Histone Methyltransferases: What we’ve learned about enzymes and substrates
The best way to describe the current state of histone methyltransferase enzymology is to admit that there is much that is simply not known. In the excitement to explore the potential for targeting HMTs therapeutically, there has been a paucity of systematic biochemical studies to carefully characterize the enzymes and the optimal conditions to support their in vitro activity. Moreover, some HMTs are multimers of four or five different proteins and some require intact nucleosomes as substrates. As many as nine proteins can be required for a fully reconstituted reaction.
All of this means that sourcing and testing reagents from different vendors can quickly become a complicated and expensive undertaking. There is no subtle way to say that this is why are developing the Transzyme Methyltransferase Assay kits, which include validated HMT enzymes and substrates combined with our Transcreener EPIGEN Methyltransferase Assay in turn-key kits. The following is some of what we have learned about HMT reagent sources and in vitro reaction methods in putting the Transzyme kits together. Most of it should be relevant regardless of the assay methods you use.
SAM. First, regardless of the assay method you will be using, it is best to use source of ultrapure SAM and to store it in a stable form, as SAM breaks down to SAH, which is inhibitory to most HMTs. We purchase it as a powder from AK Scientific, dissolve it in nuclease-free water and store it at -80. Based on analysis by HPLC, it is stable for at least six months in this format.
| Company | HMTs | Substrates |
| Abcam | No | yes (Few) |
| Active Motif | yes | yes |
| Anaspec | No | yes |
| BPS Bioscience | yes | yes |
| Cayman Chemical | yes | yes |
| EMD Millipore | yes | yes |
| New England Biolabs | yes (Few) | yes |
| ProMab | Yes | No |
| Reaction Biology | yes | yes |
| Sino Biological Inc | yes | No |
| Signal Chem | yes | Yes (Few) |
| R&D Systems | Yes | No |
| Table 1. Some commercial sources of HMT enzymes and substrates. | ||
Enzymes. After sampling purified HMT enzymes from several vendors, we decided to go with Reaction Biology as a source of enzyme for the Transzyme kits for a number of reasons. They have a great selection and are actively adding to the list, their enzymes are of high physical purity, and they generally have specific activity at least as high as enzymes from other vendors. Another important factor is that their scientific staff have a good understanding of HMT enzymology and are willing to work in a collaborative fashion. We have obtained very high quality HMT enzymes from other vendors (Table 1), but overall the consistency and the experience have been the best with Reaction Biology.
The HMTS that function as multimers are more expensive and tend to be difficult to work with. These include G9a/GLP (two proteins) EZH1 and 2 and MLL1-4 (five proteins); also SET 1A and B. Thus far, we have worked with G9a/GLP, EZH2, MLL1 and MLL4. MLL1 and EZH2 are very slow enzymes, and thus require a lot of protein to generate a good signal; we are still in the process optimizing their reaction conditions. Peptides were as effective as nucleosomes as substrates with all of the complexes; in the case of EZH2, a mono-methylated peptide is used and a trimethylated peptide serves as a “primer” for the reaction.
| HMT | Substrate |
| DOT1L | Nucleosomes |
| EZH2 | H3 Peptide (21-44) |
| G9a | H3 Peptide (1-25) |
| GLP | H3 Peptide (1-25) |
| G9a/GLP | H3 Peptide (1-25) |
| MLL4 | H3 Peptide (1-25) |
| NSD2 | Nucleosomes |
| PRMT1 | H4 Peptide (1-20) |
| PRMT3 | H4 Peptide (1-20) |
| PRMT4 | Histone H3.3 |
| PRMT8 | GST-GAR peptide |
| PRMT8 | GST-GAR peptide |
| SET7/9 | H3 Peptide (1-25) |
| SUVH391 | H3 Peptide (1-25) |
| Table 2. Optimal HMT substrates | |
Substrates. The three main types of HMT substrates are peptides, intact histones, and nucleosomes. Nucleosomes are expensive and even the highest quality preps contain some SAH, so histones or peptides are preferable. However, though some HMTs will use more than one type of substrate effectively, others have very little activity unless nucleosomes are used. Table 2 summarizes the results of our efforts to identify optimal HMT substrates for 14 HMTs; note that our studies were not exhaustive and these do not necessarily represent the best possible substrate. Of the HMTs we have worked with thus far, Dot1L and NSD2 require nucleosomes as substrate, and we have had better results with the mono- di-nucleosomes than the oligonucleosomes. Also, though we can detect activity using recombinant, reassembled nucleosomes, the native nucleosomes purified from HeLa cells have yielded the best results.
We are compiling the information we have gleaned in developing the Transzyme kits, with more detail, into a manuscript that we plan on submitting this summer. In the meantime, I hope that some of what is provided here is helpful in getting you up and running with in vitro histone methyltransferase reactions. And regardless of whether you are using BellBrook products, feel free to start a conversation about histone methyltransferase reagents or reaction conditions with our scientific staff.
