove phosphorylation modifications. This treatment should eliminate specific pThr145 signals, confirming the antibody's phospho-specificity. A peptide competition assay, where the antibody is pre-incubated with excess phospho-peptide corresponding to the Thr145 region before application to samples, serves as another critical specificity control.

For positive controls, researchers should include samples with known increased phosphorylation at Thr145, such as cells arrested in mitosis using nocodazole, as CDK1-mediated phosphorylation of H1.4 increases during this phase . Technically, researchers should perform immunoprecipitation with a control normal rabbit IgG alongside the HIST1H1E (Ab-145) Antibody to establish background binding levels . This approach has been validated in ChIP experiments with HeLa cells, where the antibody shows specific enrichment compared to IgG control .

When analyzing ChIP-seq data, include input DNA controls and use appropriate peak calling algorithms optimized for histone modifications . For Western blotting, run a lane with recombinant phosphorylated H1.4 protein as a positive control and use an antibody against total H1.4 (phosphorylation-independent) on parallel samples to normalize phospho-signal to total protein levels. When interpreting immunofluorescence results, compare the phospho-H1.4 signal distribution with total H1.4 distribution using a non-phospho-specific H1.4 antibody to distinguish specific phosphorylation patterns from total protein localization changes.

How can researchers preserve phosphorylation status during sample preparation?

Preserving phosphorylation status during sample preparation is critical for accurate detection with the HIST1H1E (Ab-145) Antibody. Phosphorylation modifications are notoriously labile and can be rapidly lost during experimental procedures. To maintain phosphorylation integrity, researchers should implement several key strategies. First, include phosphatase inhibitors in all buffers throughout the experimental workflow . A comprehensive inhibitor cocktail should contain sodium fluoride (10 mM), sodium orthovanadate (1 mM), and β-glycerophosphate (1 mM) to effectively block various phosphatase activities.

Sample processing should be performed at cold temperatures (4°C) whenever possible to reduce phosphatase activity . Minimize the time between sample collection and processing/fixation to prevent phosphorylation loss. For tissue samples, rapid freezing in liquid nitrogen immediately after collection is recommended. When preparing protein extracts for Western blotting, use specialized extraction methods that preserve phosphorylation, such as direct lysis in SDS sample buffer containing phosphatase inhibitors .

For immunofluorescence applications, optimize fixation conditions specifically for phospho-epitopes. A recommended approach is to fix cells with 3% paraformaldehyde followed by permeabilization with 0.5% Triton X-100 for 10 minutes at room temperature . This has been validated for detection of phosphorylated H1.4 in HeLa cells. For ChIP experiments, include phosphatase inhibitors in all buffers from cell collection through chromatin preparation and immunoprecipitation steps . Consider using phospho-preserving gels such as Phos-tag acrylamide gels for Western blotting, which can enhance separation and detection of phosphorylated proteins. Following these precautions ensures that the phosphorylation status of HIST1H1E at Thr145 remains intact throughout experimental procedures, allowing for reliable detection and quantification.

What are common sources of false positive and false negative results with this antibody?

Understanding potential sources of false results is crucial for accurate interpretation when using the HIST1H1E (Ab-145) Antibody. Common sources of false positive results include cross-reactivity with other phosphorylated histones, particularly other H1 variants with similar phosphorylation sites . The high sequence homology among histone H1 family members can lead to non-specific binding. Background signal from inadequate blocking can also generate false positives, especially in immunohistochemistry and immunofluorescence applications. This can be addressed by optimizing blocking conditions (5% BSA is often superior to milk-based blockers, as milk contains phosphatases that could dephosphorylate the target).

False negative results frequently stem from phosphorylation loss during sample preparation due to inadequate phosphatase inhibition or extended processing times at room temperature . The phospho-epitope at Thr145 is particularly labile and requires stringent preservation measures. Insufficient antigen retrieval in fixed tissues or cells can mask the phospho-epitope, rendering it inaccessible to the antibody. For formalin-fixed samples, optimized heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended.

Batch-to-batch variability of polyclonal antibodies can also contribute to inconsistent results . Researchers should validate each new lot against a reference sample with known phosphorylation status. Cell cycle-dependent fluctuations in H1.4 phosphorylation can lead to false negatives if samples are collected at cell cycle stages with naturally low phosphorylation levels . Synchronizing cells or accounting for cell cycle distribution can help address this variability. For ChIP applications, inadequate chromatin fragmentation or insufficient antibody amount (less than the recommended 5μg) may result in false negatives . Following validated protocols for Micrococcal Nuclease treatment and optimizing antibody concentration for each experimental system can minimize these issues.

How might HIST1H1E (Ab-145) Antibody be adapted for single-cell epigenomic analyses?

The adaptation of HIST1H1E (Ab-145) Antibody for single-cell epigenomic analyses represents a frontier in understanding chromatin regulation at unprecedented resolution. Researchers can modify traditional ChIP protocols for low-input applications by incorporating carrier chromatin (e.g., Drosophila chromatin) to improve recovery from small cell numbers . Microfluidic platforms can be integrated to process individual cells, with barcoding strategies allowing multiplexing of samples. These adaptations would enable the first single-cell maps of phosphorylated H1.4 distribution, revealing cell-to-cell variability in chromatin regulation.

Emerging techniques like CUT&Tag and CUT&RUN offer advantages over traditional ChIP for single-cell applications due to their higher sensitivity and improved signal-to-noise ratio. Adapting the HIST1H1E (Ab-145) Antibody for these methods would involve optimizing antibody concentration (typically 1:100 dilution) and incubation conditions (4-16 hours at 4°C) to maintain phospho-epitope detection specificity. These approaches could reveal how phosphorylated H1.4 distribution varies among individual cells within tissues from patients with conditions like Rahman Syndrome or lymphoma.

Integration with spatial technologies represents another promising direction. Combining the antibody with multiplexed immunofluorescence and in situ hybridization could correlate phosphorylated H1.4 patterns with specific genetic loci and gene expression in tissue context. For tissue sections from patients with HIST1H1E-related disorders, this approach could identify spatial patterns of chromatin dysregulation that contribute to tissue-specific pathology. The antibody could also be adapted for mass cytometry (CyTOF) by metal conjugation, enabling simultaneous profiling of phosphorylated H1.4 alongside multiple other chromatin marks and cellular proteins in individual cells. These emerging applications would provide unprecedented insights into how H1.4 phosphorylation contributes to cellular heterogeneity in development, aging, and disease states.

What therapeutic opportunities might emerge from studying HIST1H1E phosphorylation in disease?

Research using the HIST1H1E (Ab-145) Antibody has revealed potential therapeutic opportunities targeting histone H1.4 and its phosphorylation in various disorders. For Rahman Syndrome, understanding how frameshift mutations in the C-terminal domain affect phosphorylation patterns and chromatin compaction could lead to targeted interventions . Compounds that restore proper chromatin compaction in cells with HIST1H1E mutations could be identified through high-throughput screening using the antibody to detect changes in H1.4 phosphorylation patterns . Since cells expressing mutant HIST1H1E proteins demonstrate accelerated senescence, senescence pathway inhibitors might ameliorate premature aging phenotypes in patients .

In lymphoma, recurrent HIST1H1E mutations correlate with altered chromatin architecture and gene expression programs . The antibody facilitates evaluation of compounds that selectively affect cells with HIST1H1E mutations by monitoring phosphorylation-dependent changes in chromatin accessibility . This approach could identify synthetic lethal interactions specific to lymphoma cells with aberrant H1.4 function, leading to precision medicine strategies. Combination therapies targeting both HIST1H1E dysregulation and downstream pathways could be developed and assessed using the antibody to monitor treatment efficacy .

The antibody also enables biomarker development for patient stratification and response monitoring. Assays measuring phosphorylated H1.4 levels in liquid biopsies (circulating nucleosomes) or tissue samples could predict disease progression or treatment response. For cellular rejuvenation strategies, the antibody facilitates screening for compounds that restore youthful patterns of H1.4 phosphorylation in aging cells or prevent accelerated senescence in cells with aberrant H1.4 function . These approaches highlight the potential of targeting HIST1H1E phosphorylation as a novel intervention strategy for chromatin-related disorders ranging from rare genetic conditions to common diseases associated with epigenetic dysregulation.

How does HIST1H1E phosphorylation integrate with the broader histone code in gene regulation?

The integration of HIST1H1E phosphorylation with the broader histone code represents a complex regulatory network in gene expression control. Phosphorylation at Thr145 modulates H1.4's ability to neutralize linker DNA, influencing methylation patterns and chromatin accessibility . This modification acts as a molecular switch that coordinates with core histone modifications to regulate chromatin structure and function. Research using the HIST1H1E (Ab-145) Antibody has revealed that Thr145 phosphorylation often correlates with loss of the repressive H3K27me3 mark, suggesting a functional relationship in chromatin decompaction and gene activation.

Sequential ChIP (Re-ChIP) approaches using first the HIST1H1E (Ab-145) Antibody followed by antibodies against other histone modifications have shown co-occurrence patterns at specific genomic regions. These studies indicate that phosphorylated H1.4 preferentially associates with active chromatin regions marked by H3K4me3 and H3K36me3, while being depleted from regions with repressive marks like H3K9me3. This distribution pattern suggests phosphorylated H1.4 participates in maintaining open chromatin configurations necessary for transcriptional activity.

The temporal dynamics of H1.4 phosphorylation throughout the cell cycle provide another layer of integration with the histone code . Phosphorylation increases during S-phase and peaks during mitosis, correlating with specific changes in other histone modifications during cell cycle progression . This coordinated pattern enables proper chromatin reorganization necessary for DNA replication and cell division. In disease contexts like Rahman Syndrome and lymphoma, disruption of these integrated patterns due to HIST1H1E mutations leads to aberrant gene expression profiles . The resulting chromatin dysregulation contributes to pathological processes including accelerated senescence, intellectual disability, and malignant transformation . Understanding these integrated networks provides opportunities for developing targeted interventions that restore proper epigenetic regulation in various disease contexts.