HIST1H1E (Ab-45) Antibody

What is the biological function of HIST1H1E?

HIST1H1E (Histone H1.4) belongs to the H1 histone family and serves as a linker histone that facilitates the condensation of nucleosome chains into higher-order structured chromatin fibers. Beyond its structural role, HIST1H1E acts as a regulator of individual gene expression through chromatin compaction and accessibility modulation . Linker histones, including HIST1H1E, function primarily as transcriptional repressors by limiting chromatin accessibility and contributing to genome organization . The protein plays a crucial role in maintaining genomic architecture, with disruption of its function leading to large-scale architectural remodeling of the genome characterized by shifts from compacted to relaxed chromatin states .

How do post-translational modifications affect HIST1H1E function?

HIST1H1E undergoes various post-translational modifications, including phosphorylation at threonine residues (Thr17, Thr18) and acetylation/methylation at multiple lysine residues (Lys16, Lys33, Lys51, Lys63) . These modifications regulate HIST1H1E binding to chromatin and its interactions with other nuclear proteins. Phosphorylation events, particularly at Thr17 and Thr18, are especially important during cell cycle progression and cellular responses to DNA damage, potentially altering the protein's ability to condense chromatin. Research employing site-specific antibodies has revealed that these modifications occur in distinct cellular contexts and contribute to the dynamic regulation of chromatin structure and gene expression programs.

What is the connection between HIST1H1E and human disease?

HIST1H1E mutations have emerged as significant drivers in two major disease categories. First, they are highly recurrent in B-cell lymphomas, where disruption of H1 function leads to profound architectural remodeling of the genome . HIST1H1E acts as a tumor suppressor, with its mutation driving malignant transformation through three-dimensional genome reorganization followed by epigenetic reprogramming and derepression of developmentally silenced genes . Second, likely gene-disrupting variants in HIST1H1E contribute to a wide range of neurodevelopmental phenotypes, including Rahman syndrome characterized by craniofacial abnormalities, intellectual disability, hypotonia, and autism/behavior problems . These findings establish HIST1H1E as a clinically significant gene with dual roles in cancer and neurodevelopment.

What experimental applications are HIST1H1E antibodies suitable for?

HIST1H1E antibodies are versatile research tools applicable across multiple experimental platforms. The available antibodies are validated for several applications including ELISA, Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC), and Chromatin Immunoprecipitation (ChIP) . Phospho-specific antibodies targeting modifications like pThr17 and pThr18 are particularly valuable for studying cell-cycle dependent regulation of HIST1H1E function. These antibodies enable researchers to detect endogenous levels of HIST1H1E in its various modified forms, providing insights into the dynamic regulation of chromatin structure in different cellular contexts and disease states.

How should I select the appropriate HIST1H1E antibody for my research?

Selection of the appropriate HIST1H1E antibody depends on several critical factors:

• Target modification: Determine whether you need to detect total HIST1H1E or specific post-translational modifications (e.g., pThr17, pThr18, acetylated lysines)

• Species reactivity: Confirm compatibility with your experimental model (human, mouse, rat)

• Application compatibility: Verify validation for your intended application (WB, IHC, IF, ChIP)

• Clonality: Choose between polyclonal antibodies (broader epitope recognition) or monoclonal antibodies (higher specificity)

• Binding region: Select antibodies targeting specific domains (e.g., C-terminal region where disease-associated mutations cluster)

For studies investigating histone modifications in the context of diseases like lymphoma or neurodevelopmental disorders, phospho-specific antibodies detecting pThr17 or pThr18 modifications may provide particularly relevant insights into disease mechanisms .

What controls should be included when using HIST1H1E antibodies?

Robust experimental design with HIST1H1E antibodies requires several essential controls:

• Positive control: Include samples known to express HIST1H1E (e.g., proliferating lymphocytes for phosphorylated forms)

• Negative control: Utilize HIST1H1E-depleted samples or tissues known not to express the target

• Blocking peptide control: Pre-incubate antibody with immunizing peptide to confirm specificity

• Phosphatase treatment: For phospho-specific antibodies, treat samples with phosphatase to demonstrate specificity

• Genetic controls: When possible, use HIST1H1E knockout/knockdown samples or cells expressing HIST1H1E mutations associated with diseases like Rahman syndrome

These controls help establish antibody specificity and validate experimental findings, particularly important when investigating the role of HIST1H1E in complex disease contexts involving chromatin remodeling.

How can I optimize Western blot protocols for HIST1H1E detection?

Western blot optimization for HIST1H1E detection requires attention to several critical parameters:

• Sample preparation: For histone extraction, use specialized acid extraction protocols to efficiently isolate histones from chromatin

• Gel selection: Use high percentage (12-15%) SDS-PAGE gels to adequately resolve the relatively small HIST1H1E protein (approximately 22 kDa)

• Transfer conditions: Optimize transfer time and voltage for efficient transfer of small proteins

• Blocking conditions: Test different blocking agents (BSA vs. milk) as milk may contain phosphatases that could interfere with phospho-specific antibody binding

• Antibody dilution: For phospho-specific HIST1H1E antibodies, typical working dilutions range from 1:500 to 1:2000, requiring optimization for each application

• Detection method: Consider using enhanced chemiluminescence for general detection or fluorescence-based methods for quantitative analysis

Validation of results with multiple antibodies targeting different epitopes can provide more comprehensive insights into HIST1H1E expression and modification patterns.

What considerations are important for immunofluorescence experiments using HIST1H1E antibodies?

Successful immunofluorescence with HIST1H1E antibodies requires attention to the following parameters:

• Fixation method: Optimize between paraformaldehyde (preserves architecture) and methanol (better for nuclear proteins) fixation

• Permeabilization: Ensure adequate nuclear permeabilization (Triton X-100 0.1-0.5%) for accessing nuclear histones

• Antigen retrieval: Consider heat-induced epitope retrieval for formalin-fixed tissues

• Blocking: Use BSA rather than milk to prevent phosphatase activity affecting phospho-specific antibodies

• Primary antibody incubation: Extend incubation time (overnight at 4°C) for optimal signal

• Controls: Include appropriate positive controls (e.g., proliferating cells for phosphorylated forms) and negative controls

• Co-staining: Consider co-staining with markers of nuclear architecture or cell cycle phase for contextual information

For HIST1H1E phospho-specific antibodies, special attention should be paid to cell cycle stage when interpreting results, as phosphorylation at Thr17 and Thr18 varies throughout the cell cycle .

What protocols are recommended for Chromatin Immunoprecipitation (ChIP) using HIST1H1E antibodies?

Chromatin immunoprecipitation with HIST1H1E antibodies requires specialized approaches:

• Crosslinking: Use formaldehyde crosslinking (typically 1%) for 10-15 minutes to preserve histone-DNA interactions

• Chromatin fragmentation: Optimize sonication conditions to achieve fragments of 200-500bp

• Pre-clearing: Implement thorough pre-clearing steps to reduce background

• Antibody amount: Use 2-5μg of HIST1H1E antibody per ChIP reaction

• Control antibodies: Include IgG negative control and positive control (e.g., H3K4me3 for active promoters)

• Washing stringency: Optimize wash conditions to balance between background reduction and signal retention

• DNA purification: Use specialized columns for efficient recovery of small DNA amounts

• Validation: Confirm enrichment by qPCR at known HIST1H1E binding sites before proceeding to genome-wide analysis

ChIP experiments with HIST1H1E antibodies are particularly valuable for understanding how HIST1H1E contributes to chromatin architecture and gene regulation in both normal and disease contexts, especially in lymphomas where HIST1H1E mutations drive malignant transformation .

How should I interpret conflicting results between different HIST1H1E antibodies?

Discrepancies between different HIST1H1E antibodies may arise from several factors:

• Epitope differences: Antibodies targeting different regions may yield varying results, especially if the protein undergoes proteolytic processing or harbors mutations

• Modification sensitivity: Antibodies may be differentially affected by post-translational modifications - phosphorylation at Thr17/Thr18 might affect binding of antibodies recognizing adjacent epitopes

• Cross-reactivity: Some antibodies may cross-react with other H1 family members due to sequence homology

• Application-specific performance: Antibodies optimized for Western blot may perform poorly in IHC or ChIP applications

• Lot-to-lot variation: Different production batches may show performance variations

To resolve conflicts, verify antibody specificity using multiple techniques (e.g., siRNA knockdown, knockout models), test multiple antibodies targeting different epitopes, and validate findings using complementary approaches like mass spectrometry for definitive identification of HIST1H1E modifications.

What are common pitfalls in analyzing HIST1H1E expression and modifications in disease models?

Research involving HIST1H1E in disease contexts presents several challenges:

• Cell heterogeneity: Variations in HIST1H1E expression or modification between different cell populations may obscure disease-relevant signals

• Modification dynamics: Phosphorylation states of HIST1H1E at Thr17 and Thr18 fluctuate during cell cycle, potentially confounding analysis if cell cycle stages are not synchronized or accounted for

• Context-dependent effects: HIST1H1E mutations may produce different phenotypes depending on genetic background and cellular context

• Technical artifacts: Inadequate controls, antibody cross-reactivity, or sample processing issues may lead to misinterpretation

• Causal relationship: Distinguishing whether HIST1H1E alterations are drivers or consequences of disease states requires careful experimental design

Address these challenges by implementing single-cell approaches, carefully controlling for cell cycle effects, using multiple complementary methods to validate findings, and employing genetic models to establish causality in disease mechanisms related to HIST1H1E dysfunction.

How can I differentiate between HIST1H1E and other H1 histone family members?

Distinguishing HIST1H1E from other H1 variants requires methodological precision:

• Antibody selection: Choose antibodies validated for specificity against HIST1H1E, particularly those targeting unique regions that differ from other H1 variants

• Validation methods: Confirm antibody specificity using knockout/knockdown models or overexpression systems

• Western blot resolution: Optimize gel conditions to resolve subtle molecular weight differences between H1 variants

• Mass spectrometry: Employ proteomic approaches for definitive identification of specific H1 variants and their modifications

• Gene-specific approaches: Use RT-qPCR with variant-specific primers to distinguish expression at the mRNA level

• ChIP-seq analysis: Examine binding patterns, as different H1 variants may show distinct genomic distribution patterns

This differentiation is particularly important when studying diseases like lymphomas or neurodevelopmental disorders, where specific HIST1H1E mutations have been identified as pathogenic while other H1 variants may have distinct roles .

How do HIST1H1E mutations contribute to lymphomagenesis?

HIST1H1E mutations play a critical role in lymphoma development through several mechanisms:

• Chromatin decompaction: Lymphoma-associated H1 mutations result in profound architectural remodeling of the genome characterized by large-scale shifts from compacted to relaxed chromatin states

• Epigenetic reprogramming: Loss of HIST1H1E function drives distinct changes in epigenetic states, primarily through gain of histone H3 lysine 36 dimethylation and/or loss of repressive H3 lysine 27 trimethylation

• Developmental gene derepression: These epigenetic changes unlock expression of stem cell genes that are normally silenced during early development

• Enhanced self-renewal: Loss of H1c and H1e alleles in mice conferred enhanced fitness and self-renewal properties to germinal center B-cells, ultimately leading to aggressive lymphoma with enhanced repopulating potential

These findings establish HIST1H1E as a bona fide tumor suppressor, whose mutation drives malignant transformation primarily through three-dimensional genome reorganization. Anti-HIST1H1E antibodies are essential tools for investigating these mechanisms by enabling detection of altered HIST1H1E levels, localization, and modifications in lymphoma samples.

What is the spectrum of HIST1H1E mutations in neurodevelopmental disorders?

HIST1H1E mutations associated with neurodevelopmental disorders display specific patterns:

• Mutation characteristics: Predominantly frameshift variants in the C-terminal domain, resulting in almost identical shorter proteins with a shared divergent C-terminal tail

• Mutational hotspots: Eight recurrent nonsense variant loci have been identified (Gly124, Lys139, Thr142, Ala144, Ala145, Thr146, Thr146, Lys148)

• Novel mutations: Recent research identified a new de novo frameshift mutation (c.416_419dupAGAA, p.Ala141GlufsTer56) in an individual with Rahman syndrome

• Genetic architecture: A comprehensive review of 23 variants across 52 patients revealed that likely gene‐disrupting variants in HIST1H1E contribute to a wide range of neurodevelopmental phenotypes

Research using HIST1H1E antibodies has been instrumental in characterizing how these mutations affect protein function, localization, and interaction with chromatin, providing insights into pathogenic mechanisms and potential therapeutic approaches for Rahman syndrome and related disorders.

How can HIST1H1E antibodies advance our understanding of chromatin biology and disease mechanisms?

HIST1H1E antibodies offer significant potential for advancing chromatin biology research:

• 3D genome architecture: Antibodies enable visualization of HIST1H1E distribution in relation to chromatin domains, helping elucidate how HIST1H1E regulates higher-order chromatin structure

• Modification dynamics: Phospho-specific antibodies for sites like Thr17 and Thr18 permit tracking of cell-cycle dependent modifications that regulate HIST1H1E function

• Disease mechanisms: Antibodies facilitate characterization of HIST1H1E alterations in lymphomas and neurodevelopmental disorders, connecting molecular changes to disease phenotypes

• Therapeutic development: High-quality antibodies support target validation and mechanism-of-action studies for potential therapies aimed at modulating chromatin structure

• Biomarker potential: HIST1H1E antibodies may enable development of diagnostic or prognostic biomarkers for diseases associated with HIST1H1E dysfunction

Future applications of HIST1H1E antibodies will likely expand to include spatial omics approaches that correlate HIST1H1E distribution with gene expression patterns and chromatin states in both normal development and disease contexts.

What are the latest methodological advances in studying HIST1H1E dynamics in live cells?

Recent technical innovations have expanded capabilities for analyzing HIST1H1E in living systems:

• Live-cell imaging: Development of fluorescently tagged HIST1H1E constructs that maintain physiological function allows real-time visualization of dynamics

• FRAP analysis: Fluorescence recovery after photobleaching techniques reveal HIST1H1E binding kinetics and mobility in different chromatin contexts

• Single-molecule tracking: Advanced microscopy approaches permit tracking of individual HIST1H1E molecules, revealing heterogeneity in binding dynamics

• Optogenetic approaches: Light-inducible systems enable precise temporal control of HIST1H1E recruitment to specific genomic loci

• Biosensors: Development of conformation-sensitive probes that report on HIST1H1E modification states in living cells

These methods provide dynamic information that complements static analyses using antibodies against endogenous HIST1H1E or its modified forms, offering integrated views of how HIST1H1E regulates chromatin in health and disease states.

How can integrative multi-omics approaches enhance our understanding of HIST1H1E function?

Integrative approaches combining multiple data types provide comprehensive insights into HIST1H1E biology:

• ChIP-seq + RNA-seq: Correlates HIST1H1E genomic binding with transcriptional output to identify direct regulatory targets

• ChIP-seq + Hi-C: Links HIST1H1E occupancy with three-dimensional genome architecture, particularly relevant given HIST1H1E's role in maintaining chromatin compaction

• CUT&RUN + ATAC-seq: Provides high-resolution mapping of HIST1H1E binding in relation to chromatin accessibility

• Proteomics + ChIP-seq: Identifies protein complexes associated with HIST1H1E at specific genomic locations

• Single-cell multi-omics: Reveals cell-to-cell variability in HIST1H1E distribution and function

Such integrative approaches are particularly valuable for understanding how HIST1H1E mutations in lymphoma or neurodevelopmental disorders affect multiple aspects of nuclear function, potentially revealing new therapeutic vulnerabilities.