HIST1H1E (Ab-1) Antibody

What is the biological function of HIST1H1E (Histone H1.4) in chromatin organization?

HIST1H1E (Histone H1.4) functions as a linker histone protein critical for higher-order chromatin structure. It helps compress nucleosomes into more compact chromatin fibers, thereby regulating gene expression through chromatin accessibility modulation. The protein plays a vital role in genome organization by binding to linker DNA between nucleosomes, stabilizing the 30-nm chromatin fiber, and facilitating chromatin compaction . Recent research demonstrates that histone H1.4 participates in dysregulated gene expression when mutated, affecting genes important in neural development (such as GRIN1, GNG4, and ADY8), which explains some pathological manifestations in HIST1H1E syndrome .

What are the key post-translational modifications of HIST1H1E that can be detected by specialized antibodies?

Several post-translational modifications of HIST1H1E can be detected using specific antibodies:

These modifications regulate histone H1.4 function and interaction with chromatin and may serve as markers for specific cellular processes.

How should researchers select between phospho-specific HIST1H1E antibodies targeting Thr17 versus Thr18?

Selection between phospho-specific antibodies targeting Thr17 versus Thr18 on HIST1H1E should be guided by:

• Species compatibility: Thr18 antibodies (ABIN6256486) exhibit cross-reactivity with human, mouse, and rat samples, while Thr17 antibodies (ABIN7139625) are primarily reactive with human samples .

• Experimental application: Both can be used for ELISA, WB, and IF, but Thr18 antibodies have validated applications in IHC and ICC as well .

• Research focus: If investigating cellular stress responses or mechanical behavior coupling to chromatin organization, phosphorylation at Thr18 may be more relevant based on recent mechanistic studies .

• Specificity requirements: The phospho-Thr18 antibody detects endogenous levels of HIST1H1E only when phosphorylated at Thr18, allowing for specific detection of this modification state .

For optimal results, validation experiments comparing both antibodies in your specific experimental system would determine which provides superior signal-to-noise ratio and specificity.

What are the recommended protocols for using HIST1H1E antibodies in chromatin immunoprecipitation (ChIP) experiments?

For optimal ChIP experiments with HIST1H1E antibodies:

• Cross-linking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: Sonicate chromatin to achieve fragments of 200-500 bp, verifying fragment size by agarose gel electrophoresis.

• Antibody selection: Choose HIST1H1E antibodies validated for ChIP applications (several are listed in the search results) . Consider antibodies targeting specific modifications if investigating particular epigenetic states.

• Immunoprecipitation: Use 2-5 μg of antibody per ChIP reaction with 25-100 μg of chromatin. Include appropriate controls (IgG negative control, histone H3 positive control).

• Washing and elution: Perform stringent washing steps to reduce background. Elute protein-DNA complexes and reverse crosslinks before DNA purification.

• Analysis: Analyze enriched DNA by qPCR, sequencing, or array-based methods, comparing to input controls.

For studying HIST1H1E's role in telomere regulation, researchers should include telomeric repeat sequences in their analysis, as HIST1H1E has been implicated in telomere length regulation through RNA-RNA interactions .

How can researchers effectively use the mouse embryonic stem cell model for studying HIST1H1E mutations associated with Rahman syndrome?

To effectively use the mouse embryonic stem cell (mESC) model for studying HIST1H1E mutations:

• Model system: Utilize CRISPR/Cas9 engineered mESCs expressing human H1.4 variants under the endogenous promoter, as developed in recent studies . This approach allows for conditional expression of mutant H1.4 protein, mimicking physiological conditions.

• Visualization strategy: Take advantage of the PA-GFP and HA tag labeling system incorporated into the model to enable:

  • Monitoring of H1.4 exchange rates

  • Specific detection via immunofluorescence and western blotting

  • Pulldown assays to identify proteins interacting with mutant H1.4

• Functional assays: Implement growth curve analysis to assess proliferation effects by seeding approximately 50,000 cells and monitoring for 4 days, as described in the protocol from the mouse model study .

• Differentiation studies: Examine the impact of H1.4 mutations on neural differentiation, given the association with intellectual disability in humans with HIST1H1E syndrome.

• Chromatin analysis: Utilize the model to examine changes in chromatin organization, accessibility, and gene expression patterns resulting from the H1.4 mutation.

This model provides a valuable tool for studying the molecular mechanisms underlying HIST1H1E syndrome and potential therapeutic approaches .

What are the key phenotypic manifestations of HIST1H1E syndrome that can be investigated at the cellular level using HIST1H1E antibodies?

Key phenotypic manifestations of HIST1H1E syndrome that can be investigated at the cellular level include:

• Dysregulated gene expression: Using HIST1H1E antibodies for ChIP-seq experiments to identify alterations in chromatin binding patterns and consequent gene expression changes, particularly focusing on neural development genes such as GRIN1, GNG4, and ADY8 that are disrupted in the syndrome .

• Altered cellular proliferation: Investigate growth rates and cell cycle progression in cellular models using phospho-specific antibodies, as phosphorylation of HIST1H1E is cell-cycle regulated .

• Chromatin structural abnormalities: Employ immunofluorescence with HIST1H1E antibodies to examine changes in nuclear morphology and chromatin compaction .

• Transcriptional dysregulation: Combine HIST1H1E ChIP with RNA-seq to correlate chromatin binding patterns with gene expression changes in affected pathways.

• Cellular stress responses: Investigate potential alterations in mechanical stress responses that may be linked to the skeletal and cardiac abnormalities seen in the syndrome .

These investigations can provide insights into the molecular mechanisms underlying the intellectual disability, developmental delays, and other clinical features observed in individuals with HIST1H1E syndrome .

How does HIST1H1E interact with human telomerase RNA (hTR) and what are the implications for telomere maintenance?

HIST1H1E (or more specifically, its mRNA HIST1H1C) exhibits a novel non-coding RNA function in telomere regulation through specific interaction with human telomerase RNA (hTR):

• Interaction mechanism: The interaction occurs between the P6b stem-loop of hTR and a specific sequence in HIST1H1C mRNA called TRIAGE (located at nts 73-90). This interaction depends on sequence complementarity but also requires specific structural elements, as mutations that disrupt base-pairing abolish the interaction .

• Functional consequences:

  • Disruption of the hTR-HIST1H1C RNA association results in markedly increased telomere elongation without affecting telomerase enzymatic activity

  • Overexpression of HIST1H1C leads to telomere attrition

  • The regulatory effect occurs independently of HIST1H1C protein coding potential, revealing a non-canonical function for this mRNA

• Research methodology: This interaction can be studied using:

  • RNA antisense purification (RAP-RNA) with hTR-specific antisense oligonucleotides

  • Site-directed mutagenesis of the P6b stem-loop in hTR

  • qRT-PCR to measure pull-down efficiencies

  • Telomere length measurements using qTRAP (real-time quantitative telomeric repeat amplification protocol)

This discovery reveals an unexpected regulatory network involving HIST1H1E in telomere homeostasis beyond its traditional histone protein function and offers new avenues for investigating telomere-related disorders.

What methodological approaches should be used to distinguish the effects of different HIST1H1E isoforms in functional studies?

To distinguish the effects of different HIST1H1E isoforms in functional studies:

• Isoform-specific knockdown: Utilize siRNA or shRNA targeting unique regions of specific H1 isoforms. Research shows that individual knockdown of different H1 isoforms produces distinct phenotypes, with H1.0 being particularly crucial for fibroblast activation while H1.2 knockdown has no effect on TGF-β-induced gel contraction .

• Isoform-specific antibodies: Select antibodies that can differentiate between highly similar histone H1 variants. The search results mention various antibodies specific to HIST1H1E modifications that can be used for detection and functional studies .

• Temporal consideration in knockdown studies: Consider the timing of knockdown experiments; transient depletion may reveal roles that are compensated in germline knockouts. For example, if fibroblasts are already activated, H1.0 knockdown does not reverse TGF-β effects, but simultaneous knockdown blocks certain aspects of activation .

• Rescue experiments: Perform rescue experiments by re-expressing specific H1 isoforms in knockdown backgrounds to confirm isoform-specific functions.

• Chromatin binding analysis: Use ChIP-seq with isoform-specific antibodies to map the genomic distribution of different H1 variants and correlate with gene expression changes.

• Expression profile analysis: Quantify the relative abundance of different H1 isoforms in your experimental system, as the linker/core histone ratio can impact experimental outcomes .

These approaches can help delineate the unique roles of HIST1H1E among the histone H1 family, particularly in contexts where compensation mechanisms may mask isoform-specific functions.

What strategies can be employed to overcome low signal-to-noise ratios when using phospho-specific HIST1H1E antibodies in immunodetection methods?

To improve signal-to-noise ratios with phospho-specific HIST1H1E antibodies:

• Phosphatase inhibitors: Include comprehensive phosphatase inhibitor cocktails during sample preparation to preserve phosphorylation states. This is crucial for phospho-Thr17 and phospho-Thr18 detection .

• Antibody selection: Choose antibodies that have undergone rigorous affinity purification. For example, the phospho-Thr18 antibody (ABIN6256486) is purified via sequential chromatography on phospho- and non-phospho-peptide affinity columns, enhancing specificity .

• Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers) to identify optimal conditions. For phospho-epitopes, BSA is often preferred over milk-based blockers which contain phosphoproteins.

• Signal amplification: Consider tyramide signal amplification or polymer-based detection systems for immunohistochemistry and immunofluorescence.

• Sample enrichment: For low-abundance modifications, consider enriching phosphoproteins using phospho-protein enrichment kits before immunodetection.

• Positive controls: Include samples with known high levels of the phosphorylation (e.g., cells treated with phosphatase inhibitors or appropriate kinase activators).

• Antibody validation: Confirm specificity using peptide competition assays with phosphorylated and non-phosphorylated peptides to ensure the signal is specific to the phosphorylated form.

• Incubation conditions: Optimize antibody concentration, incubation time, and temperature. For challenging epitopes, longer incubations at 4°C may improve specific binding while reducing background.

Implementation of these strategies should significantly improve detection of phosphorylated HIST1H1E in experimental systems.

How can researchers design experiments to study the interaction between HIST1H1E modifications and cellular mechanical behaviors?

To design experiments investigating HIST1H1E modifications and cellular mechanical behaviors:

• Combined protein and mechanical analysis: Integrate techniques that measure chromatin compaction (using HIST1H1E antibodies) with those that assess cellular mechanics. Recent research shows that histone H1.0 (a related H1 isoform) regulates a wide range of mechanical behaviors including contractile force generation, cytoskeletal regulation, motility, and ECM deposition .

• Cell contraction assays: Implement collagen gel contraction assays while manipulating HIST1H1E levels or modifications. Monitor cell-generated forces using traction force microscopy .

• Cytoskeletal coupling analysis: Use co-immunoprecipitation with HIST1H1E antibodies followed by mass spectrometry to identify interactions with cytoskeletal regulatory proteins.

• Mechanical stimulation experiments:

  • Apply defined mechanical forces (stretching, compression, shear) to cells using microfluidic or substrate-based approaches

  • Measure the dynamic changes in HIST1H1E modifications using phospho-specific antibodies

  • Correlate mechanical stimuli with chromatin reorganization and gene expression changes

• Nuclear mechanics assessment: Implement nuclear deformation assays while modulating HIST1H1E levels or modifications to understand how chromatin compaction affects nuclear stiffness.

• Chromatin accessibility analysis: Combine mechanical stimulation with techniques like ATAC-seq to correlate HIST1H1E-mediated chromatin changes with mechanical responsiveness.

• Live-cell imaging: Utilize fluorescently tagged HIST1H1E constructs with targeted mutations to track dynamic changes in chromatin organization during mechanical stimulation in real-time.

This multidisciplinary approach will help elucidate how HIST1H1E modifications serve as intermediaries between cellular mechanical behaviors and chromatin regulation, potentially revealing mechanisms relevant to HIST1H1E syndrome pathophysiology .