Mono-methyl-HIST1H1C (K186) Antibody

What is Mono-methyl-HIST1H1C (K186) Antibody and what does it specifically detect?

The Mono-methyl-HIST1H1C (K186) Antibody is a rabbit-derived polyclonal antibody that specifically recognizes the mono-methylation modification at lysine 186 (K186) of histone H1.2 (HIST1H1C) protein. This antibody targets the human HIST1H1C protein, which is also known by several synonyms including H1 histone family member 2, H1.2, H1F2, and Histone H1c . The specificity of this antibody is determined by its generation against a synthetic peptide sequence surrounding the mono-methylated K186 site derived from human Histone H1.2 . Unlike antibodies that detect total HIST1H1C protein regardless of modification status, this antibody specifically binds to the protein only when mono-methylated at the K186 position, making it valuable for studying this particular post-translational modification.

What are the validated applications for Mono-methyl-HIST1H1C (K186) Antibody?

The Mono-methyl-HIST1H1C (K186) Antibody has been validated for multiple research applications:

These applications have been experimentally validated , making this antibody a versatile tool for investigating mono-methylated HIST1H1C in various experimental contexts.

What is the biological significance of histone H1.2 methylation?

Histone H1.2 methylation represents an important regulatory mechanism in chromatin biology. Linker histones such as H1.2 influence nucleosome positioning, chromatin compaction, chromosome structural integrity during mitosis, and higher-order chromatin structure maintenance . Specifically, HIST1H1C (H1.2) methylation has been implicated in:

• Regulation of gene expression through altered chromatin accessibility

• Cellular differentiation processes

• Development of certain cancer phenotypes, particularly stemness features

Recent research has demonstrated that methylation of histone H1 by the methyltransferase WHSC1 (albeit at K85 rather than K186) induces stem cell-like features in squamous cell carcinoma of the head and neck (SCCHN) . This suggests that histone H1 methylation may play crucial roles in determining cell fate and contributing to disease progression. The specific role of K186 mono-methylation is still being investigated, but it likely participates in similar regulatory mechanisms affecting chromatin structure and gene expression patterns.

How does HIST1H1C methylation contribute to chromatin organization and gene regulation?

HIST1H1C methylation significantly impacts chromatin organization through several mechanisms:

The binding of histone H1 to linker DNA typically leads to more compacted chromatin, decreasing accessibility to regulatory proteins, chromatin remodeling factors, and histone modifiers . Methylation of HIST1H1C can alter this binding affinity, thereby modulating chromatin compaction states. Research suggests that methylated H1 histones may have differential binding properties compared to their unmethylated counterparts.

At the gene expression level, HIST1H1C methylation has been shown to influence transcription in a context-dependent manner. While histone H1 is traditionally associated with transcriptional repression through chromatin compaction, studies indicate that specific methylation patterns can also lead to transcriptional activation of certain genes . For example, WHSC1-mediated mono-methylation of histone H1.4 (a related H1 variant) at K85 has been shown to induce transcriptional activation of OCT4 and stemness features in SCCHN cells .

The specific effects of K186 mono-methylation may differ from other methylation sites, emphasizing the importance of site-specific antibodies like the Mono-methyl-HIST1H1C (K186) Antibody for distinguishing these unique modification patterns.

What methodological considerations are crucial for ChIP experiments using Mono-methyl-HIST1H1C (K186) Antibody?

When performing Chromatin Immunoprecipitation (ChIP) with the Mono-methyl-HIST1H1C (K186) Antibody, researchers should consider several critical factors:

Recent ChIP protocols have demonstrated successful immunoprecipitation of methylated histone H1 variants using buffers containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100 for chromatin preparation, followed by washes with increasing salt concentrations .

How does WHSC1-mediated methylation of histone H1 impact cellular phenotypes?

Research has revealed that WHSC1 (Wolf-Hirschhorn syndrome candidate 1), a protein lysine methyltransferase, can mono-methylate histone H1 at K85 . This specific methylation has significant implications for cellular phenotypes:

• Stem cell-like features: WHSC1-mediated H1 methylation induces stemness characteristics in squamous cell carcinoma of the head and neck (SCCHN) cells .

• Transcriptional activation: Unlike the generally repressive role of unmodified H1, WHSC1-methylated H1 contributes to the transcriptional activation of certain genes, particularly OCT4, a key pluripotency factor .

• Cancer progression: The methylation of H1 by WHSC1 may contribute to cancer progression by promoting stem cell-like features that enhance tumor aggressiveness and treatment resistance .

While the studies cited specifically examined K85 methylation rather than K186, they demonstrate the principle that specific methylation events on histone H1 can dramatically alter cellular phenotypes. The Mono-methyl-HIST1H1C (K186) Antibody enables researchers to investigate whether similar phenotypic consequences result from methylation at the K186 position, potentially revealing novel regulatory mechanisms.

How should I validate the specificity of Mono-methyl-HIST1H1C (K186) Antibody in my experimental system?

Validating antibody specificity is crucial for reliable research findings. For the Mono-methyl-HIST1H1C (K186) Antibody, consider implementing these validation strategies:

• Peptide competition assay: Pre-incubate the antibody with excess:

  • Mono-methylated K186 peptide (should eliminate signal)

  • Unmethylated K186 peptide (should not affect signal)

  • Peptides with other methylation states (di/tri) or at different lysine positions

• Genetic approaches:

  • siRNA/shRNA knockdown of HIST1H1C (should reduce signal)

  • Overexpression of wild-type vs. K186A mutant HIST1H1C (mutant should show no signal)

  • Knockdown of methyltransferases that target HIST1H1C

• Mass spectrometry correlation: Confirm the presence of mono-methylation at K186 using mass spectrometry and correlate with antibody detection.

• Positive and negative controls:

  • Cell lines known to have high vs. low levels of HIST1H1C K186 methylation

  • Recombinant methylated vs. unmethylated proteins

A validation strategy similar to that used for WHSC1-mediated H1 methylation can be adapted, where researchers generated stable cell lines expressing FLAG-tagged wild-type H1.4 versus K85A mutant H1.4 to confirm methylation site specificity .

What are optimal Western blot protocols for detecting mono-methylated HIST1H1C (K186)?

For optimal detection of mono-methylated HIST1H1C (K186) by Western blot:

Sample preparation:

• Extract nuclear proteins using a dedicated nuclear extraction kit (e.g., Active Motif)

• Include protease inhibitors, phosphatase inhibitors, and deacetylase inhibitors

• Add 5-10 mM sodium butyrate to preserve histone modifications

• Use acid extraction protocols (0.2N HCl) for enrichment of histone proteins

Gel and transfer conditions:

• Use 15% SDS-PAGE gels to properly resolve histone proteins (~30 kDa for H1)

• Transfer to PVDF membranes at lower voltage (30V) overnight at 4°C

• Fix proteins on membrane with 0.2% glutaraldehyde in PBS for 30 minutes before blocking

Blocking and antibody incubation:

• Block with 5% BSA (not milk) in TBST

• Dilute primary antibody 1:500 to 1:2000 in blocking buffer

• Incubate overnight at 4°C with gentle rocking

• Use 3-5 TBST washes (10 minutes each)

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000)

Controls and verification:

• Run recombinant methylated and unmethylated standards

• Include positive control lysates from cells known to express the modification

• Consider stripping and reprobing with total HIST1H1C antibody to normalize signals

Using these optimized conditions will maximize sensitivity and specificity for detecting mono-methylated HIST1H1C (K186).

What controls are essential for immunofluorescence experiments with this antibody?

When performing immunofluorescence (IF) with the Mono-methyl-HIST1H1C (K186) Antibody, include these essential controls:

• Primary antibody controls:

  • Omission control (secondary antibody only)

  • Isotype control (non-specific IgG from same species)

  • Peptide competition (pre-incubation with mono-methylated K186 peptide)

  • Concentration gradient to determine optimal dilution (typically 1:100-1:500)

• Biological controls:

  • Cells with HIST1H1C knockdown

  • Cells treated with methyltransferase inhibitors

  • Cells expressing K186A mutant HIST1H1C (negative control)

  • Cells with known high expression of methylated HIST1H1C (positive control)

• Technical controls:

  • Nuclear counterstain (DAPI or Hoechst)

  • Additional histone markers for co-localization studies

  • Multiple fixation methods comparison (4% PFA vs. methanol)

• Validation approaches:

  • Parallel Western blot to confirm specificity

  • Z-stack imaging to confirm nuclear localization

  • Super-resolution microscopy to examine subnuclear distribution patterns

For optimal results, use 4% paraformaldehyde fixation for 15 minutes followed by 0.1% Triton X-100 permeabilization for 10 minutes, as this preserves nuclear architecture while allowing antibody access to nuclear proteins.

Why might I observe non-specific bands when using this antibody in Western blot?

Non-specific bands are a common challenge when working with histone modification antibodies. Potential causes and solutions include:

When comparing knockdown experiments with WHSC1 siRNA, researchers observed a significant reduction in the specific bands corresponding to methylated H1, confirming antibody specificity . Similar approaches can validate the mono-methyl-HIST1H1C (K186) Antibody.

How can I study the relationship between HIST1H1C methylation and gene expression?

To investigate how HIST1H1C methylation affects gene expression:

• Genome-wide approaches:

  • ChIP-seq using Mono-methyl-HIST1H1C (K186) Antibody to map genomic binding sites

  • RNA-seq following manipulation of methyltransferases that target HIST1H1C K186

  • CUT&RUN or CUT&Tag for higher resolution of binding sites with less starting material

• Gene-specific analysis:

  • ChIP-qPCR at promoters/enhancers of interest

  • RT-qPCR to measure expression of candidate genes

  • Reporter assays with wild-type vs. K186A mutant HIST1H1C

• Functional studies:

  • Generate K186 methylation-deficient mutants (K186A or K186R)

  • Identify and manipulate expression of methyltransferases targeting K186

  • Use MS/MS approaches to identify proteins interacting with methylated vs. unmethylated HIST1H1C

• Integrative analysis:

  • Correlate HIST1H1C K186 methylation with chromatin accessibility (ATAC-seq)

  • Examine co-occurrence with other histone modifications

  • Determine cell-type specific patterns of HIST1H1C methylation

Previous studies demonstrated that WHSC1-mediated H1 methylation affected transcriptional activation of specific genes like OCT4 . Similar approaches can be applied to study K186 methylation effects.

How does HIST1H1C methylation pattern change during cellular differentiation or disease progression?

Investigating dynamic changes in HIST1H1C methylation requires temporal analysis across cellular states:

• Cellular differentiation models:

  • Track HIST1H1C K186 methylation during embryonic stem cell differentiation

  • Compare methylation patterns in primary cells vs. terminal differentiated cells

  • Examine induced pluripotent stem cell reprogramming changes

• Disease progression analysis:

  • Compare normal tissue vs. tumor samples for methylation levels

  • Analyze progressive stages of cancer for changes in methylation patterns

  • Correlate methylation with patient outcomes or treatment response

• Quantitative approaches:

  • Develop ELISA-based assays for high-throughput quantification

  • Use mass spectrometry for precise quantitation of methylation stoichiometry

  • Implement multiplexed immunofluorescence imaging

• Time-course experiments:

  • Synchronized cell populations at different cell cycle stages

  • Drug-induced differentiation with temporal sampling

  • Stress response patterns in methylation

Research has shown that histone H1 plays vital roles in embryonic development and cellular differentiation . Specifically, WHSC1-mediated histone H1 methylation was linked to stemness features in squamous cell carcinoma, suggesting that H1 methylation patterns change during cancer progression and may contribute to disease phenotypes .

What are the interplays between different post-translational modifications on HIST1H1C?

Histone H1.2 undergoes multiple post-translational modifications (PTMs) beyond methylation, including phosphorylation, acetylation, and ubiquitination. Understanding the interplay between these modifications is crucial:

• PTM crosstalk mechanisms:

  • Sequential modifications (one modification facilitating or inhibiting another)

  • Competitive modifications at the same residue (methylation vs. acetylation)

  • Cooperative modifications enhancing reader protein binding

• Analytical approaches:

  • Mass spectrometry to identify co-occurring modifications

  • Sequential ChIP (Re-ChIP) to detect co-occurrence on the same molecules

  • Antibodies detecting specific PTM combinations

• Known interactions:

  • H1 phosphorylation, which mimics H1 depletion and results in chromatin decondensation, has been reported to halt progression to mitosis

  • Specific PTM patterns may define functional subtypes of HIST1H1C

Integrated analysis of multiple modifications will provide deeper insights into the "histone code" as it applies to linker histones like HIST1H1C.

How can I adapt ChIP-seq protocols specifically for Mono-methyl-HIST1H1C (K186) Antibody?

ChIP-seq with histone H1 variant-specific antibodies presents unique challenges requiring specific adaptations:

• Chromatin preparation optimization:

  • Dual crosslinking with DSG/EGS followed by formaldehyde

  • Optimized MNase digestion instead of sonication for certain applications

  • Careful titration of crosslinking time and reagent concentration

• IP conditions:

  • Higher antibody concentrations (4-5 μg per reaction)

  • Extended incubation times (overnight at 4°C)

  • Low-salt washing buffers to preserve interactions

• Library preparation considerations:

  • Input normalization strategies

  • Specialized adapters for low-input samples

  • PCR cycle optimization to minimize amplification bias

• Bioinformatic analysis adaptations:

  • H1-specific peak calling parameters

  • Integrative analysis with nucleosome positioning data

  • Correlation with chromatin accessibility datasets

When performing immunoprecipitation of methylated histone H1, researchers have successfully used specific buffer compositions (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100) for optimal results .

What future directions might research on HIST1H1C methylation take?

Research on HIST1H1C methylation is poised for significant advances in several areas:

• Technological innovations:

  • Development of site-specific methyltransferase inhibitors

  • CRISPR-based approaches for precise modification of specific residues

  • Single-cell analysis of histone modifications

  • Live-cell imaging of methylation dynamics

• Biological questions:

  • Cell-type specific functions of HIST1H1C methylation

  • Evolutionary conservation of methylation patterns across species

  • Role in 3D genome organization and phase separation

  • Contribution to aging and cellular senescence

• Clinical applications:

  • Diagnostic potential of methylation patterns in disease

  • Therapeutic targeting of methyltransferases in cancer

  • Biomarker development for treatment response

• Methodological advances:

  • Improved antibody specificity through recombinant approaches

  • Multiplexed detection of multiple modifications

  • Integration with spatial transcriptomics

As demonstrated by research on WHSC1-mediated H1 methylation in cancer, targeting histone H1 methylation may hold therapeutic potential . The development of WHSC1 inhibitors highlights the possibility that enzymes targeting K186 of HIST1H1C might similarly become therapeutic targets.

How can computational approaches enhance research on histone H1 methylation?

Computational approaches offer powerful tools for advancing histone H1 methylation research:

• Predictive modeling:

  • Machine learning algorithms to predict methylation sites

  • Structure-based modeling of reader protein interactions

  • Simulation of chromatin structural changes upon methylation

• Integrative data analysis:

  • Multi-omics integration (ChIP-seq, RNA-seq, ATAC-seq)

  • Network analysis of methylation-dependent interactions

  • Pathway enrichment for genes affected by HIST1H1C methylation

• Evolutionary approaches:

  • Comparative genomics of histone H1 variants across species

  • Evolutionary conservation of methylation sites

  • Phylogenetic analysis of methyltransferases

• Clinical correlations:

  • Analysis of methylation patterns in patient databases

  • Correlation with clinical outcomes and treatment responses

  • Identification of subtype-specific methylation signatures