September 1, 2016 (Vol. 36, No. 15)

Mary Anne Jelinek Ph.D. Senior Research Scientist Active Motif

Choosing the Right Substrate Is Critical

Biochemical assays are a common approach used to identify inhibitors of epigenetic-modifying enzymes. Challenges that arise when designing these assays are centered on choosing histone substrates that are affordable, perform robustly, and most closely mimic the cellular environment in which the small molecule will ultimately function. This tutorial discusses substrate options for epigenetic enzyme activity assays and demonstrates the importance of substrate selection in the development of successful biochemical assays.

In the past decade there has been tremendous progress in our understanding of the mechanisms that lead to changes in cellular epigenetic landscapes and how these changes influence cell development, differentiation, and disease. Post-translational modification (PTM) of the unstructured histone tails is one of the principle epigenetic mechanisms that regulate chromatin structure and gene expression through a highly regulated process that requires the participation of enzymes that deposit and remove these modifications, as well as the effector proteins that bind the PTMs (also known as the “writers”, “erasers”, and “readers”, respectively).

Together, these formulate the histone patterns that dictate alterations in chromatin structure and, consequently, the interactions with underlying DNA that ultimately affect gene transcription. Although there are over 80 different known histone modifications, the most widely studied modifications are acetylation, which is associated with active chromatin and positively correlated with gene expression, and methylation, which, depending on the amino acid residue being modified and the degree of methylation, can be associated with either active or repressed chromatin.

In addition to influencing gene expression, it has more recently been observed that alterations in histone modifications are correlated with disease. There is growing evidence in the literature demonstrating a link between aberrant histone modification patterns and the onset and progression of several human pathologies, including autoimmune, neurological, inflammatory, and neoplastic disorders.

In cancer, mutations have been identified not only in histones, but in all classes of epigenetic-modifying enzymes, including histone methyltransferases (HMTs), histone demethylases (HDMs), histone acetyltransferases (HATs), and histone deacetylases (HDACs). The presence of these mutations and associated aberrant epigenetic landscapes in cancer has gained the attention of the pharmaceutical industry, resulting in significant investment in scientific resources to develop small molecule therapeutics that target the epigenetic machinery in order to inhibit or reverse cancer progression.

Efforts to develop epigenetic drug therapies have proven highly successful. Along with the discovery of inhibitors within all epigenetic enzyme classes, a novel class of inhibitors have also been identified that block nonenzymatic effector proteins, such as BRD4, from interacting with their modified lysine targets. There are currently 5 FDA approved drugs on the market that have epigenetic modes of action and greater than 15 more in single or combination clinical trials.

The effort to develop new and improved epigenetic inhibitors continues, and one of the common approaches utilized to identify inhibitors are homogeneous biochemical assays that use recombinant enzymes and substrates to screen for changes in enzymatic activity. Assays are typically configured to either detect an altered substrate, such as the gain or a loss of a histone modification, or to detect a cofactor by-product of the enzymatic reaction.

Both assay readout types present unique challenges. The former requires specific reagents, typically antibodies, capable of discriminating between subtle changes in the modification status of the substrate (e.g., the gain or loss of a CH3- methyl group). Therefore, the choice of substrate may affect assay performance, as antibody directed detection of a modification might differ between peptide, protein, or multi-protein substrates.

By contrast, assays that measure the conversion of a cofactor will not have detection variations due to substrate selection but may have more complex readouts that require coupling to additional enzymatic reactions. Additionally, these types of assays are generally less sensitive and require higher enzyme concentrations.

Substrate Choices

An additional challenge to developing these assays is the choice of substrate. The biological targets of the epigenetic enzymes within the cell are not free histones. Rather, histone targets are organized into nucleosomes that are further packaged into high order structures within chromatin. The nucleosome consists of histone octamers made up of two copies each of histones H2A, H2B, H3, and H4, around which DNA is wound. In regions containing actively expressed or transcribed genes, chromatin exists in an open and accessible structure, while, in transcriptionally repressed regions, chromatin is more condensed.

The complexity and structural variability of chromatin as the natural substrate represents a unique drug discovery challenge for these classes of enzymes because it is difficult to reproduce the chromatin structure in vitro. Therefore, alternative substrates, such as short modified peptides that can effectively mimic the histone tail, are often used in high-throughput screens.

However, peptides do not reproduce the complexity of the native biological substrate and do not always interact with epigenetic enzymes in a way that yields a productive reaction. Other types of substrates have been shown to perform better with specific enzymes and include 1) modified or unmodified full-length histones, 2) histone octamers, 3) mononucleosomes, and 4) oligonucleosomes (Figure 1).

Additionally, many of these substrates can be generated to include histone variants, such as H3.1 vs H3.3, or histone H3 containing cancer-associated mutations, such as lysine 27 to methionine (K27M), glycine 34 to arginine/valine (G34R/V) or lysine 36 to methionine (K36M) mutations that occur in some childhood cancers. Therefore, critical decisions need to be made during assay development with regard to what substrate choice will perform best with its associated enzyme in the assay.

Figure 1. Substrate choices for epigenetic enzymes.

Enzyme-Dependent Substrate Preferences

To illustrate how substrate choice may influence assay results, we chose to examine the activity of two histone methyltransferases, PRC2 and SETD2, in the presence of either short histone H3 or histone H4 peptides, full-length recombinant histone H3, octamers and nucleosomes (Figure 2). EZH2, the catalytic component of the multiprotein PRC2 complex, methylates histone H3 lysine 27, while SETD2 is a methyltransferase that catalyzes the methylation of histone H3 lysine 36.

The substrate preferences of the PRC2 and SETD2 methyltransferases were examined using an assay that measures the production of S-Adenosyl-L-homocysteine (SAH) from the methyl group donor S-Adenosyl methionine (SAM), a cofactor used by all methyltransferases. Measurement of SAH production as a catalytic by-product enables an unbiased comparison of substrate preference since SAH production is a reflection of catalytic activity and is not affected by substrate differences.

The data presented in Figure 2A shows that the preferred substrate for PRC2 is the mononucleosome. While full-length histone, assembled octamers, and peptide substrate are effective as substrates, they are less efficient substrates of the PRC2 complex, suggesting that PRC2 activity is regulated by nucleosomal environment in vivo so as to reduce the nonspecific activities on free histones prior to chromatin integration. 

The result of the SETD2 assay in Figure 2B also shows that enzyme activity is highly dependent on substrate selection. For SETD2, the nucleosome is the only effective substrate. This result is expected, as SETD2 is known to require intact nucleosomes and bind to histone H4 in the context of the nucleosome while depositing a methyl group on histone H3 lysine 36.

Figure 2. (A) PRC2 favors mononucleosomes over other substrate alternatives. Activity of PRC2, a complex of EZH2, EED, SUZ12, and RbAp46/48, toward a variety of substrates was measured using an HTRF assay detecting conversion of SAM to SAH. This assay format enables direct comparison of the various substrates but requires more enzyme. (B) SETD2 has an absolute requirement for nucleosome substrates. Activity of SETD2 toward a variety of substrates was measured using an HTRF assay.


The data presented above demonstrate that there are clear differences in epigenetic enzyme activities in biochemical assays depending on which substrate is provided. Having insight into the substrate requirements of an enzyme along with an understanding of the strengths and limitations of the selected assay platform are essential for developing successful and robust assays for epigenetic drug discovery.

Mary Anne Jelinek, Ph.D. is senior research
scientist at Active Motif.

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