Histones are a group of positively charged proteins that are the main building blocks of chromatin, the complex of DNA and proteins that form the structure of chromosomes in eukaryotic cells. By compacting DNA while modulating its access to enzymes, histones play a crucial dual role in regulating gene expression and maintaining genome integrity.

There are five major classes of histones: H1, H2A, H2B, H3 and H4. Each of these classes has multiple isoforms with slightly different properties. Histones H2A, H2B, H3 and H4 form the core of the nucleosome, which is the basic repeating unit of chromatin. The nucleosome consists of two copies of H2A, H2B, H3 and H4, around which the DNA is wound into a left superhelix. Histone H1, which binds to the linker DNA between the nucleosomes, helps to further condense the chromatin into higher order structures, such as the 30 nm fibre and the chromosome.

Digging into our past, histones can be traced back to phylogenetic groups of unicellular Archaea, and the histone doublet was probably a key intermediate in the transition from archaeal to eukaryotic histones (1). Constraints on single histone folding may have been too great to allow the diversification seen in the eukaryotic histone octamer.

But despite their high conservation, a combination of genetic, environmental, and lifestyle factors can contribute to the alteration of histones through post translational modifications.

Histone modifications refer to covalent modifications of histone proteins and include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, glycosylation, butyrilation, crotonylation, propionylation, succinylation, malonylation, glutarylation, palmitoylation, hydroxylation, citrullination and amino-acid mutation.

The specific epigenetic modulations can regulate various cellular processes, such as gene expression, DNA replication and DNA repair, and may vary according to cell type and cellular context. Their functions may also be influenced by other factors such as other post-translational modifications, chromatin structure and interactions with other proteins.

The addition of one (Ac) or two (Ac2) acetyl groups to lysine residues on histone proteins, which is associated with transcriptional activation. The most common acetylation modifications on histones occur on the lysine residues of the N-terminal tails of histone proteins.

The addition of one or more ADP-ribose molecules to specific amino acid residues on histones, typically on glutamate or aspartate residues. This modification is catalyzed by enzymes known as poly(ADP-ribose) polymerases (PARPs), which transfer ADP-ribose units from NAD+ onto histones. PARPs are activated in response to DNA damage, and histone ADP-ribosylation is a crucial event in the DNA damage response and repair pathways.

The addition of one (mono) or two (di) butyryl groups to lysine residues on histone proteins. This modification is catalyzed by histone acetyltransferases (HATs) that can also perform acetylation reactions. However, some specific HATs are known to preferentially perform butyrylation over acetylation.

Also known as histone deimination, is a post-translational modification that involves the conversion of the amino acid arginine to citrulline in histone proteins. This modification is catalyzed by a family of enzymes known as peptidylarginine deiminases (PADs), which are calcium-dependent enzymes.

The addition of one (mono) or two (di) a crotonyl groups (-CH=CH-CO-) to lysine residues on histones. Crotonylation is a relatively new histone modification, and its biological functions are still being investigated.

Like histone crotonylation, propionylation, succinylation, and malonylation, glutarylation involves the addition of one (mono) or two (di) chemical groups. Specifically, a glutaryl group (-CH2CH2CH2CO-) is added to lysine residues on histones.

Glycosylation is a post-translational modification of histones that involves the addition of carbohydrate molecules to specific amino acid residues. Glycosylation of histones is not as well studied as other post-translational modifications such as acetylation and methylation. There are two types of histone glycosylation, including O-GlcNAcylation, which involves the addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues on histones, and O-fucosylation, which involves the addition of O-linked fucose to serine and threonine residues.

Unlike other histone modifications, hydroxylation involves the addition of a hydroxyl group (-OH) to lysine and arginine residues on histones.

Like histone crotonylation, propionylation, and succinylation, malonylation involves the addition of one (mono) or two (di) chemical groups. Specifically, a malonyl group (-CH2CO-) is added to lysine residues on histones.

The addition of one (me), two (me2) or three (me3) methyl groups to lysine or arginine residues on histone proteins, which can be associated with both transcriptional activation and repression, depending on the specific lysine or arginine residue that is modified.

Involves the addition of a lipid molecule, palmitate, to cysteine residues on histones. Studies have shown that palmitoylation levels are elevated during neuronal differentiation, and that palmitoylated histones may promote the expression of genes involved in neuronal development.

Like histone crotonylation, propionylation involves the addition of one (mono) or two (di) chemical groups. Specifically, a propionyl group (-CH2CH2CO-) is added to lysine residues on histones.

The addition of a phosphate group to serine, thyrosine or threonine residues on histone proteins, which can be associated with both transcriptional activation and repression, depending on the specific serine or threonine residue that is modified.

The addition of a single (mono) or several (multi) ubiquitin molecules to lysine residues on histone proteins, which is associated with transcriptional repression.

Like histone crotonylation and propionylation, succinylation involves the addition of one (mono) or two (di) chemical groups. Specifically, a succinyl group (-CH2CH2CO-CH2CO-) is added to lysine residues on histones.

The addition of a single (mono) or several (multi) small ubiquitin-like modifier (SUMO) molecules to lysine residues on histone proteins, which can be associated with both transcriptional activation and repression, depending on the specific lysine residue that is modified.

DNA sequence that encodes this amino acid has been altered. Depending on the type of mutation, this can result in a change to the amino acid itself or to its surrounding environment, potentially affecting the way the histone interacts with other proteins and DNA.

Understanding the roles and functions of the different histone modifications is a rapidly developing field of research and has implications for various fields, including oncology research and drug development for various diseases.

Aberrant histone modifications have been linked to numerous diseases, including cancer, neurological disorders, and immunological disorders (e.g. diabetes). For example, the global loss of histone acetylation has been observed in cancer cells, leading to transcriptional silencing of tumor suppressor genes.

For the most well known modifications, the example of H3K36 methylation is quite telling. H3K36 methylation is a type of histone modification that occurs on lysine 36 of the histone H3 protein. This epigenetic modification is known to play a crucial role in gene expression and chromatin structure, and dysregulation of H3K36 methylation has been associated with various diseases, including cancer.

In particular, H3K36 methylation has been shown to have a positive impact on transcription elongation, which is the process by which RNA polymerase moves along the DNA template to synthesize RNA. This modification recruits proteins that promote RNA polymerase movement and helps to prevent RNA polymerase from stalling or falling off the DNA template during transcription.

Additionally, H3K36 methylation is involved in DNA repair and genome stability. The modification can recruit proteins involved in DNA damage response and repair, and loss of H3K36 methylation has been linked to defects in DNA repair and increased genomic instability.

Furthermore, H3K36 methylation can also influence alternative splicing, which is the process by which different forms of mRNA are generated from a single gene. The modification can recruit splicing factors that regulate alternative splicing and affect the choice of splicing sites.

One specific example of a mutated H3K18 residue is H3K18Q, which stands for a substitution of lysine (K) with glutamine (Q) at position 18 of histone H3. This mutation has been linked to a type of pediatric brain cancer called diffuse intrinsic pontine glioma (DIPG), as well as other cancers, and has been shown to promote tumorigenesis and alter gene expression patterns.

Histone modifications can be detected using a variety of techniques, including chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq), which allows for the identification of genomic regions that are bound by specific histone modifications, and western blotting, which allows for the detection of specific histone modifications in protein samples. Specific monoclonal antibodies can be used for the detection of histone modifications by these techniques.

The detection of histone modifications is crucial for understanding their role in various cellular processes and disease states. One method for detecting histone modifications is through the use of specific monoclonal antibodies.

Nevertheless, it is known to be extremely difficult to generate antibodies against such complex antigens:

• Given their crucial role in the regulation of gene expression in all eukaryotes, histones are among the most conserved proteins in all organisms. A naive or synthetic library approach to antibody generation, therefore, seems impossible. If we focus on an in-vivo approach to antibody generation, we need to use technologies that can break tolerance mechanisms on almost 100% homologous antigens.

• The different histone isoforms - H2A, H2B, H3, and H4 - share a high degree of sequence similarity, which can make it difficult to generate antibodies that distinguish between them.

• In the context of chromatin, histones are tightly bound to DNA, which can mask some of the epitopes on the histones. We will also add that post-translational modifications alter histone conformation and accessibility to the epitope. This can make it very difficult for antibodies to bind to histones due to steric hindrance.

• Histones are highly abundant proteins in cells, and their abundance can interfere with the generation of antibodies that specifically recognize them. 

• We also believe at SYnAbs that the immune system rapidly sets up a control loop to prevent the generation of DNA-interfering autoantibodies, automatically excluding and destroying any splenocytes that might pose a risk to the maintenance of the host's genetic integrity. B-cell populations secreting antibodies specific to histone modifications are therefore extremely rare.

SYnAbs antibodies are highly specific for the modified histone residues and can be used in a variety of applications, including western blotting (WB), immunoprecipitation, and chromatin immunoprecipitation (ChIP).

Our mouse and rat monoclonal antibodies to histone post-translational modifications offer high specificity, simplicity, visual representation, and cost-effectiveness for studying individual histone modifications:

• H3K18Ac: specific monoclonal antibody to acetylation of lysine 18 on histone H3

• H3K27me2me3: specific monoclonal antibody with cross-reaction to di and triméthyl groups on Histone 3 without cross-reactivity to monomethyl group on Histone 3

• H3K27me3: specific monoclonal antibody to triméthyl group on Histone 3 without cross-reactivity to monomethyl or dimethyl group on Histone 3

• H3K36me3: specific monoclonal antibody to triméthyl group of lysine 36 on histone H3, without cross-reaction to monomethyl or dimethyl group on Histone 3

• panH3: specific monoclonal antibody to detect Histone 3

• panH4: specific monoclonal antibody to detect Histone 4

• pan-Nucléosome: specific monoclonal antibody to detect all histones

Antibodies to epigenetic modifications have the potential to revolutionize disease monitoring by providing a new level of specificity in targeting epigenetic changes associated with various pathologies. By recognizing specific modifications on histones, these antibodies can be used to selectively target cells that have aberrant epigenetic patterns, such as cancer cells or cells affected by autoimmune disorders and so shed light on the mechanisms underlying different diseases.

Epigenetic therapy - such as EZH2, LSD1, HDAC, DNMT or BET inhibitors - has the potential to convert a tumour from an immune cold state to an immune hot one by modulating the immune composition of the tumour microenvironment.

In fact, these drugs can deplete myeloid-derived suppressor cells (MDSCs) and regulation of macrophage M1/M2 polarization. Additionally, they increase the number of effector T cells. For instance, G protein-coupled CXC-chemokine receptor 3 (CXCR3) and CC-chemokine receptor 7 (CCR7) are epigenetically regulated in antigen-specific T cells, ensuring the transition from a naive to effector phenotype state.

(1) Fahrner RL, Cascio D, Lake JA, Slesarev A. An ancestral nuclear protein assembly: crystal structure of the Methanopyrus kandleri histone. Protein Sci. 2001 Oct;10(10):2002-7. doi: 10.1110/ps.10901. PMID: 11567091; PMCID: PMC2374223.