HIST1H1C (Ab-80) Antibody

What is HIST1H1C and what are its primary functions in cellular biology?

HIST1H1C (Histone H1.2) is a linker histone variant that interacts with DNA between nucleosomes and mediates chromatin compaction into higher-order structures. Unlike core histones (H2A, H2B, H3, and H4), which form the nucleosome octamer, H1 variants like HIST1H1C bind to linker DNA and play key roles in:

• Maintenance of higher-order chromatin structure

• Regulation of gene expression

• Modulation of interferon-β (IFN-β) production during viral infections

• Epigenetic regulation through post-translational modifications

Recent findings demonstrate that HIST1H1C specifically interacts with influenza virus NS2 protein via its C-terminal domain in the nucleus, affecting viral replication. H1N1 influenza virus replicates more efficiently in HIST1H1C knockout A549 cells compared to wild-type cells, primarily due to HIST1H1C's regulation of interferon-β .

How does HIST1H1C differ from other H1 histone variants?

H1 histone variants show distinct characteristics despite structural similarities:

• HIST1H1C (H1.2) exhibits lower abundance than other variants at transcription start sites of inactive genes

• Promoters enriched in HIST1H1C tend to be repressed, differing from those enriched in other H1 variants

• HIST1H1C is uniquely enriched at chromosomal domains with low GC content

• HIST1H1C shows strong association with lamina-associated domains (LADs)

• HIST1H1C's genome-wide distribution pattern differs significantly from other variants, including H1.0 and H1X (the two structurally most distant variants within the somatic H1 family)

These differences suggest non-redundant, specialized functions for HIST1H1C compared to other H1 variants.

What are the recommended experimental protocols for HIST1H1C detection in different applications?

Based on validated methodologies from multiple sources, the following protocols are recommended:

Western Blotting (WB):

• Dilution range: 1:500-1:3000

• Expected molecular weight: 32-33 kDa (despite calculated MW of 21 kDa)

• Best results observed in Jurkat cells, L02 cells, MCF-7 cells, and human testis tissue

Chromatin Immunoprecipitation (ChIP):

• For HIST1H1C-specific ChIP-qPCR: Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Buffer composition: Standard ChIP buffers containing protease and phosphatase inhibitors

• Cross-linking recommendation: 1% formaldehyde for 10 minutes at room temperature

• Sonication parameters: Optimize to achieve fragments of 200-500 bp

Immunohistochemistry (IHC):

• Dilution range: 1:100-1:600

• Antigen retrieval: TE buffer pH 9.0 (alternatively citrate buffer pH 6.0)

• Best results observed in human ovary tumor tissue and normal colon tissue

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Dilution range: 1:50-1:500

• Positive detection validated in HeLa cells, HepG2 cells, and MCF-7 cells

How can researchers generate and validate HIST1H1C knockout cell lines for functional studies?

The CRISPR/Cas9 system has been successfully employed to generate HIST1H1C knockout cell lines. Based on published methodologies:

• Guide Sequence Design:

  • Example successful guide sequence: 5'-AACCAATGTCACCGGCGCCGGCC-3' (forward)

  • Example successful guide sequence: 5'-TTGGTTACAGTGGCCGCGGCCGG-3' (reverse)

• Transfection and Selection Protocol:

  • Transfect target cells (e.g., A549) with px335-H1C plasmid

  • Culture transfected cells for 2 days

  • Repeat transfection

  • Digest cells, dilute highly, and culture in multi-well plates until monolayer growth

  • Analyze cells by PCR and Western blotting to confirm knockout

• Validation Methods:

  • PCR verification of genetic modification

  • Western blot confirmation of protein absence (multiple antibodies recommended)

  • Functional assays relevant to research question

  • Rescue experiments by reintroducing wild-type HIST1H1C

The H1C-KO A549 cell line has demonstrated increased susceptibility to influenza virus replication, with the virus proliferating more robustly compared to wild-type cells. This phenotype can be reversed by reintroducing HIST1H1C expression .

How should researchers address the challenges of HIST1H1C antibody specificity and cross-reactivity?

HIST1H1C antibody specificity presents significant challenges due to:

• High sequence similarity among H1 variants

• Various post-translational modifications affecting epitope recognition

• Limited evolutionary conservation compared to core histones

Recommended validation approaches:

• Multiple antibody validation:

  • Test antibodies targeting different regions (N-terminal, middle region, C-terminal)

  • Compare results from at least two different antibodies against the same target

  • Use antibodies from different host species when possible

• Knockout controls:

  • Include HIST1H1C knockout cell lysates as negative controls

  • Compare staining/signal patterns between wild-type and knockout samples

  • Example: Western blot validation shows absence of band at ~32-33 kDa in HIST1H1C KO HeLa lysate

• Specificity testing:

  • Test reactivity against recombinant H1 variants

  • Perform peptide competition assays

  • Analyze predicted cross-reactivity: Most HIST1H1C antibodies show 85-93% predicted reactivity with mouse, rat, cow, dog, guinea pig, horse, and rabbit orthologs

• Application-specific validation:

  • For ChIP applications: Perform sequential ChIP or ChIP-reChIP

  • For IF/IHC: Include peptide blocking controls

  • For WB: Run gradient gels to ensure separation of similarly sized H1 variants

What post-translational modifications of HIST1H1C are relevant for research, and how can they be detected?

HIST1H1C undergoes various post-translational modifications that affect its function in chromatin regulation and immune responses:

Key PTMs with functional significance:

• Phosphorylation:

  • T146 phosphorylation decreases IFN-β expression

  • Detection: Anti-H1-T146p antibody (e.g., Abcam ab3596)

• Methylation:

  • K34 and K187 methylation mutants increase IFN-β by promoting IRF3 binding to the IFN-β promoter

  • K45 methylation affects chromatin binding

  • Detection: Anti-HIST1H1C (meLys45), Anti-HIST1H1C (meLys96), Anti-HIST1H1C (meLys186) antibodies

• Acetylation:

  • Multiple acetylation sites (K16, K62, K84, K96) with distinct functions

  • Detection: Modification-specific antibodies such as anti-HIST1H1C (acLys16), anti-HIST1H1C (acLys62), anti-HIST1H1C (acLys84), anti-HIST1H1C (acLys96)

Methodological considerations for PTM detection:

• For site-specific modification detection, use PTM-specific antibodies in WB, ChIP, or IF

• Mutational studies (K→A or T→A) can help determine functional significance

• Mass spectrometry provides comprehensive PTM profiling but has limitations with histone H1 analysis

• ChIP-seq with PTM-specific antibodies can map genomic locations of modified HIST1H1C

Research has shown that HIST1H1C phosphorylation mutant (T146A) decreases IFN-β, while methylation mutants (K34A, K187A) increase IFN-β by releasing the nucleosome and promoting IRF3 binding to the IFN-β promoter .

How does HIST1H1C contribute to antiviral immune responses and interferon regulation?

Recent research has uncovered a novel role for HIST1H1C in regulating interferons and antiviral immunity:

• HIST1H1C regulation of IFN-β:

  • HIST1H1C significantly upregulates IFN-β expression

  • This effect is enhanced by K34A and K187A mutations

  • T146A mutation significantly decreases this upregulation

  • H1C knockout cells show increased susceptibility to influenza virus infection

• Mechanistic pathway:

  • HIST1H1C affects IFN-β stimulated by RIG-I, MAVS, TBK-1, IKK-ξ, and IRF3

  • HIST1H1C specifically regulates IFN-β via IRF3, not IRF7

  • NS2 protein from influenza virus interacts with HIST1H1C, reducing H1C-IRF3 interaction

  • This interaction inhibits IFN-β enhancement by HIST1H1C

• Impact on other cytokines:

  • HIST1H1C significantly increases TNF-α expression

  • HIST1H1C expression increases CXCL10 levels

  • Limited effect on MX1, OASL, IL-8, and IL-6 production

These findings suggest HIST1H1C plays multifaceted roles in immune regulation beyond structural chromatin functions.

What are the implications of combined HIST1H1C and H1.4 depletion for cancer research?

Combined depletion of HIST1H1C (H1.2) and H1.4 produces dramatic effects not observed with individual knockdowns:

• Interferon response activation:

  • Strong upregulation of interferon-stimulated genes (ISGs)

  • Activation of IFN signaling transducers

  • Involvement of cytosolic nucleic acid receptors

  • Induction of IFN synthesis

• Chromatin effects:

  • Upregulation of satellites and endogenous retroviruses

  • Increased cytosolic RNA

  • Compromised heterochromatin integrity

• Cancer relevance:

  • Pancreatic carcinomas with constitutively induced IFN response express low levels of several H1 variants

  • Strong deleterious effect on cancer cell growth when both variants are depleted

  • Potentially targetable vulnerability in certain cancer types

These findings demonstrate the importance of histone H1 variants in maintaining heterochromatin integrity and preventing potentially growth-inhibiting IFN responses in cancer cells.

Why do HIST1H1C antibodies often detect proteins at approximately 32-33 kDa instead of the calculated 21 kDa in Western blotting?

This common observation represents a technical challenge in HIST1H1C research:

Factors contributing to the molecular weight discrepancy:

• Post-translational modifications:

  • Multiple phosphorylation, methylation, and acetylation sites increase apparent molecular weight

  • Different cell types may exhibit different modification patterns

• Structural properties:

  • Highly basic proteins like histones often migrate aberrantly on SDS-PAGE

  • The unstructured N- and C-terminal tails affect electrophoretic mobility

  • Histone H1 variants are known to migrate at approximately 30-33 kDa despite their calculated masses of ~21 kDa

• Technical validation:

  • Multiple independent antibodies detect the same 32-33 kDa band (e.g., ab17677 and ab4086)

  • This band is absent in HIST1H1C knockout cell lysates

  • Similar migration patterns are observed across different cell types and tissues

Recommended approaches:

• Include recombinant protein standards of known molecular weight

• Use HIST1H1C knockout cells as negative controls

• Consider alternative percentage gels (15-18%) for better separation

• Validate with at least two different antibodies targeting separate epitopes

How can researchers effectively map HIST1H1C genomic distribution and distinguish it from other H1 variants?

Given the challenges in distinguishing H1 variants in genomic studies, these methodological approaches have proven effective:

• ChIP-seq optimization:

  • Use highly specific antibodies validated for ChIP applications

  • Include appropriate controls (input DNA, IgG controls)

  • Consider spike-in normalization for quantitative comparisons

  • Apply stringent peak calling parameters

• Alternative approaches:

  • HA-tagged recombinant H1 variants expressed in cell lines

  • DamID technology for mapping genomic locations

  • CUT&RUN or CUT&Tag for higher resolution and lower background

• Bioinformatic analysis:

  • Focus on differential enrichment patterns between variants

  • Analyze association with specific chromatin features (LADs, GC content)

  • Compare with transcriptional status of associated genes

  • Examine correlation with histone modifications

Research using these approaches has revealed that HIST1H1C enrichment correlates most closely with gene repression, structural domains of chromatin such as lamina-associated domains (LADs), and regions of low GC content, distinguishing it from other H1 variants .