HIST1H1C (Ab-45) Antibody

What is HIST1H1C and what cellular functions does it regulate?

HIST1H1C (H1.2) is an important variant of the linker histone H1 family that plays multifunctional roles in cellular processes. Research has demonstrated that HIST1H1C regulates several critical pathways:

• Immune response regulation through direct effects on interferon-β (IFN-β) expression

• Autophagy modulation via interactions with ATG proteins

• Epigenetic regulation through maintenance of H4K16 deacetylation status via SIRT1 and HDAC1 pathways

• Inflammatory response regulation, with effects on TNF-α and CXCL10

• Chromatin structure maintenance and DNA accessibility control

The protein contains specific domains that mediate nuclear interactions, with the C-terminal region being particularly important for interaction with viral proteins such as influenza NS2 . Recent studies show HIST1H1C upregulation under diabetic conditions and stress responses, suggesting its involvement in pathological processes .

How does HIST1H1C (Ab-45) Antibody recognize its target?

HIST1H1C (Ab-45) Antibody recognizes specific epitopes in the histone H1.2 protein with high specificity. The antibody binding characteristics include:

• Recognition of native and denatured forms of HIST1H1C protein

• Specific binding to unique sequences not shared with other H1 histone variants

• Ability to detect both unmodified and certain post-translationally modified forms

For optimal detection, researchers should note that antibody recognition may be affected by certain post-translational modifications of HIST1H1C, particularly those occurring at K34, K187, and T146 positions. These residues have been identified as critical for HIST1H1C functions in interferon regulation . Researchers should validate antibody specificity using appropriate controls, including HIST1H1C knockout cells as demonstrated in studies using A549-H1C-KO cell lines.

What experimental techniques can utilize HIST1H1C (Ab-45) Antibody?

HIST1H1C (Ab-45) Antibody can be employed across multiple research techniques:

Research shows this antibody is particularly valuable for investigating HIST1H1C interactions with viral proteins during infection and for examining its role in autophagy regulation under diabetic conditions .

What controls should be included when using HIST1H1C (Ab-45) Antibody?

Rigorous experimental design requires appropriate controls:

• Negative controls: HIST1H1C knockout cell lines (A549-H1C-KO) have been successfully generated using CRISPR/Cas9 and serve as excellent negative controls

• Knockdown controls: siRNA treatment specifically targeting HIST1H1C (with Si-NC and Si-GAPDH as appropriate controls)

• Overexpression controls: Plasmid vectors expressing HA-tagged HIST1H1C to confirm antibody specificity

• Cell type controls: Compare expression across multiple cell lines (documented in both rMC-1 and 293T cells)

• Loading controls: Standard housekeeping proteins for normalization in western blotting

Researchers should verify knockdown or overexpression efficiency through RT-PCR and western blotting as demonstrated in published protocols .

How can HIST1H1C (Ab-45) Antibody be used to study viral infection mechanisms?

HIST1H1C (Ab-45) Antibody serves as a critical tool for investigating host-pathogen interactions, particularly with influenza virus:

• Co-immunoprecipitation studies: HIST1H1C has been shown to interact with influenza virus NS2 via its C-terminal domain in the nucleus. The antibody can help isolate these protein complexes to study interaction mechanisms .

• Viral replication impact: Research demonstrates that H1N1 influenza virus replicates more efficiently in H1C knockout A549 cells compared to wild-type cells. The antibody can help quantify HIST1H1C levels and correlate them with viral protein expression .

• Signaling pathway analysis: HIST1H1C regulates antiviral IFN-β production by affecting IRF3 binding to the IFN-β promoter. The antibody can be used in ChIP assays to examine this regulatory interaction .

When designing experiments to study HIST1H1C in viral infections, researchers should:

• Examine both endogenous HIST1H1C and viral protein levels at multiple time points post-infection

• Compare results between wild-type and HIST1H1C-mutant cells (particularly K34A, K187A, and T146A mutants)

• Investigate downstream effects on antiviral cytokines including IFN-β, TNF-α, and CXCL10

What role does HIST1H1C play in autophagy regulation and how can the antibody help investigate this?

HIST1H1C has emerged as a critical regulator of autophagy, particularly in the context of diabetic retinopathy:

• Upregulation pattern: HIST1H1C and autophagy-related (ATG) proteins are both upregulated in the retinas of diabetic rodents and high-glucose cultured cells .

• Mechanistic pathway: HIST1H1C overexpression upregulates SIRT1 and HDAC1, maintaining H4K16 deacetylation status, which leads to increased ATG protein expression and enhanced autophagy .

• Downstream effects: HIST1H1C-induced autophagy is associated with inflammation, glial activation, and neuron loss in diabetic retinopathy models .

The HIST1H1C (Ab-45) Antibody can be used to:

• Quantify HIST1H1C expression levels in diabetic vs. normal tissues

• Track nuclear/cytoplasmic distribution of HIST1H1C under autophagy-inducing conditions

• Perform co-immunoprecipitation to identify HIST1H1C-binding partners in the autophagy pathway

• Monitor HIST1H1C levels in response to autophagy modulators

Experimental protocols should include autophagy flux assessment using chloroquine (CQ) or bafilomycin A1 (BafA1) treatments alongside HIST1H1C detection, as well as monitoring LC3B-I to LC3B-II conversion and SQSTM1/p62 degradation .

How do post-translational modifications of HIST1H1C affect its function and antibody detection?

Post-translational modifications (PTMs) dramatically alter HIST1H1C functions, and researchers should consider these when using antibodies:

Research demonstrates that:

• The HIST1H1C phosphorylation mutant (T146A) decreases IFN-β production

• HIST1H1C methylation mutants (K34A, K187A) increase IFN-β by releasing the nucleosome and promoting IRF3 binding to the IFN-β promoter

• These modifications directly impact viral replication, with K34A and K187A mutations significantly enhancing the inhibitory effect of HIST1H1C on virus replication

When studying HIST1H1C PTMs, researchers should consider using modification-specific antibodies alongside the general HIST1H1C (Ab-45) Antibody to comprehensively characterize the protein's functional state.

How can conflicting data on HIST1H1C function be reconciled in experimental design?

Researchers may encounter seemingly contradictory findings about HIST1H1C functions across different experimental systems:

• Cell-type specific effects: HIST1H1C functions differently in immune cells versus other cell types. In A549 cells, HIST1H1C inhibits viral replication through IFN-β regulation, while in retinal cells, it promotes autophagy and inflammation .

• Context-dependent functions: HIST1H1C can be:

  • Protective: Inhibiting viral replication through IFN-β upregulation

  • Pathological: Promoting inflammation and cell toxicity in diabetic retinopathy

• Distinguishing direct vs. indirect effects: When HIST1H1C influences multiple pathways (IFN-β, TNF-α, CXCL10), determining causality requires careful experimental design .

To reconcile these complexities:

• Use multiple cell types in parallel experiments

• Employ both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches

• Utilize HIST1H1C mutants (T146A, K34A, K187A) to dissect specific modification-dependent effects

• Conduct time-course experiments to distinguish between early and late effects of HIST1H1C modulation

• Employ pathway-specific inhibitors to delineate which downstream effects are direct or indirect

This comprehensive approach will help researchers distinguish context-dependent functions from core mechanistic roles of HIST1H1C.

What optimization strategies improve HIST1H1C (Ab-45) Antibody performance in immunoprecipitation?

Optimizing immunoprecipitation (IP) protocols for HIST1H1C requires attention to several parameters:

• Lysis buffer composition:

  • Use low-salt buffers (150mM NaCl) to preserve nuclear protein interactions

  • Include phosphatase inhibitors to maintain modification status of T146

  • Add deacetylase inhibitors if studying interactions with SIRT1/HDAC1

• Cross-linking considerations:

  • Formaldehyde cross-linking (1-2%) can preserve transient interactions

  • DSP (dithiobis[succinimidyl propionate]) cross-linking may better preserve nuclear complexes

• IP protocol refinements:

  • Pre-clear lysates with protein A/G beads for 1 hour at 4°C

  • Use 2-5μg antibody per 500μg protein lysate

  • Extend incubation time to overnight at 4°C with gentle rotation

  • Include detergent modulation (0.1-0.5% NP-40) to balance solubilization and interaction preservation

• Elution strategies:

  • For downstream functional assays: mild elution with competing peptide

  • For mass spectrometry: more stringent SDS elution

These optimizations are particularly important when studying HIST1H1C interactions with viral proteins like NS2 or cellular factors such as IRF3, which have been demonstrated to be functionally relevant in infection models .

How should the subcellular localization of HIST1H1C be investigated?

Proper investigation of HIST1H1C subcellular localization is critical given its context-dependent functions:

• Nuclear vs. cytoplasmic distribution: While primarily nuclear, HIST1H1C can translocate to the cytoplasm under certain conditions, though this was not observed in high glucose treatment or overexpression conditions in rMC-1 cells .

• Recommended fractionation protocol:

  • Harvest cells in isotonic buffer (10mM HEPES, pH 7.9, 10mM KCl, 1.5mM MgCl₂)

  • Swell cells on ice for 10 minutes

  • Add NP-40 to 0.3% final concentration

  • Vortex for 10 seconds

  • Centrifuge at 16,000g for 30 seconds

  • Collect cytoplasmic fraction (supernatant)

  • Wash nuclear pellet with isotonic buffer

  • Extract nuclear proteins with high-salt buffer (20mM HEPES, pH 7.9, 420mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 25% glycerol)

• Verification methods:

  • Immunofluorescence microscopy (1:200-1:500 dilution of HIST1H1C antibody)

  • Western blot analysis of fractionated samples

  • Include proper controls (Lamin B for nuclear fraction; GAPDH for cytoplasmic fraction)

Research has shown that nuclear/cytoplasmic fractionation assays and immunofluorescence staining consistently demonstrate nuclear enrichment of HIST1H1C in rMC-1 and 293T cells under normal and high glucose conditions .

What troubleshooting approaches resolve common issues with HIST1H1C (Ab-45) Antibody?

Researchers may encounter several technical challenges when working with HIST1H1C antibodies:

When troubleshooting HIST1H1C detection in experiments studying its role in IFN-β regulation or autophagy pathways, researchers should consider:

• Using mutant constructs (T146A, K34A, K187A) as controls to understand modification-specific detection issues

• Validating results with multiple detection methods (western blot, IF, RT-PCR)

• Including appropriate controls (knockout cells, siRNA knockdown)

How can ChIP-seq be optimized for studying HIST1H1C genome-wide binding patterns?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for HIST1H1C requires special considerations:

• Chromatin preparation:

  • Formaldehyde fixation time: optimize between 5-15 minutes at room temperature

  • Sonication conditions: aim for fragments between 200-500bp

  • Pre-clearing: extended pre-clearing with protein A/G beads (2 hours) reduces background

• Immunoprecipitation optimization:

  • Antibody amount: 5-10μg per ChIP reaction

  • Incubation time: overnight at 4°C with rotation

  • Washing stringency: gradually increase salt concentration in wash buffers

• Sequencing considerations:

  • Input normalization: sequence input chromatin at >10% depth of ChIP samples

  • Read depth: aim for minimum 20 million uniquely mapped reads

  • Replicate design: perform at least 3 biological replicates

• Data analysis pipeline:

  • Peak calling: use MACS2 with appropriate parameters for histone proteins

  • Motif analysis: examine co-occurrence with transcription factors like IRF3

  • Integration: correlate with RNA-seq data from HIST1H1C knockdown/overexpression experiments

This approach would be particularly valuable for studying how HIST1H1C regulates IFN-β by controlling IRF3 binding to the IFN-β promoter, as suggested by the research on its role in influenza virus inhibition .

What is known about HIST1H1C's role in interferon regulation during viral infection?

HIST1H1C plays a critical role in interferon regulation during viral infection:

• Antiviral mechanism: HIST1H1C enhances IFN-β production through interaction with IRF3, a key transcription factor in the interferon pathway. This provides a novel mechanism for host defense against influenza virus .

• Pathway specificity: While HIST1H1C strongly regulates IFN-β via IRF3, it has minimal effect on IFN-β activation by IRF7, demonstrating pathway selectivity .

• Viral counteraction: Influenza virus NS2 protein interacts with HIST1H1C via its C-terminal domain in the nucleus, which reduces HIST1H1C-IRF3 interaction and inhibits IFN-β production. This represents a viral evasion strategy .

• Modification-dependent regulation: The effects of HIST1H1C on IFN-β are modulated by specific post-translational modifications:

  • Phosphorylation at T146 decreases IFN-β production

  • Methylation at K34 and K187 increases IFN-β by releasing the nucleosome and promoting IRF3 binding to the IFN-β promoter

This regulatory mechanism functions across multiple signaling components, as HIST1H1C modulates IFN-β induction by RIG-I, MAVS, TBK-1, IKK-ξ, and IRF3, but not IRF7 .

How does HIST1H1C influence autophagy in diabetic retinopathy models?

HIST1H1C is a critical regulator of autophagy in diabetic retinopathy development:

• Expression pattern: Both HIST1H1C and ATG proteins are upregulated in retinas of diabetic rodents and in high-glucose cultured cells .

• Mechanistic pathway:

  • HIST1H1C overexpression upregulates SIRT1 and HDAC1

  • This maintains deacetylation status of H4K16

  • Leads to upregulation of ATG proteins

  • Promotes autophagy in cultured retinal cells

• Functional consequences: HIST1H1C-induced autophagy promotes:

  • Inflammation (increased CCL2, IL6 expression)

  • Glial activation (increased GFAP expression)

  • Reduced cell viability

  • Neuron loss in retinal tissue

• Interventional potential: Knockdown of histone Hist1h1c by siRNA in the retinas of diabetic mice significantly attenuated diabetes-induced autophagy, inflammation, glial activation, and neuron loss, suggesting therapeutic potential .

Importantly, HIST1H1C promotes cell death through a mechanism different from its previously reported function as an apoptosis mediator. In diabetic retinopathy models, HIST1H1C remains enriched in the nuclei rather than translocating to the cytoplasm, suggesting a distinct pathological mechanism .

What is the significance of HIST1H1C knockout/knockdown models in research?

HIST1H1C knockout and knockdown models provide critical insights into its functions:

These models reveal that:

• HIST1H1C knockout significantly enhances virus proliferation in A549 cells compared to wild-type cells

• Reintroduction of HIST1H1C in knockout cells restores inhibition of viral replication

• K34A and K187A mutations significantly enhance the inhibitory ability of HIST1H1C on virus replication

• T146A mutation relieves this inhibition

• In diabetic retinopathy models, HIST1H1C knockdown attenuates pathological autophagy and inflammation

These findings establish HIST1H1C as a potential therapeutic target for both viral infections and diabetic retinopathy.

How do experimental conditions affect HIST1H1C-dependent cellular responses?

The cellular responses regulated by HIST1H1C are highly dependent on experimental conditions:

Key variables affecting experimental outcomes:

• Cell type (A549 respiratory cells vs. rMC-1 retinal cells)

• Duration of stress conditions (acute vs. chronic)

• HIST1H1C post-translational modification status

• Presence of specific interaction partners (NS2, IRF3)