O-GlcNAcyl-HIST1H4A (S47) Antibody

What is the significance of O-GlcNAcylation at serine 47 of histone H4?

Histone H4 O-GlcNAcylation at serine 47 (S47) represents a critical component of the histone code that regulates chromatin structure and gene expression. S47 is strategically located on the lateral surface of the nucleosome where it makes indirect interactions with DNA . This site is particularly significant because:

• It resides in loop 1 within the histone-fold domain, influencing nucleosome stability and DNA interactions

• Unlike many histone modifications that occur on N-terminal tails, S47 modification directly affects the nucleosome core particle structure

• O-GlcNAcylation at this site changes dynamically during cellular processes including mitosis and in response to stress conditions such as heat shock

• The modification exhibits cross-talk with other histone post-translational modifications, particularly phosphorylation

Understanding this specific modification provides insights into the dynamic regulation of chromatin and transcriptional control mechanisms.

How is O-GlcNAcylation of histones detected and verified experimentally?

Multiple orthogonal approaches are necessary to confidently detect and verify histone O-GlcNAcylation:

Immunological Detection Methods:

• Western blotting with O-GlcNAc-specific antibodies (CTD110.6, RL2)

• Competition assays with free GlcNAc (typically 1M) to confirm specificity

• Immunoprecipitation followed by immunoblotting

Enzymatic and Chemical Methods:

• Chemoenzymatic labeling using mutant galactosyltransferase (mGalT1) to tag O-GlcNAc with azido-modified galactosamine (GalNAz)

• Biotin alkyne reaction via Huisgen cycloaddition (click chemistry)

• Metabolic labeling with UDP-[³H]-Gal as donor substrate

• β-elimination assay using mild alkaline conditions (55 mM NaOH, 16h at 40°C) to confirm O-GlcNAc linkages

Mass Spectrometry Approaches:

• Enrichment of O-GlcNAcylated peptides followed by LC-MS/MS analysis

• Mild β-elimination with Michael addition using DTT to create a stable marker for site mapping

• Higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) MS methods for precise site identification

Verification requires combining multiple techniques, as each has limitations and potential artifacts when used alone.

What controls should be included when using O-GlcNAcyl-HIST1H4A (S47) antibodies?

Proper experimental controls are essential for accurate interpretation of results with site-specific O-GlcNAc antibodies:

Essential Controls:

• Peptide Competition: Include controls where the antibody is pre-incubated with the antigenic peptide (RGGVKRIS(O-GlcNAc)GLIYEE)

• GlcNAc Competition: Perform parallel immunoblots with 1M free GlcNAc to verify O-GlcNAc specificity

• Enzymatic Treatments:

  • OGA (O-GlcNAcase) inhibitor (e.g., PUGNAc) to increase O-GlcNAcylation levels

  • In vitro OGT reactions to confirm antibody recognition of enzymatically added O-GlcNAc

• Genetic Controls:

  • OGT knockdown/knockout samples to demonstrate loss of signal

  • Alanine mutants (S47A) of histone H4 to confirm site specificity

• Cross-reactivity Assessment: Test against other O-GlcNAcylated histones (H2A T101, H2B S36) to ensure site specificity

Control Sample Preparation Table:

What are the optimal conditions for using O-GlcNAcyl-HIST1H4A (S47) antibody in immunoblotting?

Successful immunoblotting with site-specific O-GlcNAc antibodies requires careful attention to sample preparation and experimental conditions:

Sample Preparation:

• Extract histones using acid extraction (0.2N HCl) to effectively isolate histones while preserving O-GlcNAc modifications

• Treat cells with OGA inhibitors (e.g., PUGNAc, Thiamet-G) for 6-24 hours before extraction to increase O-GlcNAc signal if detection is challenging

• Process samples quickly and maintain cold conditions to prevent loss of the labile O-GlcNAc modification

Immunoblotting Conditions:

• Blocking: 3-5% BSA in TBS (not milk, which contains glycoproteins that may interfere)

• Primary antibody dilution: Start with 1:1000 and optimize as needed

• Buffer: TBS with 0.05% Tween-20 (PBS may contain phosphate groups that can interfere with some O-GlcNAc antibodies)

• Secondary antibody: Anti-rabbit HRP (for rabbit polyclonal antibodies)

• Detection: Enhanced chemiluminescence with longer exposure times may be necessary due to potentially low stoichiometry of O-GlcNAcylation

Special Considerations:

• Run appropriate molecular weight markers (15-20 kDa range for histones)

• Include glycosylated protein controls to verify antibody functionality

• Consider using alternative gel systems such as Acetic acid/Urea (AU) gels to separate differentially modified histone forms

• For two-dimensional separation, combine AU gel electrophoresis with SDS-PAGE to resolve histones based on both charge and size

How can researchers perform ChIP experiments using O-GlcNAcyl-HIST1H4A (S47) antibodies?

Chromatin immunoprecipitation (ChIP) with O-GlcNAc-specific antibodies requires modifications to standard protocols:

Optimized ChIP Protocol:

• Crosslinking and Chromatin Preparation:

  • Standard formaldehyde crosslinking (1% for 10 minutes at room temperature)

  • Include OGA inhibitors (e.g., PUGNAc) in all buffers to preserve O-GlcNAc modifications

  • Sonicate to achieve fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use magnetic Dynabeads for immunoprecipitation

  • Incubate antibody with beads in TBS (preferred over PBS)

  • Include O-GlcNAc-specific controls in parallel experiments

  • Extend incubation time (overnight at 4°C) to improve capture of potentially low-abundance modifications

• Washing and Elution:

  • Include OGA inhibitors in wash buffers

  • Elute bound proteins by boiling in Laemmli Sample Buffer

• Analysis:

  • Perform parallel ChIP with total H4 antibody for normalization

  • Consider sequential ChIP (Re-ChIP) with other histone modification antibodies to assess co-occurrence of modifications

  • Analyze enrichment by qPCR or next-generation sequencing

Special Considerations:

• O-GlcNAcylation may exhibit lower abundance than other histone modifications, requiring optimization of antibody amounts and incubation conditions

• The dynamic nature of O-GlcNAc may necessitate stabilization with OGA inhibitors throughout the protocol

• Compare results with ChIP using general O-GlcNAc antibodies (RL2, CTD110.6) to validate findings

What techniques can be used to determine the stoichiometry of O-GlcNAcylation at histone H4 S47?

Determining the stoichiometry of histone O-GlcNAcylation remains challenging but several approaches can be combined:

• Mass Spectrometry-Based Quantification:

  • Label-free quantification comparing modified and unmodified peptides

  • Targeted MS methods such as Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM)

  • Develop standard curves using synthetic peptides with and without O-GlcNAc modification

• Radiometric Approaches:

  • Metabolic labeling with UDP-[³H]-Gal and quantification of incorporated radioactivity

  • Compare labeling efficiency to standards with known modification rates

• Immunological Methods:

  • Use paired antibodies that recognize the same site with and without O-GlcNAc

  • Quantitative western blotting with purified standards

  • Enzyme-linked immunosorbent assay (ELISA) with recombinant histones

• Chemical Methods:

  • Glycosite-to-cysteine mutagenesis with S-GlcNAcylation can achieve higher stoichiometry (>70%) for functional studies

  • Chemical installation of O-GlcNAc analogs at specific sites

Comparative Analysis Table for Stoichiometry Determination:

How does O-GlcNAcylation at histone H4 S47 interact with other histone modifications?

O-GlcNAcylation at H4 S47 participates in complex cross-talk with other histone modifications, forming an integral part of the histone code:

Cross-talk with Acetylation:

• Two-dimensional gel electrophoresis (AU/SDS-PAGE) has demonstrated that O-GlcNAcylation can coexist with acetylation on histones

• The presence of O-GlcNAc may influence histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities through structural changes or protein-protein interactions

Relationship with Phosphorylation:

• O-GlcNAcylation and phosphorylation often exhibit reciprocal relationships, competing for the same or adjacent serine/threonine residues

• Immunoprecipitation experiments with phospho-specific histone antibodies show reduced O-GlcNAc signal compared to total histone immunoprecipitates, suggesting these modifications may be mutually exclusive at some sites

Impact on Methylation:

• OGT activity influences histone methylation patterns, particularly H3K27me3

• This relationship appears to be mediated through O-GlcNAcylation of methyltransferases like EZH2 rather than direct effects of histone O-GlcNAcylation

Spatial Organization:

• H4 S47 is positioned on the lateral surface of the nucleosome where it makes indirect contacts with DNA

• This positioning suggests O-GlcNAcylation at this site could affect nucleosome stability and DNA accessibility

• The bulky O-GlcNAc modification likely alters the local chromatin structure, potentially influencing recruitment of reader proteins

Temporal Dynamics:

• O-GlcNAcylation changes during mitosis and in response to stress conditions like heat shock

• These changes may coordinate with phosphorylation events to regulate chromatin condensation and gene expression

What is the role of H4 S47 O-GlcNAcylation in regulating transcription and cell cycle progression?

H4 S47 O-GlcNAcylation appears to play important regulatory roles in both transcriptional control and cell cycle progression:

Transcriptional Regulation:

• H4 S47 makes indirect contacts with DNA, suggesting O-GlcNAcylation at this site could affect DNA accessibility

• Changes in O-GlcNAcylation during heat shock correlate with altered transcriptional programs

• OGT and its activity have been found in complex with transcriptional repressors like mSin3A/HDAC1, suggesting a role in gene silencing

Cell Cycle Regulation:

• O-GlcNAcylation levels change during the cell cycle, particularly during mitosis

• Impaired removal of O-GlcNAc inhibits efficient cell cycle transition from G2 to mitosis

• O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control

• Constitutively elevated O-GlcNAcylation by OGA disruption impairs cell proliferation and results in mitotic defects

Mitotic Effects:

• OGA loss leads to specific mitotic defects including:

  • Cytokinesis failure and binucleation

  • Increased lagging chromosomes

  • Micronuclei formation

• These findings suggest an important role for O-GlcNAc cycling in maintaining genomic stability

Developmental Importance:

• Genetic disruption of OGA results in constitutively elevated O-GlcNAcylation in embryos and leads to neonatal lethality with developmental delay

• O-GlcNAcylation influences cell differentiation programs, including erythropoietic lineage commitment

How can researchers experimentally manipulate H4 S47 O-GlcNAcylation for functional studies?

Several approaches can be employed to manipulate H4 S47 O-GlcNAcylation for mechanistic studies:

Enzymatic Manipulation:

• OGT/OGA Modulation:

  • OGT knockdown using siRNA/shRNA to reduce global O-GlcNAcylation

  • OGA inhibitors (PUGNAc, Thiamet-G) to increase O-GlcNAcylation

  • CRISPR-Cas9 genome editing for complete knockout of OGT or OGA

  • In-nucleo OGT assays using radiolabeled UDP-GlcNAc to assess dynamic changes in histone targeting

• Site-Directed Mutagenesis:

  • Generate S47A mutants to prevent O-GlcNAcylation at the specific site

  • Express mutant histones with FLAG tags for immunoprecipitation and functional studies

  • Create glutamic acid substitutions (S47E) to mimic the negative charge but not the steric effects of O-GlcNAc

• Advanced Chemical Biology Approaches:

  • Glycosite-to-cysteine mutagenesis (S47C) combined with thioglycoligase-mediated S-GlcNAcylation

  • This approach allows installation of a GlcNAc analog that resists OGA hydrolysis, enabling study of constitutive modification effects

• Inducible Systems:

  • Tetracycline-inducible expression of OGT or OGA

  • Auxin-inducible degron (AID) tags on OGT/OGA for rapid protein depletion

  • Optogenetic control of OGT/OGA activity for temporal and spatial manipulation

Experimental Design for Functional Studies:

What are common challenges when working with O-GlcNAcyl-HIST1H4A (S47) antibodies and how can they be addressed?

Site-specific O-GlcNAc antibodies present several technical challenges:

Challenge 1: Low Signal Intensity

• Cause: Low stoichiometry of O-GlcNAcylation at specific sites

• Solutions:

  • Treat cells with OGA inhibitors (PUGNAc, Thiamet-G) before sample collection

  • Optimize extraction methods to preserve labile O-GlcNAc modifications

  • Use enhanced chemiluminescence with longer exposure times

  • Consider signal amplification methods like tyramide signal amplification

Challenge 2: Specificity Concerns

• Cause: Cross-reactivity with other O-GlcNAcylated histones or proteins

• Solutions:

  • Always include competition controls with free GlcNAc (1M)

  • Include S47A mutant controls to confirm site specificity

  • Perform parallel experiments with general O-GlcNAc antibodies (RL2, CTD110.6)

  • Consider pre-clearing antibodies with non-specific O-GlcNAcylated proteins

Challenge 3: Inconsistent Results

• Cause: Dynamic nature of O-GlcNAcylation that changes with cellular conditions

• Solutions:

  • Standardize growth conditions, particularly glucose concentration

  • Control for cell cycle phase (synchronization if necessary)

  • Document and control environmental stressors that influence O-GlcNAcylation

  • Consider rapid sample processing or direct lysis in SDS sample buffer

Challenge 4: Background in Immunofluorescence

• Cause: Non-specific binding or high background with nuclear staining

• Solutions:

  • Use BSA or commercial protein-free blockers (not milk)

  • Include 0.1-0.3% Triton X-100 in blocking and antibody incubation steps

  • Pre-absorb antibodies with fixed, permeabilized cells lacking the target

  • Include appropriate peptide competition controls

Challenge 5: Reproducibility Issues

• Cause: Batch-to-batch antibody variation or inconsistent modification levels

• Solutions:

  • Validate each new antibody lot with known positive controls

  • Combine orthogonal detection methods to verify results

  • Consider generating stable cell lines with OGT/OGA modulation as consistent controls

  • Document detailed experimental conditions in publications

How can researchers distinguish between cause and consequence when studying H4 S47 O-GlcNAcylation effects?

Determining causality in histone modification studies requires specialized approaches:

Strategy 1: Temporal Manipulation and Analysis

• Use rapid induction/inhibition systems (e.g., auxin-inducible degron-tagged OGT/OGA)

• Establish time courses to determine order of events

• Combine with techniques like ChIP-seq and RNA-seq at multiple time points to track chromatin and transcriptional changes

Strategy 2: Site-Specific Manipulation

• Express H4 S47A mutants to prevent O-GlcNAcylation specifically at this site

• Use glycosite-to-cysteine mutagenesis with S-GlcNAcylation to artificially maintain the modification

• Compare phenotypes with global OGT/OGA manipulation to identify site-specific effects

Strategy 3: Targeted Recruitment Approaches

• Employ CRISPR-dCas9 systems to recruit OGT or OGA to specific genomic loci

• Use MS2-tagged RNA to recruit OGT/OGA to specific nascent transcripts

• Compare effects of local versus global O-GlcNAcylation changes

Strategy 4: Separation of Function Mutations

• Create OGT mutants that specifically affect histone targeting without disrupting other functions

• Design H4 mutations that affect O-GlcNAcylation without altering other modifications or functions

• Use these tools to dissect specific activities from pleiotropic effects