HIST1H1E (Ab-3) Antibody

What is HIST1H1E and what cellular functions does it regulate?

HIST1H1E, also known as Histone H1.4, belongs to the linker histone H1 family and plays a crucial role in chromatin organization and compaction. It functions as a structural component that binds to nucleosomes and facilitates higher-order chromatin structure formation. Recent research has demonstrated that HIST1H1E is involved in regulating genome-wide chromatin organization that impacts cellular processes including transcriptional regulation, stress response, and cellular senescence .

The protein participates in DNA compaction by binding to linker DNA between nucleosomes, thereby stabilizing higher-order chromatin structures. Notably, studies have shown that mutations affecting the C-terminal tail of HIST1H1E can disrupt proper DNA compaction, leading to aberrant nuclear architecture and gene expression patterns . This disruption has been linked to accelerated cellular senescence and premature aging phenotypes in affected individuals.

What are the key specifications and properties of HIST1H1E (Ab-3) polyclonal antibody?

The HIST1H1E (Ab-3) polyclonal antibody is derived from rabbit hosts and specifically targets human Histone H1.4 (HIST1H1E). Its key specifications include:

• Antibody Type: Primary, polyclonal antibody

• Host Species: Rabbit

• Reactivity: Human (Homo sapiens)

• Isotype: IgG

• Label: Unconjugated

• Immunogen: Peptide sequence around the site of Threonine-3 (Thr3) from Human Histone H1.4

• Purification Method: Antigen affinity purified

The antibody recognizes the HIST1H1E protein, which is also known by several synonyms including H1 histone family member 4, H1.4, H14_HUMAN, H1E, H1F4, Histone 1 H1e, Histone cluster 1 H1e, and Histone H1B . This antibody has been validated for multiple applications including ELISA, Western Blotting, Immunohistochemistry, and Immunofluorescence techniques .

What experimental applications is the HIST1H1E (Ab-3) antibody suitable for?

The HIST1H1E (Ab-3) antibody has been validated for multiple laboratory applications, making it a versatile tool for histone research. The primary applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of HIST1H1E in solution samples.

• Western Blotting (WB): For detecting HIST1H1E protein in cell or tissue lysates, allowing analysis of protein expression levels and post-translational modifications.

• Immunohistochemistry (IHC): For visualizing HIST1H1E localization within tissue sections, enabling studies of expression patterns in different cell types and tissues.

• Immunofluorescence (IF): For high-resolution visualization of HIST1H1E localization within cells and subcellular compartments .

While the antibody has been validated for these applications, researchers should perform optimization experiments to determine ideal conditions for their specific experimental setups, including appropriate dilutions, incubation times, and blocking conditions.

How should proper sample preparation be conducted when working with HIST1H1E antibodies?

Effective sample preparation is crucial for successful experiments using HIST1H1E antibodies. The following methodological approaches are recommended:

For protein extraction and Western blotting:

• Extract histones using specialized acid extraction protocols to efficiently isolate histones from chromatin

• Include protease and phosphatase inhibitors during extraction if studying phosphorylated forms (such as pThr3, pThr17, or pThr18)

• Carefully control protein loading amounts (typically 10-20 μg per lane)

• Use fresh samples or properly stored aliquots to avoid freeze-thaw cycles

For immunofluorescence and immunohistochemistry:

• Fix cells or tissues with 4% paraformaldehyde for optimal preservation

• Consider mild permeabilization methods to maintain nuclear structure while allowing antibody access

• Include antigen retrieval steps when working with formalin-fixed paraffin-embedded tissues

• Block adequately with appropriate sera or BSA solutions to minimize non-specific binding

For all applications, including appropriate positive and negative controls is essential for validating experimental results and determining antibody specificity .

How can researchers distinguish between different post-translationally modified forms of HIST1H1E?

Distinguishing between different post-translationally modified forms of HIST1H1E requires careful experimental design and selection of appropriate antibodies. Several phosphorylated forms of HIST1H1E have been characterized, including phospho-Thr3, phospho-Thr17, and phospho-Thr18 .

To effectively distinguish between these modifications:

• Select modification-specific antibodies: Use antibodies that specifically recognize individual phosphorylation sites, such as anti-HIST1H1E (phospho-T3), anti-HIST1H1E (pThr17), or anti-HIST1H1E (pThr18) . These antibodies are designed to recognize the specific phosphorylated residue and surrounding sequence.

• Employ phosphatase controls: Treat parallel samples with lambda phosphatase to remove phosphate groups, confirming the signal is phosphorylation-dependent.

• Sequential immunoprecipitation approach: First, immunoprecipitate with a pan-HIST1H1E antibody, then probe with modification-specific antibodies to determine the proportion of the protein carrying specific modifications.

• Two-dimensional gel electrophoresis: Separate proteins first by isoelectric point and then by molecular weight to resolve differently modified forms of HIST1H1E.

• Mass spectrometry validation: Use mass spectrometry-based proteomics to confirm the presence and location of specific modifications, providing unambiguous identification of the modified residues .

When publishing results involving post-translational modifications, researchers should clearly document which specific modification is being detected and validate findings using complementary approaches.

What approaches should be used to investigate HIST1H1E's role in chromatin compaction and genome organization?

Investigating HIST1H1E's role in chromatin compaction and genome organization requires advanced methodological approaches that can detect changes in chromatin structure at different scales. Based on recent research findings, the following approaches are recommended:

• Chromatin Immunoprecipitation (ChIP) analysis: Use HIST1H1E antibodies for ChIP followed by sequencing (ChIP-seq) to map genome-wide distribution patterns. This helps identify which genomic regions are preferentially bound by HIST1H1E and how this binding correlates with gene expression or chromatin states .

• Chromosome Conformation Capture techniques: Methods like Hi-C, 4C, or Micro-C can reveal how HIST1H1E affects three-dimensional genome organization, including changes in topologically associating domains (TADs) and chromatin loops .

• ATAC-seq (Assay for Transposase-Accessible Chromatin): This technique can determine how HIST1H1E affects chromatin accessibility genome-wide. Comparing ATAC-seq profiles between wild-type and HIST1H1E-depleted or mutant cells can reveal regions where HIST1H1E regulates chromatin compaction .

• Live-cell imaging with fluorescently tagged HIST1H1E: This approach allows visualization of HIST1H1E dynamics in living cells and can be combined with techniques like FRAP (Fluorescence Recovery After Photobleaching) to study HIST1H1E mobility and binding kinetics.

• Electron microscopy of reconstituted chromatin: In vitro systems with purified components can be used to directly visualize how HIST1H1E affects chromatin fiber structure and compaction at the molecular level.

These approaches have revealed that HIST1H1E significantly contributes to genome compaction and organization, with mutations in its C-terminal tail disrupting proper DNA compaction and leading to altered gene expression profiles .

How should researchers troubleshoot non-specific binding or weak signals when using HIST1H1E antibodies?

When encountering non-specific binding or weak signals with HIST1H1E antibodies, systematic troubleshooting approaches should be implemented:

For non-specific binding issues:

• Optimize blocking conditions: Increase blocking time or try alternative blocking agents (5% BSA, 5% non-fat dry milk, or commercial blocking buffers).

• Adjust antibody concentration: Titrate the antibody to find the optimal dilution that maximizes specific signal while minimizing background.

• Increase washing stringency: Use higher concentrations of detergent (0.1-0.3% Tween-20 or Triton X-100) or increase the number and duration of washing steps.

• Pre-absorb the antibody: Incubate with negative control lysates to remove antibodies that might bind non-specifically.

• Consider cross-reactivity: Verify if the antibody cross-reacts with other histone H1 family members, particularly in multi-species studies.

For weak signal issues:

• Sample preparation: Ensure proper extraction of nuclear proteins, as inadequate extraction can result in low histone yields.

• Antigen retrieval: For fixed tissues or cells, optimize antigen retrieval methods (heat-induced or enzymatic) to improve epitope accessibility.

• Antibody incubation conditions: Try longer incubation times or incubating at different temperatures (4°C overnight versus room temperature).

• Detection system: Switch to more sensitive detection methods like enhanced chemiluminescence (ECL) Plus for Western blots or tyramide signal amplification for immunohistochemistry.

• Sample storage: Verify sample integrity, as degradation or improper storage can reduce antigen availability .

Documenting optimization steps and including appropriate positive controls (such as cells known to express high levels of HIST1H1E) will help validate the specificity and sensitivity of the antibody in your experimental system.

What is the relationship between HIST1H1E mutations and cellular senescence or premature aging?

Recent research has revealed a critical connection between HIST1H1E mutations, particularly frameshift mutations affecting the C-terminal tail, and accelerated cellular senescence leading to premature aging. This relationship involves several molecular mechanisms:

• Chromatin structure disruption: Frameshift mutations in the C-terminal tail of HIST1H1E result in proteins that still bind to chromatin but fail to properly compact DNA. This disruption in chromatin architecture leads to altered gene expression profiles and genomic instability .

• Cell cycle dysregulation: Cells expressing mutant HIST1H1E proteins show dramatically reduced proliferation rates and competence. These cells struggle to enter the S phase of the cell cycle, suggesting that proper HIST1H1E function is required for normal cell cycle progression .

• Accelerated senescence: The expression of mutant HIST1H1E triggers premature cellular senescence, a state where cells remain metabolically active but cease to divide. This accelerated senescence phenotype is accompanied by characteristic changes in cell morphology and gene expression patterns .

• Specific methylation profiles: HIST1H1E mutations are associated with distinct DNA methylation patterns, suggesting that the protein plays a role in regulating epigenetic modifications that influence gene expression and cellular aging .

• Clinical manifestations: In human patients, heterozygous frameshift mutations in HIST1H1E have been identified as causing a disorder characterized by premature aging features. This finding establishes a direct link between aberrant chromatin remodeling due to HIST1H1E dysfunction and accelerated aging phenotypes .

These discoveries highlight the importance of proper histone function in maintaining normal cellular aging processes and suggest that HIST1H1E might serve as a potential therapeutic target for age-related conditions.

How should controls be designed when using phospho-specific HIST1H1E antibodies?

When working with phospho-specific HIST1H1E antibodies, proper control design is crucial for result validation. The following control strategies are recommended:

• Phosphatase treatment controls:

  • Divide your sample into two portions

  • Treat one portion with lambda phosphatase to remove phosphate groups

  • The phospho-specific antibody should show reduced or no signal in the phosphatase-treated sample

  • This confirms signal specificity for the phosphorylated form

• Stimulation/inhibition controls:

  • Include samples from cells treated with phosphorylation-inducing stimuli (e.g., mitotic arrest for many histone phosphorylations)

  • Include samples treated with kinase inhibitors that target the relevant kinase

  • These manipulations should show respective increases or decreases in phospho-signal

• Genetic controls:

  • Where possible, include samples from cells expressing phospho-mutant HIST1H1E (e.g., T3A, T17A, or T18A)

  • These mutants should not be recognized by the corresponding phospho-specific antibody

  • This confirms epitope specificity

• Cross-validation:

  • Compare results using multiple phospho-specific antibodies targeting the same modification from different suppliers

  • Use complementary methods like mass spectrometry to validate phosphorylation status

  • This strengthens confidence in the observed results

Documenting these controls in publications is essential for demonstrating antibody specificity and result reliability, especially when making claims about phosphorylation-dependent functions of HIST1H1E.

What approaches are recommended for optimizing immunofluorescence protocols with HIST1H1E antibodies?

Optimizing immunofluorescence protocols for HIST1H1E antibodies requires careful attention to several parameters to achieve specific nuclear staining with minimal background. Based on research applications, the following optimization approaches are recommended:

• Fixation methods optimization:

  • Compare different fixatives (4% paraformaldehyde, methanol, or combination protocols)

  • Fixation time should be carefully controlled (typically 10-15 minutes)

  • For phospho-specific antibodies, phosphatase inhibitors should be included in fixation buffers

  • Crosslinking fixatives may better preserve nuclear architecture

• Permeabilization strategy:

  • Optimize detergent type and concentration (0.1-0.5% Triton X-100 or 0.1-0.3% Tween-20)

  • Consider detergent incubation time (5-15 minutes)

  • For dense heterochromatin regions, increased permeabilization may be necessary to allow antibody access

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Optimize blocking duration (1-2 hours at room temperature)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific binding

• Antibody dilution and incubation:

  • Create a dilution series to determine optimal antibody concentration

  • Compare room temperature (1-2 hours) versus 4°C overnight incubation

  • For co-staining experiments, verify antibody compatibility (same species issues)

• Signal amplification considerations:

  • For weak signals, consider tyramide signal amplification

  • Biotin-streptavidin systems can enhance detection sensitivity

  • Adjust exposure settings to prevent photobleaching during imaging

These methodological refinements should be systematically documented to enable reproducibility and reliable comparison between experimental conditions when studying HIST1H1E localization and dynamics.

How can researchers differentiate between different histone H1 variants in experimental settings?

Differentiating between histone H1 variants presents a significant challenge due to their sequence similarity. Researchers should employ a combination of techniques for reliable discrimination:

• Selection of highly specific antibodies:

  • Use antibodies raised against unique peptide regions of each H1 variant

  • Validate antibody specificity using knockout/knockdown controls or overexpression systems

  • Consider monoclonal antibodies for higher specificity when available

• Mass spectrometry approaches:

  • Employ liquid chromatography-tandem mass spectrometry (LC-MS/MS) to distinguish variants based on unique peptide signatures

  • Use isotopically labeled standards for absolute quantification of different variants

  • This approach provides the highest resolution for variant identification

• Genetic manipulation strategies:

  • Use CRISPR/Cas9 to tag specific variants with epitope tags or fluorescent proteins

  • Generate knockout/knockdown systems for individual variants to confirm antibody specificity

  • This allows tracking of specific variants without relying solely on antibody specificity

• Chromatin immunoprecipitation specificity controls:

  • Include peptide competition assays using synthetic peptides corresponding to the unique regions of each variant

  • Cross-validate ChIP results using multiple antibodies or tagged variants

  • Analyze enrichment patterns as different H1 variants may show distinct genomic distributions

• Expression pattern analysis:

  • Leverage the fact that different H1 variants show distinct tissue and developmental expression patterns

  • Use carefully selected cell types or developmental stages where certain variants predominate

  • This contextual approach can help isolate the functions of specific variants

Research has shown that while individual histone H1 isoforms may be dispensable for normal mouse development, combined loss of multiple variants (such as H1.3, H1.4, and H1.5) leads to developmental abnormalities, highlighting the importance of maintaining proper linker/core histone ratios through compensatory mechanisms .

How is HIST1H1E involved in cellular stress response mechanisms?

Recent research has revealed that HIST1H1E plays a significant role in cellular stress response pathways through its impact on chromatin organization and gene expression regulation. The molecular mechanisms include:

• Chromatin reorganization during stress:

  • HIST1H1E participates in genome-wide chromatin reorganization in response to cellular stressors

  • This reorganization facilitates transcriptional changes in cytoskeletal and extracellular matrix (ECM) genes

  • The protein appears to be particularly important for fibroblast activation in response to cytokine stimulation

• Genome compaction regulation:

  • HIST1H1E acts both locally to promote formation of more compact chromatin fibers and globally to condense the genome

  • This compaction dynamic regulates cellular deformability and mechanical properties

  • Under stress conditions, these changes in chromatin architecture influence cellular resilience and adaptability

• Transcriptional reprogramming:

  • HIST1H1E is required for cytokine-induced reprogramming of activating chromatin modifications, particularly histone H3 lysine 27 acetylation (H3K27Ac)

  • This epigenetic regulation affects the expression of genes involved in stress response pathways

  • The binding pattern of HIST1H1E may shift during stress, leading to altered gene accessibility and expression

• Cellular senescence induction:

  • Under certain stress conditions, HIST1H1E dysfunction can accelerate cellular senescence

  • This premature senescence represents a cellular response to genomic or epigenetic stress

  • The process involves global changes in chromatin organization and gene expression patterns

These findings highlight HIST1H1E as a critical link between chromatin structure, cellular mechanical properties, and stress response mechanisms, suggesting potential therapeutic targets for conditions involving aberrant stress responses or premature cellular aging.

What methodological approaches are used to study the role of HIST1H1E in chromatin dynamics?

Investigating HIST1H1E's role in chromatin dynamics requires specialized methodological approaches that span from molecular to cellular levels. Currently employed techniques include:

• Chromatin Accessibility Assays:

  • ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) to map open chromatin regions

  • DNase-seq or MNase-seq to assess nucleosome positioning and occupancy

  • These techniques help determine how HIST1H1E influences chromatin compaction and accessibility

• Chromosome Conformation Capture Technologies:

  • Hi-C to analyze genome-wide chromatin interactions and topologically associating domains (TADs)

  • 4C-seq to examine interactions between a specific locus and the rest of the genome

  • Micro-C for high-resolution nucleosome-level interaction maps

  • These approaches reveal how HIST1H1E affects three-dimensional genome organization

• Live Cell Imaging Techniques:

  • Fluorescence Recovery After Photobleaching (FRAP) to study HIST1H1E mobility and binding dynamics

  • Single-molecule tracking to analyze HIST1H1E behavior at the molecular level

  • These techniques provide insights into the kinetics of HIST1H1E-chromatin interactions

• Biochemical Reconstitution Systems:

  • In vitro chromatin assembly with purified components

  • Atomic Force Microscopy (AFM) or Electron Microscopy (EM) visualization of reconstituted chromatin fibers

  • These approaches allow direct observation of HIST1H1E's effect on chromatin structure

• Integrative Genomics Approaches:

  • ChIP-seq for HIST1H1E combined with RNA-seq to correlate binding with gene expression

  • Integration of histone modification data (H3K27Ac, H3K9me3, etc.) with HIST1H1E occupancy

  • Computational modeling of chromatin states based on multi-omics data

  • These integrative approaches provide comprehensive views of HIST1H1E's genomic functions

These methodologies have revealed that HIST1H1E is required for proper genome compaction and plays a crucial role in controlling cellular mechanical behaviors through chromatin organization, with implications for both normal development and disease states .

What is known about the interaction between HIST1H1E mutations and epigenetic modifications?

The relationship between HIST1H1E mutations and epigenetic modifications represents an emerging area of research with significant implications for understanding chromatin regulation and disease mechanisms:

• DNA methylation alterations:

  • Frameshift mutations in the C-terminal tail of HIST1H1E are associated with specific DNA methylation profiles

  • These altered methylation patterns may contribute to the aberrant gene expression seen in affected cells

  • The connection suggests that proper HIST1H1E function is required for maintaining normal DNA methylation landscapes

• Histone modification crosstalk:

  • HIST1H1E is required for proper reprogramming of histone H3 lysine 27 acetylation (H3K27Ac) in response to cytokine stimulation

  • This indicates a functional interplay between linker histones and core histone modifications

  • Disruption of this crosstalk through HIST1H1E mutations may lead to dysregulation of gene expression programs

• Chromatin accessibility changes:

  • Mutant HIST1H1E proteins that bind to chromatin but fail to properly compact DNA lead to altered chromatin accessibility

  • These accessibility changes affect the deposition and maintenance of histone modifications

  • The resulting epigenetic landscape changes contribute to cellular dysfunction and accelerated senescence

• Impact on heterochromatin formation:

  • HIST1H1E appears to play a role in heterochromatin formation and maintenance

  • Mutations may disrupt the establishment of repressive chromatin domains

  • This disruption could lead to inappropriate gene expression and genomic instability

• Developmental epigenetic programming:

  • The role of histone H1 variants in development suggests that HIST1H1E is involved in establishing tissue-specific epigenetic patterns

  • Mutations affecting this function could disrupt normal developmental programming

  • This disruption may contribute to the developmental abnormalities observed in affected individuals

These findings highlight the complex interplay between HIST1H1E function, epigenetic modifications, and cellular phenotypes, particularly in the context of premature aging and developmental disorders caused by HIST1H1E mutations .

What are the emerging applications for HIST1H1E antibodies in aging and senescence research?

HIST1H1E antibodies are becoming increasingly valuable tools in aging and cellular senescence research, with several promising applications emerging from recent discoveries. The connection between HIST1H1E mutations and premature aging has opened new avenues for investigating fundamental mechanisms of cellular aging and developing potential therapeutic interventions.

Emerging applications include monitoring HIST1H1E dynamics during cellular senescence, investigating the protein's role in age-related chromatin reorganization, and studying its interactions with other aging-associated factors. Research suggests that HIST1H1E antibodies will be crucial for mapping changes in histone variant distribution across the genome during aging and for understanding how these changes contribute to age-related cellular dysfunction .

Furthermore, the ability to detect specific post-translational modifications of HIST1H1E using specialized antibodies provides opportunities to investigate how these modifications change during aging and senescence. This could reveal new regulatory mechanisms and potentially identify therapeutic targets for age-related conditions .

What are the key considerations for designing experiments to investigate HIST1H1E in disease models?

When designing experiments to investigate HIST1H1E in disease models, researchers should consider several critical factors to ensure robust and reproducible results:

• Model system selection: Choose appropriate cell lines or animal models that recapitulate the specific disease features under investigation. For studying HIST1H1E mutations associated with premature aging, patient-derived cells or CRISPR-engineered models with the relevant frameshift mutations would be most informative .

• Experimental controls: Include wild-type controls alongside disease models, and consider using isogenic cell lines that differ only in HIST1H1E status to minimize confounding variables. For animal studies, littermate controls are essential .

• Temporal considerations: Design time-course experiments to capture the progressive nature of HIST1H1E-associated phenotypes, particularly for senescence studies where temporal dynamics are critical .

• Multi-omics approach: Combine techniques that assess chromatin structure (ATAC-seq, Hi-C), gene expression (RNA-seq), and epigenetic modifications (ChIP-seq) to obtain a comprehensive picture of HIST1H1E's role in disease contexts .

• Functional validation: Include functional assays that assess cellular phenotypes relevant to the disease, such as proliferation assays, senescence markers, or tissue-specific functional tests .