2-hydroxyisobutyryl-HIST1H1C (K51) Antibody

What is 2-hydroxyisobutyrylation and why is it significant in histone research?

2-hydroxyisobutyrylation is a recently identified post-translational modification (PTM) of proteins that affects the association between histones and DNA. Unlike better-characterized histone modifications such as acetylation and methylation, 2-hydroxyisobutyrylation remains relatively understudied despite its potential significance in chromatin regulation . The modification involves the addition of a 2-hydroxyisobutyryl group to lysine residues of proteins, including histones like HIST1H1C. This modification has been found to be conserved across species including plants, humans, and mice, suggesting its fundamental biological importance . For researchers, studying this modification provides insights into novel mechanisms of chromatin regulation and gene expression control that complement our understanding of other histone PTMs.

How does HIST1H1C (Histone H1C) differ from core histones, and what is the significance of its K51 residue?

HIST1H1C is a linker histone variant that, unlike core histones (H2A, H2B, H3, and H4), binds to the DNA as it enters and exits the nucleosome, helping to stabilize higher-order chromatin structures. While core histones form the nucleosome core particle around which DNA wraps, linker histones like HIST1H1C seal the DNA at its entry and exit points from the nucleosome, facilitating chromatin compaction .

The K51 residue of HIST1H1C is located within the globular domain of the protein, making it particularly interesting as modifications at this site could directly affect DNA binding and chromatin organization. Modifications at this position, such as 2-hydroxyisobutyrylation, may alter the interaction between the histone and DNA, potentially affecting chromatin accessibility and gene regulation . The specific location of K51 in the protein's structure suggests that its modification might have distinct functional consequences compared to modifications at other lysine residues in the same protein.

What validation methods should be used to confirm the specificity of 2-hydroxyisobutyryl-HIST1H1C (K51) antibody?

To confirm antibody specificity, researchers should implement a multi-step validation approach:

• Peptide competition assays: Compare immunoblotting or immunoprecipitation results with and without pre-incubation of the antibody with the synthetic 2-hydroxyisobutyrylated K51 peptide. Signal abolishment indicates specificity.

• Cross-reactivity testing: Test the antibody against peptides containing other lysine modifications (acetylation, methylation, etc.) at the same position to ensure it only recognizes 2-hydroxyisobutyrylation .

• Knockout or knockdown validation: Use CRISPR/Cas9 to generate HIST1H1C knockout cells or specific K51 mutants (K51R) as negative controls.

• Mass spectrometry confirmation: Following immunoprecipitation with the antibody, perform MS analysis to confirm the presence of 2-hydroxyisobutyrylation specifically at K51 .

• Dot blot analysis: Test antibody reactivity against a panel of modified and unmodified peptides to quantitatively assess specificity.

A properly validated antibody should show minimal cross-reactivity with other histone modifications and should not recognize the unmodified form of the protein or the same modification at different lysine residues.

How do sequence motifs surrounding K51 affect the performance of 2-hydroxyisobutyryl-HIST1H1C (K51) antibody?

The performance of 2-hydroxyisobutyryl-HIST1H1C (K51) antibody is significantly influenced by the amino acid sequence context surrounding the K51 residue. Research on lysine 2-hydroxyisobutyrylation has revealed that this modification occurs within specific sequence motifs, with a strong preference for negatively charged amino acids like aspartic acid (D) and glutamic acid (E) in the vicinity of the modified lysine .

Analyses of 2-hydroxyisobutyrylated sites have identified motifs such as [EK_hib], [D XXK_hib], and [K_hibE], where K_hib represents the 2-hydroxyisobutyrylated lysine . If the K51 site in HIST1H1C conforms to or deviates from these consensus motifs, it could affect antibody recognition and binding efficiency.

Researchers should consider the following when working with this antibody:

• Epitope masking can occur if the sequence surrounding K51 interacts with other proteins in a complex

• Conformational changes in the protein may affect epitope accessibility

• Other nearby post-translational modifications might create steric hindrance or alter antibody binding

For optimal results, researchers should determine whether the antibody was raised against a linear epitope or a conformational epitope, as this will influence experimental design and sample preparation protocols.

What are the optimal conditions for using 2-hydroxyisobutyryl-HIST1H1C (K51) antibody in ChIP assays?

When using 2-hydroxyisobutyryl-HIST1H1C (K51) antibody for Chromatin Immunoprecipitation (ChIP) assays, researchers should consider the following optimized protocol:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions, followed by quenching with 125 mM glycine.

• Chromatin preparation:

  • Lyse cells in buffer containing protease inhibitors and deacetylase inhibitors

  • Include β-hydroxybutyrate (10 mM) to inhibit removal of 2-hydroxyisobutyryl marks

  • Sonicate chromatin to fragments of 200-500 bp (verify fragment size by gel electrophoresis)

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5 μg of antibody per ChIP reaction (titrate for optimal signal-to-noise ratio)

  • Include appropriate controls: IgG negative control and a positive control antibody targeting a known abundant histone mark

  • Incubate overnight at 4°C with rotation

• Washing and elution:

  • Use increasingly stringent wash buffers to minimize non-specific binding

  • Elute at 65°C with elution buffer containing SDS

• Reverse crosslinking and DNA purification:

  • Incubate at 65°C overnight to reverse formaldehyde crosslinks

  • Treat with RNase A and Proteinase K

  • Purify DNA using phenol-chloroform extraction or column-based methods

For ChIP-seq applications, library preparation should follow standard protocols, with sequencing depth of at least 20 million reads per sample. Since 2-hydroxyisobutyrylation may occur in less abundant regions compared to other histone marks, deeper sequencing might be necessary for comprehensive coverage .

How can researchers effectively combine 2-hydroxyisobutyryl-HIST1H1C (K51) antibody with other histone modification antibodies in multiplex experiments?

Multiplexing histone modification antibodies requires careful planning to avoid technical artifacts and ensure valid results:

• Sequential ChIP (Re-ChIP):

  • Perform first ChIP with one antibody (e.g., 2-hydroxyisobutyryl-HIST1H1C (K51))

  • Elute under mild conditions to preserve epitopes (avoid harsh elution buffers)

  • Use the eluted material for a second round of ChIP with a different antibody

  • This approach reveals genomic regions containing both modifications simultaneously

• Co-immunoprecipitation followed by immunoblotting:

  • Use 2-hydroxyisobutyryl-HIST1H1C (K51) antibody for IP

  • Probe the immunoprecipitate with antibodies against other modifications

  • Alternatively, perform sequential immunoblotting after careful stripping

• Multiplexed fluorescence imaging:

  • Use primary antibodies from different species

  • Select secondary antibodies with non-overlapping fluorescence spectra

  • Include appropriate controls for antibody cross-reactivity

  • Apply spectral unmixing algorithms if bleed-through occurs

• Mass spectrometry-based approach:

  • Immunoprecipitate with 2-hydroxyisobutyryl-HIST1H1C (K51) antibody

  • Analyze the precipitated histones by mass spectrometry to identify co-occurring modifications

  • This method can identify combinations of modifications that may not be detectable by antibody-based methods

When designing multiplex experiments, researchers should validate that antibodies do not compete for overlapping epitopes and that the presence of one modification does not hinder detection of another. Pre-testing antibody combinations on known standards is essential before proceeding with complex samples.

How do patterns of 2-hydroxyisobutyrylation at HIST1H1C K51 differ between cell types and physiological conditions?

Analysis of 2-hydroxyisobutyrylation patterns at HIST1H1C K51 reveals distinct cell-type and condition-specific profiles:

These patterns suggest that 2-hydroxyisobutyrylation at K51 may function as a metabolic sensor that integrates cellular energy status with chromatin structure. The modification likely works in concert with other PTMs like citrullination, which has been shown to displace H1 from chromatin to promote open chromatin states during cellular reprogramming to pluripotency .

Researchers should note that these patterns may vary across experimental systems and should be validated in their specific cell types of interest. The dynamic nature of this modification requires careful consideration of sample collection timing, particularly in cell-cycle synchronized experiments.

What is the functional interplay between 2-hydroxyisobutyrylation at K51 and other post-translational modifications on HIST1H1C?

The functional interplay between 2-hydroxyisobutyrylation at K51 and other PTMs on HIST1H1C represents a complex regulatory network:

• Competitive modifications: 2-hydroxyisobutyrylation at K51 may compete with acetylation at the same residue. While acetylation of H1.4 (HIST1H1E) at K51 has been studied and is associated with specific cellular functions , the presence of 2-hydroxyisobutyrylation at this site would preclude acetylation, potentially creating a regulatory switch.

• Sequential modifications: Evidence from studies on other histones suggests that 2-hydroxyisobutyrylation may occur in sequence with other modifications. For example, prior phosphorylation of nearby residues might create a favorable environment for enzymes that catalyze 2-hydroxyisobutyrylation.

• Cross-talk with adjacent modifications: The status of nearby residues can influence K51 modification. Studies have shown that phosphorylation of H1.4 at Ser27 inhibits the binding of heterochromatin protein 1 (HP1) to methylated Lys26 . Similar interactions may exist involving the K51 residue.

• Modification readers: Different PTMs recruit specific "reader" proteins. The 2-hydroxyisobutyrylation mark likely recruits a different set of reader proteins compared to acetylation or other modifications at the same site, leading to distinct downstream effects.

• Metabolic influence: Unlike acetylation, which is directly linked to acetyl-CoA levels, 2-hydroxyisobutyrylation is connected to 2-hydroxyisobutyryl-CoA metabolism . This suggests that these modifications might respond to different metabolic cues, adding another layer of regulation.

Researchers investigating this interplay should employ techniques such as mass spectrometry to identify co-occurring modifications and implement genetic approaches to manipulate specific modifications and observe the effects on others.

What are the most effective strategies for identifying and characterizing enzymes responsible for adding and removing 2-hydroxyisobutyryl marks on HIST1H1C K51?

Identifying the "writers" and "erasers" of 2-hydroxyisobutyrylation at HIST1H1C K51 requires a multi-faceted approach:

• Candidate enzyme screening:

  • Test known histone acetyltransferases (HATs) for potential 2-hydroxyisobutyrylation activity

  • Screen sirtuin family members and histone deacetylases (HDACs) for eraser activity

  • Perform in vitro enzyme assays with recombinant HIST1H1C and candidate enzymes

• Unbiased proteomics approaches:

  • Conduct SILAC-based mass spectrometry to identify proteins that interact with 2-hydroxyisobutyrylated HIST1H1C

  • Use BioID or APEX proximity labeling with 2-hydroxyisobutyrylated HIST1H1C as bait

  • Perform whole-genome CRISPR screens looking for changes in 2-hydroxyisobutyrylation levels

• Metabolic dependency analysis:

  • Manipulate cellular metabolism to alter 2-hydroxyisobutyryl-CoA levels

  • Monitor resulting changes in K51 2-hydroxyisobutyrylation

  • Identify metabolic enzymes that influence modification levels

• Evolutionary analysis:

  • Compare 2-hydroxyisobutyrylation patterns across species

  • Identify conserved enzyme families that correlate with the presence of this modification

  • Use phylogenetic approaches to predict potential enzymes

Given that 2-hydroxyisobutyrylation is a relatively newly identified modification, researchers should be open to discovering novel enzymes rather than restricting investigations to known writers and erasers of other histone modifications. The characterization of these enzymes will be crucial for developing targeted interventions to manipulate this modification for experimental or therapeutic purposes.

How can computational approaches enhance our understanding of 2-hydroxyisobutyryl-HIST1H1C (K51) function in chromatin organization?

Computational methods offer powerful tools for elucidating the function of 2-hydroxyisobutyryl-HIST1H1C (K51) in chromatin organization:

• Molecular dynamics simulations:

  • Model the structural impact of K51 2-hydroxyisobutyrylation on HIST1H1C

  • Simulate interactions between modified HIST1H1C and DNA

  • Predict how this modification affects linker histone binding and dissociation kinetics

• Integrative genomics analysis:

  • Correlate ChIP-seq data for 2-hydroxyisobutyryl-HIST1H1C (K51) with other genomic datasets

  • Use machine learning to identify genomic features associated with this modification

  • Integrate with transcriptomics data to establish functional correlations

• Network analysis:

  • Construct protein-protein interaction networks centered on 2-hydroxyisobutyrylated HIST1H1C

  • Identify hub proteins and potential signaling pathways influenced by this modification

  • Model the dynamics of histone modification networks including 2-hydroxyisobutyrylation

• Sequence motif and structural prediction:

  • Analyze the sequence context of K51 in relation to known 2-hydroxyisobutyrylation motifs

  • Predict structural accessibility of K51 in different chromatin states

  • Develop algorithms to predict sites of 2-hydroxyisobutyrylation based on sequence and structural features

Recent studies have shown that 2-hydroxyisobutyrylation sites display distinct sequence motifs with a preference for negatively charged amino acids (D, E) in their vicinity . Computational approaches can extend these findings to predict how the modification at K51 specifically might influence HIST1H1C function in different cellular contexts and chromatin environments.