Histone modifications are dynamically added and removed from histone proteins by specific enzymes. The balance between these writers and erasers dictates which marks are present on histones, and at what levels, to ultimately control whether specific genetic programs and the cellular processes they orchestrate, are turned on or off.

The major categories of histone writers and erasers:

For more details on the readers, writers, and erasers of histone modifications take a look at our epigenetic modification’s poster.

Identifying modification pathways and the specific writers and erasers at play can reveal:

• Relevant cellular pathways, genetic programs and physiological effects for further investigation. For example, histone deacetylases (HDACs) activate immune developmental pathways, while histone acetyltransferases (HATs) play a crucial role in differentiation and proliferation.
• Imbalances between writers and erasers that alter genetic programming and underlie disease processes. Characterizing such imbalances, and the specific enzymes involved, can provide insights into disease pathology, from cancers to autoimmune disorders.
• New drug targets and therapeutic strategies. Once an imbalance is identified, drugs can be developed to impact the activity of these enzymes and correct the imbalance, offering new therapeutic strategies against diseases that have thus far evaded medical efforts. For example, many HDAC inhibitors are in development as novel drugs against cancers and inflammatory diseases like arthritis and type I diabetes.

For drug development efforts, compounds can easily be screened for their impact on writer and eraser activity.

In general, histone methyltransferase (HMT) assays are challenging to develop, and most have several drawbacks due to assay design. Typical HMT assays utilize 3H-SAM as a methyl donor and measure S-adenosylhomocysteine (SAH) as a general by-product of the methylation reaction. However, this requires

• Handling radioactive material
• High sensitivity to overcome low k_cat (turnover typically < 1 min^-1) and K_M values for the methyl donor, SAM
• Prior purification of enzyme/protein complexes to assess activity of specific HMTs

Abcam HMT activity assays overcome these difficulties, assessing the activity of specific HMTs with antibodies that detect the specific methylated product, providing:

• Easy colorimetric or fluorometric detection, without radioactivity
• Compatibility with nuclear extracts, or purified proteins (assay is specific for the modification of interest)
• Data in 3 hours

Find out more about our histone methylation assays.

Histone demethylase activity assays typically measure the formation of formaldehyde, a by-product of demethylation. They are therefore susceptible to interference from detergents, thiol groups and a range of ions. Similar to methylation assays, these assays are not specific for any demethylase and can only be performed with purified protein.

Abcam’s histone demethylase assays circumvent these issues by directly measuring the formation of the demethylated product, providing:

• Increased sensitivity (20–1,000 fold) over formaldehyde-based assays
• More accurate data without interference from thiols, detergents or ions
• Compatibility with nuclear extracts or purified protein (due to the assay’s specificity for the modification of interest)
• Measures demethylase activity from a broad range of species including mammalian cells/tissues, plants, and bacteria
• Fast microplate format with simple colorimetric or fluorometric readouts
• Data in 3 hours

Find out more about our histone demethylase assays.

Abcam offers kits to analyze overall, as well as H4-specific, HAT activity. These assays measure the HAT-catalyzed transfer of acetyl groups from the Acetyl-CoA donor to histone peptides, which generates the acetylated peptide and CoA-SH. The CoA-SH byproduct is then be measured via colorimetric or fluorometric methods:

• Colorimetric assays- CoA-SH serves as an essential coenzyme for producing NADH, which reacts with soluble tetrazolium dye to generate a product that can be detected spectrophotometrically. This assay is ideal for kinetic studies, with continuous detection.
• Fluorometric assays- CoA-SH reacts with a developer and Probe to generate a product that is detected fluorometrically.

HDAC proteins fall into four major groups (class I, class IIA, class IIB, class III, class IV) based on function and DNA sequence similarity. Classes I, IIA, and IIB are considered "classical" HDACs whose activities are inhibited by trichostatin A (TSA), whereas class III is a family of NAD⁺-dependent proteins (sirtuins (SIRTs)) not affected by TSA. Class IV is considered an atypical class on its own, based solely on DNA sequence similarity to the others.

Each of these classes are associated with different cellular programs and may be assayed individually with various fluorometric assays. For example, SIRTs are typically associated with cancers and neurological diseases. Detecting SIRT activity, or identifying drugs that impact SIRT activity, may point to novel diagnostics or therapeutic strategies for these diseases.

Fluorometric assays utilize an acetylated peptide substrate with a fluorophore and quencher at its amino and carboxyl terminals. Once the substrate is deacetylated, it can be cleaved by a peptidase, releasing the fluorophore from the quencher. The subsequent increase in fluorescence intensity of the fluorophore is directly proportional to deacetylase activity.

It can be useful to inhibit these modifying enzymes using small molecules and then assess downstream consequences to probe the involvement and biological functions of histone modifications. Thus, inhibitors of writers and erasers are vital tools for understanding the roles of epigenetic modification pathways. They are also essential for the validation of “druggable” targets in the context of pre-clinical studies both in academic and industry contexts.

Find out more about our range of histone methyltransferase and demethylase inhibitors.