HIST1H2BC (Ab-36) Antibody

What is HIST1H2BC and what role does Serine 36 phosphorylation play in cellular processes?

HIST1H2BC (also known as H2BC4, H2BFL) is a core component of nucleosomes that helps wrap and compact DNA into chromatin. Nucleosomes regulate DNA accessibility to cellular machinery required for transcription, replication, and repair processes. Histone H2B Serine 36 phosphorylation (H2B-Ser36p) is a critical post-translational modification that fluctuates during cellular differentiation .

This modification is mediated primarily by AMPK (AMP-activated protein kinase) during processes like adipogenesis, with levels of H2B-Ser36p dramatically increasing 24-48 hours after differentiation initiation before receding . Phosphorylation at this site plays important roles in gene expression regulation as part of the complex "histone code" that dictates chromatin structure and function .

How specific is the HIST1H2BC (Ab-36) antibody for detecting H2B-Ser36 phosphorylation?

The HIST1H2BC (Ab-36) antibody is a polyclonal antibody raised in rabbits against a peptide sequence surrounding Serine 36 of Histone H2B type 1-C/E/F/G/I . This antibody demonstrates high specificity for the phosphorylated Ser36 epitope as evidenced in research applications.

What applications has the HIST1H2BC (Ab-36) antibody been validated for?

The HIST1H2BC (Ab-36) antibody has been validated for several research applications including:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of H2B-Ser36 phosphorylation levels .

• IHC (Immunohistochemistry): With recommended dilutions of 1:10-1:100 .

• Western Blotting: Successfully used to monitor temporal changes in H2B-Ser36 phosphorylation during cellular differentiation .

• Immunoprecipitation (IP): Effective for isolating H2B and analyzing its modification status .

The antibody has been particularly valuable in studying the inverse relationship between H2B-Ser36 phosphorylation and H2B ADP-ribosylation during adipocyte differentiation .

How does the interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation regulate gene expression?

The functional relationship between these adjacent modifications represents a sophisticated regulatory mechanism. Research has revealed an inverse correlation between H2B ADP-ribosylation (H2B-PAR) and H2B-Ser36 phosphorylation during cellular differentiation . When one modification is enriched, the other is depleted.

Mechanistically, ADP-ribosylation at Glu35 directly inhibits AMPK-mediated phosphorylation of the adjacent Ser36 residue . This inhibition is specifically driven by PARP-1/NMNAT-1-catalyzed modification of H2B-Glu35, not by PARP-1 binding or PAR linked to PARP-1 itself . In vitro studies conclusively demonstrated that:

• NMNAT-1-dependent PARylation of H2B inhibits phosphorylation of Ser36 by AMPK

• Mutation of Glu35 to alanine (E35A) restores AMPK's ability to phosphorylate Ser36

• Order-of-addition experiments confirmed that direct PARylation of H2B-Glu35 drives the inhibition

This molecular switch between modifications likely controls the temporal accessibility of chromatin regions during differentiation processes, ensuring proper gene expression program execution .

What enzymatic machinery regulates H2B-Ser36 phosphorylation and H2B-Glu35 ADP-ribosylation?

The regulatory enzymes involved in these modifications form a complex network:

For H2B-Ser36 phosphorylation:

• AMPK (AMP-activated protein kinase) is the primary kinase responsible during adipogenesis

• S6K1 has also been identified as capable of phosphorylating this site in certain contexts

For H2B-Glu35 ADP-ribosylation:

• PARP-1 (Poly(ADP-ribose) polymerase 1) catalyzes the addition of ADP-ribose to H2B-Glu35

• NMNAT-1 (Nicotinamide mononucleotide adenylyltransferase 1) acts as an essential cofactor for PARP-1-mediated ADP-ribosylation

• Unlike genotoxic stress conditions, HPF1 (Histone PARylation Factor 1) is not required for H2B PARylation during normal cellular processes like adipogenesis

Notably, NMNAT-1 can activate PARP-1 activity toward histone H2B in an ATP-dependent manner but independent of its own catalytic activity . This interaction facilitates the formation of a PARP-1/histone H2B/NMNAT-1 complex that specifically targets Glu/Asp residues for ADP-ribosylation, distinct from the Ser-directed activity promoted by HPF1 .

How can HIST1H2BC (Ab-36) antibody be used to study chromatin dynamics during cellular differentiation?

The HIST1H2BC (Ab-36) antibody serves as a powerful tool for tracking temporal changes in chromatin state during differentiation processes. Researchers can implement several approaches:

• Time-course experiments: Monitor H2B-Ser36p levels at different stages of differentiation using Western blotting, as demonstrated in 3T3-L1 adipogenesis where phosphorylation peaks at 24-48 hours post-induction .

• ChIP-seq (Chromatin Immunoprecipitation sequencing): Map the genome-wide distribution of H2B-Ser36p during differentiation to identify genes regulated by this modification.

• Co-IP (Co-Immunoprecipitation) studies: Use the antibody to isolate H2B and associated proteins to identify novel interaction partners during differentiation.

• Dual modification analysis: Implement sequential immunoprecipitation protocols to study the mutually exclusive relationship between H2B-Ser36p and H2B-PAR .

This experimental approach has revealed that H2B-Ser36p levels increase during differentiation precisely when nuclear NAD+ synthesized by NMNAT-1 and PARP-1 catalytic activity reach minimum levels, establishing a mechanistic link between these cellular events .

What are the optimal conditions for Western blotting using HIST1H2BC (Ab-36) antibody?

For optimal Western blotting results with HIST1H2BC (Ab-36) antibody, researchers should consider the following protocol elements:

Sample preparation:

• Extract histones using acidic extraction methods (0.2N HCl) to specifically enrich for histones

• Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in all buffers to preserve phosphorylation status

• Use freshly prepared samples when possible, as freeze-thaw cycles may affect phospho-epitope integrity

Blotting conditions:

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for phospho-epitopes)

• Blocking: Use 5% BSA in TBST rather than milk (milk contains phosphoproteins that can interfere)

• Primary antibody: Incubate at 1:1000 dilution in 5% BSA overnight at 4°C

• Secondary antibody: Anti-rabbit HRP at 1:5000 for 1 hour at room temperature

Controls to include:

• Positive control: Extracts from differentiating 3T3-L1 cells (24-48h post-induction)

• Negative control: Samples treated with lambda phosphatase

• Specificity control: Peptide competition assay using the immunizing peptide

This approach has successfully detected the inverse relationship between H2B-PAR and H2B-Ser36p throughout adipocyte differentiation .

How can HIST1H2BC (Ab-36) antibody be used in combination with PARP inhibitors to study modification crosstalk?

PARP inhibitors provide an excellent experimental approach to investigate the crosstalk between ADP-ribosylation and phosphorylation of H2B. The following methodology has been validated:

Experimental design:

• Treat cells with PARP-1-selective inhibitors (e.g., BYK204165 or Niraparib)

• Monitor changes in:

  • Bulk PAR levels (should decrease)

  • Phospho-AMPKα levels (typically increase)

  • H2B-Ser36p levels (dramatically increase)

  • H2B-Ser32p levels (should remain unchanged as a specificity control)

Data collection and analysis:

• Perform Western blotting with antibodies against PAR, phospho-AMPKα, H2B-Ser36p, and H2B-Ser32p

• Quantify band intensities using image analysis software

• Plot the inverse correlation between PAR and H2B-Ser36p levels

Complementary approaches:

• siRNA-mediated knockdown of PARP-1 should produce similar effects as PARP inhibitors

• siRNA against NMNAT-1 would also affect this pathway

This experimental strategy revealed that PARP inhibition causes a dramatic increase in H2B-Ser36p levels while specifically not affecting H2B-Ser32p, highlighting the site-specific nature of this regulatory mechanism .

What methodologies can detect both H2B-Ser36 phosphorylation and H2B-Glu35 ADP-ribosylation simultaneously?

Detecting these mutually exclusive modifications simultaneously presents technical challenges but can be accomplished through several approaches:

Sequential immunoprecipitation:

• First IP: Use anti-PAR antibodies to isolate ADP-ribosylated proteins

• Western blot: Probe with H2B antibody to confirm H2B presence

• Second IP: Use HIST1H2BC (Ab-36) antibody on the non-bound fraction

• Western blot: Determine the levels of H2B-Ser36p in this fraction

Mass spectrometry-based approach:

• Enrich for histones using acid extraction

• Perform tryptic digestion under controlled conditions

• Analyze peptides using LC-MS/MS with electron transfer dissociation (ETD) fragmentation

• Quantify the relative abundance of modified peptides containing Glu35 and Ser36

Proximity ligation assay (PLA):
For cellular localization studies, combining:

• HIST1H2BC (Ab-36) antibody

• Anti-PAR antibody

• Appropriate PLA probes

This approach can visualize where these modifications occur in relation to each other within the nucleus and confirm their mutually exclusive nature observed in biochemical studies .

How should researchers interpret changes in H2B-Ser36 phosphorylation in relation to genotoxic stress?

When interpreting H2B-Ser36 phosphorylation data in the context of genotoxic stress, researchers should consider these key findings:

• HPF1 (Histone PARylation Factor 1) is required for H2B PARylation in response to genotoxic stress (e.g., H₂O₂ treatment) but not for H2B PARylation under normal conditions .

• H2B-Ser36p levels remain largely unaffected by H₂O₂ treatment or HPF1 knockdown, suggesting this modification responds primarily to developmental or metabolic signals rather than DNA damage .

• The regulation of H2B-Ser36p during normal cellular processes (e.g., adipogenesis) involves different mechanisms than those activated during genotoxic stress.

When analyzing experimental data showing changes in H2B-Ser36p:

• Determine whether changes correlate with cellular differentiation programs

• Examine AMPK activation status, as AMPK is the primary kinase for H2B-Ser36

• Consider metabolic state changes that might influence AMPK activity

• Distinguish between stress-induced ADP-ribosylation (HPF1-dependent) and metabolic state-induced ADP-ribosylation (NMNAT-1-dependent)

This interpretation framework helps differentiate between developmental regulation and stress response pathways affecting histone modifications.

What are the potential pitfalls when analyzing mutant H2B constructs with the HIST1H2BC (Ab-36) antibody?

When using mutant H2B constructs to study phosphorylation dynamics, researchers should be aware of several potential pitfalls:

Antibody recognition issues:

• The H2B-Ser36p antibody recognizes Glu35Ala (E35A) mutant H2B peptides with reduced affinity compared to wild-type

• This means the effects of mutating Glu35 on H2B-Ser36 phosphorylation may be underestimated in Western blot analyses

Control requirements:

• Each mutant construct should be validated for expression level compared to wild-type

• Include phospho-deficient controls (S36A mutant) to confirm antibody specificity

• For E35A mutants, calibrate detection sensitivity using purified phosphopeptides

Interpretation guidelines:

• When working with E35A mutants, normalization to total H2B is critical

• Consider complementary techniques (mass spectrometry) to validate antibody-based results

• Remember that point mutations may alter nucleosome structure or stability, potentially affecting multiple histone modifications

This guidance is particularly important when investigating the mechanistic link between Glu35 ADP-ribosylation and Ser36 phosphorylation, as demonstrated in order-of-addition experiments with PARP-1/NMNAT-1 and AMPK .

How can researchers differentiate between direct and indirect effects on H2B-Ser36 phosphorylation?

Distinguishing direct from indirect effects on H2B-Ser36 phosphorylation requires careful experimental design:

In vitro reconstitution assays:
Researchers should follow the approach demonstrated in published work :

• Perform in vitro ADPRylation assays on H2B (full-length or N-terminal peptide 10-59)

• Follow with AMPK-mediated phosphorylation

• Include order-of-addition controls:

  • Unmodified PARP-1 alone

  • PARylated PARP-1 without further catalytic activity

  • PARP-1/NMNAT-1 in the presence of a PARP inhibitor

These controls revealed that direct PARylation of H2B-Glu35 by PARP-1 with NMNAT-1, not PARP-1 binding to H2B or PAR linked to PARP-1, drives the inhibition of AMPK-mediated H2B-Ser36 phosphorylation .

Site-directed mutagenesis approach:
Create a panel of mutants to isolate specific effects:

• S36A (phospho-deficient)

• E35A (ADPRylation-deficient)

• Double mutants

The observation that PARylation occurred with an H2B peptide in which Ser36 was mutated (S36A), but was lost with an H2B peptide in which Glu35 was mutated (E35A), provided critical evidence for the site-specificity of this regulation .

Cellular approaches:
Use drug treatments with appropriate controls:

• PARP inhibitors (BYK204165, Niraparib)

• AMPK activators (AICAR, A-769662)

• AMPK inhibitors (Compound C)