Crotonyl-HIST1H1C (K158) Antibody

What is Crotonyl-HIST1H1C (K158) Antibody and what does it target?

Crotonyl-HIST1H1C (K158) Antibody is a polyclonal antibody that specifically recognizes the crotonylation post-translational modification at lysine 158 of human Histone H1.2 (HIST1H1C). This antibody is derived from rabbit hosts and is typically generated using a synthetic peptide sequence surrounding the crotonylated lysine 158 site of human Histone H1.2 as the immunogen . The target protein, Histone H1.2, is a linker histone that helps maintain higher-order chromatin structure by binding to nucleosome entry and exit sites, facilitating chromatin compaction. HIST1H1C is also known by several alternative names including H1 histone family member 2, H1.a, H12_HUMAN, H1F2, H1s-1, and Histone cluster 1 H1c . The antibody exhibits high specificity for the crotonylated form of K158, distinguishing it from other post-translational modifications that may occur at this or nearby residues.

What applications can Crotonyl-HIST1H1C (K158) Antibody be used for?

The Crotonyl-HIST1H1C (K158) Antibody has been validated for multiple experimental applications in epigenetic and chromatin-related research. According to the product information, this antibody can be effectively used in:

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of crotonylated HIST1H1C in solution

• Immunocytochemistry (ICC) - For visualizing the cellular localization of crotonylated HIST1H1C in fixed cells

• Immunofluorescence (IF) - For fluorescent detection and localization studies of the modified histone

• Chromatin Immunoprecipitation (ChIP) - For investigating genomic regions associated with crotonylated HIST1H1C

While not specifically validated for the crotonylated K158 form, related HIST1H1C antibodies targeting other modifications have shown utility in Western blotting (WB) and immunoprecipitation (IP), suggesting potential broader applications . When using this antibody, researchers should consider appropriate positive controls, such as human cell lines known to express HIST1H1C with K158 crotonylation, including Jurkat, MCF-7, and HeLa cells .

What is the biological significance of histone H1.2 (HIST1H1C) in cellular functions?

Histone H1.2 (HIST1H1C) serves critical functions in chromatin organization and gene expression regulation. Unlike core histones that form the nucleosome octamer, H1.2 functions as a linker histone that binds to nucleosomal DNA at entry and exit sites, facilitating higher-order chromatin compaction. Recent research has revealed several important biological roles:

• Autophagy Regulation: HIST1H1C/H1.2 has been implicated in autophagy regulation, particularly in the context of diabetic retinopathy. Overexpression of HIST1H1C upregulates SIRT1 and HDAC1, maintaining H4K16 deacetylation status, which leads to increased expression of autophagy-related proteins and enhanced autophagy .

• Inflammatory Response: Research indicates that HIST1H1C overexpression may promote inflammation and cellular toxicity in vitro , suggesting its potential role in inflammatory pathways.

• Epigenetic Regulation: As a linker histone, HIST1H1C influences chromatin accessibility and thereby affects gene expression patterns. Post-translational modifications, including crotonylation at K158, likely alter its binding properties and regulatory functions.

• Disease Relevance: Aberrant expression or modification of HIST1H1C has been associated with pathological conditions, including diabetic complications . The observed molecular weight of HIST1H1C in experimental settings (32-33 kDa) differs from its calculated weight (21 kDa), suggesting extensive post-translational modifications that may be physiologically relevant .

How does crotonylation at K158 differ from other post-translational modifications on HIST1H1C?

Crotonylation at K158 represents one of several post-translational modifications (PTMs) that can occur on Histone H1.2. This specific modification differs from others in several key aspects:

Crotonylation differs chemically from acetylation by having a four-carbon chain with a double bond, making it bulkier and potentially creating different binding interfaces for chromatin regulators. While acetylation is often recognized by bromodomain-containing proteins, crotonylation may recruit distinct "reader" proteins. The specific placement of crotonylation at K158, which lies within the C-terminal domain of HIST1H1C, suggests it may particularly affect DNA binding properties and interactions with other chromatin components.

Evidence from studies of other histone crotonylations indicates this modification may be especially associated with active enhancers and promoters of genes involved in cellular differentiation and stress responses, distinguishing it functionally from other modifications .

How can I optimize Chromatin Immunoprecipitation (ChIP) protocols using Crotonyl-HIST1H1C (K158) Antibody?

Optimizing ChIP protocols for Crotonyl-HIST1H1C (K158) antibody requires careful consideration of several experimental parameters:

• Crosslinking Optimization: For linker histones like HIST1H1C, standard 1% formaldehyde crosslinking (10 minutes at room temperature) may not be optimal. Consider dual crosslinking with 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) for 30 minutes followed by 1% formaldehyde for 10 minutes to better preserve linker histone-DNA interactions.

• Sonication Parameters: Crotonylated histones may reside in regions with distinct chromatin accessibility. Optimize sonication to generate fragments between 200-500 bp, using 15-20 cycles (30 seconds ON/30 seconds OFF) with appropriate amplitude settings. Always verify fragment size distribution by agarose gel electrophoresis.

• Antibody Amount and Incubation: For optimal results, use 2-5 μg of Crotonyl-HIST1H1C (K158) antibody per ChIP reaction containing chromatin from approximately 1-3 × 10^6 cells. Extend the antibody incubation to overnight at 4°C with gentle rotation .

• Washing Stringency: Balance between reducing background and maintaining specific interactions. A recommended washing series includes:

  • Low Salt Wash Buffer (150 mM NaCl) - twice

  • High Salt Wash Buffer (500 mM NaCl) - twice

  • LiCl Wash Buffer (250 mM LiCl) - once

  • TE Buffer - twice

• Controls: Include the following essential controls:

  • Input sample (5-10% of chromatin used for IP)

  • Negative control using non-specific IgG from the same species (rabbit)

  • Positive control using antibody against a well-established histone mark (e.g., H3K4me3)

• Data Analysis: For qPCR analysis of ChIP DNA, normalize to input and compare enrichment to IgG control. For ChIP-seq, include spike-in controls for normalization across samples and use appropriate peak-calling algorithms optimized for histone modifications.

Maximizing signal-to-noise ratio may require exploring buffer modifications, such as adding crotonyl-lysine (1-5 mM) to blocking buffers to reduce non-specific binding, particularly when investigating systems with low abundance of the K158 crotonylation mark .

What are the best experimental approaches to study the role of HIST1H1C K158 crotonylation in autophagy regulation?

Given the emerging connection between HIST1H1C and autophagy regulation , investigating the specific role of K158 crotonylation requires multifaceted experimental approaches:

• Site-Specific Mutagenesis: Generate K158R and K158Q mutants of HIST1H1C to mimic non-crotonylated and constitutively crotonylated states, respectively. Express these constructs in appropriate cell models and assess autophagy markers.

• Modulation of Cellular Crotonylation:

  • Increase global crotonylation by supplementing cells with crotonate (2-5 mM) or inhibiting decrotonylases

  • Measure changes in autophagy flux using LC3-II/LC3-I ratio, p62 degradation, and autophagic vesicle formation

• ChIP-seq with Crotonyl-HIST1H1C (K158) Antibody: Identify genomic regions associated with K158-crotonylated HIST1H1C and correlate with:

  • Expression of autophagy-related genes

  • Other histone modifications associated with active transcription

  • Chromatin accessibility (ATAC-seq)

• Proximity Ligation Assays: Investigate protein-protein interactions between crotonylated HIST1H1C and autophagy regulators like SIRT1 and HDAC1 .

• Quantitative Proteomics: Use stable isotope labeling (SILAC) combined with IP using Crotonyl-HIST1H1C (K158) antibody to identify protein complexes specifically associated with this modification under basal and autophagy-inducing conditions.

• Functional Assays in Relevant Models:

  • For diabetic retinopathy studies , use retinal cell lines treated with high glucose

  • Monitor autophagy markers including ATG proteins

  • Assess inflammation markers and cell viability

These approaches should be combined to establish both correlation and causation between HIST1H1C K158 crotonylation and autophagy regulation .

How can I validate the specificity of Crotonyl-HIST1H1C (K158) Antibody against other histone post-translational modifications?

Rigorous validation of antibody specificity is crucial for histone PTM research. For Crotonyl-HIST1H1C (K158) antibody, consider these comprehensive validation strategies:

• Peptide Competition Assays: Pre-incubate the antibody with:

  • Crotonylated K158 peptide (specific competitor)

  • Unmodified K158 peptide (negative control)

  • Peptides with other modifications at K158 (acetylation, methylation)

  • Peptides with crotonylation at other lysine residues of HIST1H1C

  Monitor signal reduction in immunoblotting or immunofluorescence to assess specificity.

• PTM-Specific Knockdown/Knockout Models:

  • Use CRISPR/Cas9 to generate K158R mutants

  • Employ siRNA knockdown of enzymes responsible for crotonylation

  • Test antibody reactivity in these models versus controls

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with the Crotonyl-HIST1H1C (K158) antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirm the presence of K158 crotonylation and assess enrichment of other modifications

• Cross-Reactivity Panel Testing: Test the antibody against a panel of synthetic peptides containing various modifications at K158 and crotonylation at other lysine residues in HIST1H1C, including:

  • K16, K45, K62, K84, K96, and K186 sites known to undergo other modifications

• Dot Blot Analysis: Create a systematic array of modified peptides at various concentrations to determine:

  • Detection limit for K158 crotonylation

  • Potential cross-reactivity threshold with other modifications

• Orthogonal Detection Methods: Compare results using:

  • Alternative antibodies targeting the same modification

  • Chemical labeling of crotonylated proteins

  • Recombinant reader domains specific for crotonylation

Carefully document all validation experiments with appropriate positive and negative controls. This comprehensive validation approach ensures that observed signals truly represent K158 crotonylation of HIST1H1C rather than cross-reactivity with similar modifications .

What are the recommended experimental controls when working with Crotonyl-HIST1H1C (K158) Antibody in immunofluorescence studies?

For rigorous immunofluorescence experiments using Crotonyl-HIST1H1C (K158) antibody, implement these essential controls:

• Primary Antibody Controls:

  • Negative control: Omit primary antibody but include all other steps and reagents

  • Isotype control: Use non-specific rabbit IgG at the same concentration

  • Peptide competition: Pre-incubate antibody with crotonylated K158 peptide before application

  • Dilution series: Test antibody at multiple concentrations (1:50-1:500) to determine optimal signal-to-noise ratio

• Sample Controls:

  • Positive control: Cell lines known to express HIST1H1C with K158 crotonylation (e.g., HeLa, HepG2)

  • Negative control: Cells treated with crotonylation inhibitors or CRISPR-modified cells with K158R mutation

  • Treatment controls: Include cells with enhanced crotonylation (e.g., crotonate treatment) or histone deacetylase inhibitors

• Fixation and Permeabilization Optimization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • Compare different permeabilization agents (0.1-0.5% Triton X-100, 0.1-0.5% saponin)

  • Ensure optimal epitope accessibility while preserving cellular architecture

• Co-localization Studies:

  • Nuclear markers (DAPI, Hoechst)

  • Euchromatin/heterochromatin markers to determine chromatin context of K158 crotonylation

  • Co-staining with antibodies against proteins involved in crotonylation/decrotonylation

• Technical Controls:

  • Secondary antibody only control to assess non-specific binding

  • Autofluorescence control (unstained cells)

  • Channel bleed-through controls in multi-color experiments

When analyzing IF results, perform quantitative assessment using digital image analysis with appropriate background subtraction and signal normalization. Include at least 50-100 cells per condition for statistical analysis and report both signal intensity and subcellular distribution patterns .

How does crotonylation at K158 of HIST1H1C relate to other histone modifications in the context of gene regulation?

Crotonylation at K158 of HIST1H1C exists within a complex network of histone modifications that collectively regulate chromatin structure and gene expression. Understanding these relationships requires investigation of several dimensions:

• Co-occurrence Patterns: Sequential ChIP (re-ChIP) experiments combining Crotonyl-HIST1H1C (K158) antibody with antibodies against other histone modifications reveal:

  • Positive correlation with active chromatin marks (H3K4me3, H3K27ac)

  • Potential mutual exclusivity with repressive marks (H3K9me3, H3K27me3)

  • Co-occurrence with other acylation marks (acetylation, butyrylation)

• Crosstalk Mechanisms: Crotonylation at K158 may influence or be influenced by:

  • Acetylation at neighboring sites (K62, K84, K96) through competition for same residues or by affecting enzyme accessibility

  • Methylation at K45 or K186 through conformational changes that alter enzyme recruitment

  • Modifications on core histones (particularly H4K16 deacetylation) through protein-protein interactions involving SIRT1 and HDAC1

• Reader Proteins: Crotonylation creates binding interfaces for:

  • YEATS domain-containing proteins that preferentially recognize crotonylated lysines

  • Regulatory complexes involved in transcription initiation or elongation

  • Factors with dual recognition capabilities for multiple acyl modifications

• Genomic Distribution: ChIP-seq studies suggest K158 crotonylation may be enriched at:

  • Transcriptionally active regions

  • Cell-type specific enhancers

  • Developmentally regulated genes

  • Autophagy-related gene loci

• Dynamic Regulation: The establishment and removal of K158 crotonylation appears regulated by:

  • Cellular metabolic state (crotonyl-CoA levels)

  • Activity of writer enzymes (p300/CBP with crotonylation activity)

  • Decrotonylases (class I HDACs and sirtuins)

  • Stimulus-responsive signaling pathways

Studies of HIST1H1C in diabetic retinopathy models suggest that its modifications, potentially including K158 crotonylation, influence autophagy by regulating SIRT1/HDAC1-dependent deacetylation of H4K16. This represents a specific example of crosstalk between different histone modifications across different histone proteins that collectively regulate critical cellular processes .

What are the known interactions between HIST1H1C K158 crotonylation and SIRT1/HDAC1 pathways?

The interaction between HIST1H1C K158 crotonylation and SIRT1/HDAC1 pathways represents an emerging area of research at the intersection of epigenetic regulation and cellular homeostasis. While the specific relationship between K158 crotonylation and these deacetylases is still being elucidated, several mechanistic connections can be outlined:

• Enzymatic Regulation: SIRT1 and HDAC1 are known deacetylases that may also possess decrotonylase activity:

  • SIRT1 (a NAD⁺-dependent deacetylase) can remove crotonyl groups from histones, but with lower efficiency than acetyl groups

  • HDAC1 (a zinc-dependent deacetylase) shows some activity toward crotonylated substrates

  • The balance between writers (crotonyl-transferases) and erasers (decrotonylases) likely determines K158 crotonylation levels

• HIST1H1C-Mediated Regulation: Research indicates that HIST1H1C overexpression upregulates SIRT1 and HDAC1, which:

  • Maintains H4K16 in a deacetylated state

  • Leads to upregulation of autophagy-related genes

  • Promotes autophagy in retinal cells, potentially contributing to diabetic retinopathy

• Metabolic Connections: Crotonylation depends on crotonyl-CoA availability, which is linked to:

  • Fatty acid metabolism pathways

  • NAD⁺ levels, which also regulate SIRT1 activity

  • Cellular energy status, creating potential for coordinated regulation

• Signaling Integration: The HIST1H1C-SIRT1/HDAC1 axis may integrate multiple cellular signals:

  • Stress responses (oxidative stress in diabetic conditions)

  • Nutrient availability (affecting both crotonyl-CoA and NAD⁺ levels)

  • Inflammatory stimuli (relevant to diabetic complications)

• Proposed Mechanistic Model:

  • Under normal conditions, balanced K158 crotonylation contributes to regulated gene expression

  • In diabetic conditions, altered HIST1H1C levels or modifications disrupt this balance

  • Upregulated SIRT1/HDAC1 changes the histone modification landscape

  • This leads to dysregulated autophagy and inflammation

  • K158 crotonylation may serve as either a trigger or a consequence in this pathway

Further research using Crotonyl-HIST1H1C (K158) antibody in combination with SIRT1/HDAC1 modulation will be crucial to fully elucidate this complex regulatory network and its implications for diseases like diabetic retinopathy .