br Results and Discussion The

Results and Discussion The Set7 sequence is homologous to the other identified histone methyltransferases in S. pombe (Figure S1) and also shares similarity with human HMTases, H3K9 GLP/Ga9, and H4K20 SUV420H1 (Figure S1). Set7 fused with the GFP (Set7-GFP) localized in the nucleus and cytoplasm in an asynchronous culture (Figure 1A), leading us to hypothesize that Set7 may function as a chromatin modifier. In vitro assays revealed that Set7 methylates histone H3.1 and H3.1-peptide (amino acids 32–39) containing K36 and K37 (Figure 1B). H3K36 is a well-studied epigenetic histone mark, while H3K37 has been recently identified (Szerlong et al., 2010, Yu et al., 2017). H3K37 mark is involved in target of rapamycin complex 1 signaling regulation in Saccharomyces cerevisiae (Chen et al., 2013, Chen et al., 2016), but the H3K37 mark remain uncharted in S. pombe and humans. Furthermore, no methyltransferase targeting H3K37 has been discovered in eukaryotes so far. MALDI-TOF mass spectrometry on histone H3.1 incubated with Set7 ruled out H3K36 methylation but suggested H3K37 methylation by Set7 in vitro (Figures 1C and S1). In addition, methylation of H4K20, a key mark in chromatin dynamics, was not detected (Figure 1C). The methylation specificity of HMTases may depend on the nature of the substrate. However, Set7 consistently methylates H3K37 on a H3-peptide, histone H3.1 as well as nucleosome substrates (Figures 1B, 1D, 1F, S1, and S2). Levels of H3K37me1-3 increased in a Set7 dose-dependent manner in vitro (Figure 1D) and the level of all H3K37me1-3, but not H3K36me1-3, decreased in set7Δ Pam2CSK4 (Figure 1E). Set2 is known as the sole H3K36 methyltransferase in S. pombe (Morris et al., 2005). H3K36 methylation was abolished in set2Δ and set2Δset7Δ cells, and H3K37 methylation levels appeared to be affected in set2Δ and set2Δset7Δ cells (Figure 1E), implying that H3K36 methylation may play a role in K37 modification. One possible explanation for residual H3K37 methylation seen in set7Δ cells is the existence of additional HMTase(s) that can methylate H3K37 in S. pombe. Nonetheless, we could not exclude the possibility of a cross-reactivity issue with antibodies. To further validate Set7 as a bona fide H3K37 methyltransferase, we introduced point mutations on nine amino acids, R20A, E58A, Y59A, Y61A, N79A, H80A, W88A, Y115A, and W120A, which we predicted to be essential for Set7 enzymatic activity based upon a structural overlay with the Paramecium bursaria chlorella virus (PBCV-1) vSET H3K27 methyltransferase (discussed below) (Finn et al., 2015, Qian et al., 2006). Point mutations, Y59A, Y61A, N79A, H80A, W88A, and Y115A, completely abolished Set7 enzymatic activity on H3K37 mono-methylation in vitro (Figure S2). Set7Y61A failed to methylate K37 on histone H3.1 and nucleosomes in vitro (Figure 1F). Overexpression of Set7Y61A partially rescued the reduction in H3K37 methylation in set7Δ cells, compared with the excessive level of H3K37 methylation by wild-type (WT) Set7 overexpression (Figure 1G). These results suggest that H3K37, but not H3K36, is the target of Set7 (Figure 1). Thus, Set7 is the only identified methyltransferase responsible for H3K37 methylation in eukaryotes. Set7 is a short 147-amino acid histone methyltransferase with its catalytic SET domain starting on the N-terminal region and spanning over 70% of the protein (Figure 2A), which is similarly observed in the PBCV-1 vSET H3K27 methyltransferase structure (Finn et al., 2015, Qian et al., 2006). In S. pombe, the SET domain of Clr4 (PDB: 1MVX) is the only known structure of a histone methyltransferase, so far (Min et al., 2002). Here, we solved the Set7 crystal structure at 2.0 Å and discovered that the structure of Set7 is homo-dimeric (Table 1, Figures 2A and S3). Each Set7 monomer bears a canonical SET domain fold with three β sheets located around a pseudo-knot structural arrangement (Figure 2A) (Aravind and Iyer, 2003). Set7 is structurally homologous to PBCV-1 vSET (PDB: 3KMT), with a Cα root-mean-square deviation of only 1.1 Å, while sharing only 23% sequence identity (Figures 2B, 2C, S1, and S3). Both Set7 and PBCV-1 vSET monomers share a highly similar fold with an arrangement of 3 α helices and 10 β sheets. Structural comparison with other known human HMTases such as MLL3 (PDB: 5F59) and SUV420H2 (PDB: 3RQ4) reveals a nearly identical fold of their SET domains, despite different histone-lysine substrate preferences (Figure S3).