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To confirm that dG9a has HMTase activity, it was incubated with H3_-S-adenosyl

methionine (SAM) and different substrates (Figure 4.19). Drosophila G9a methylated

H3 and H4 present as free histones but had no detectable activity on nucleosomal arrays.

Recombinant as well as native H3 and H4 were methylated by dG9a (Figure 4.19.A). Mouse G9a methylated H3 alone and H3 in a mixture of recombinant and native histones and had a very low activity on reconstituted nucleosomes (Figure 4.19.B).

Figure 4.19 Characterization of recombinant dG9a. (A)Invitro methylation reactions using dG9a

(lanes 1-6), no enzyme (lane 7) and SU(VAR)3-9 (lane 8). In the reaction 1 µg of different histones were used: recombinant histone H3 (lane 1), recombinant histone H4 (lane 2), recombinant (lane 3) and native histone octamer (lane 5) and recombinant and native nucleosomes (lane 4 and 6) reconstituted on circular pBS(KS) from equimolar amounts of histones. The upper panel shows Coomassie stained gel and the lower panel the autoradiograph. (B) Activity of recombinant mouse G9a expressed in

baculovirus infected cells (a kind gift from S. Pradhan). HMTase activity on 1 µg of different histone substrates: recombinant histone H3 (lane 1), recombinant histone H4 (lane 2), recombinant and native histone octamers (lane 3 and 5) and recombinant and native nucleosomes (lane 4 and 6). Mock control (lane 7) is incubation of recombinant octamer without enzyme. The Coomassie gel is shown at the top and the corresponding autoradiograph at the bottom.

Mouse G9a methylated only H3 whereas dG9a methylated H3 and H4. In order to exclude that the activity towards H4 was due to a contaminating activity co-purifying with dG9a, a corresponding enzyme carrying a H1536K mutation within the conserved SET domain was expressed. The same mutation in SU(VAR)3-9 abolished its enzymatic activity (Figure 4.7). The mutated enzyme had no activity towards H3 and H4 indicating that both were methylated by dG9a (Figure 4.20). It cannot be excluded that the H4 activity may be due to the deletion of the N-terminus of dG9a.

Figure 4.20. The H1536 mutation abolished the enzymatic activity of dG9a. Activity ofFlag-dG9a

wild type versus dG9a containing a H1536K mutation in the conserved region of the SET domain. The upper panel shows a western blot of the two proteins. Recombinant octamer (2 μg) was used as substrate for 25, 50 and 100 ng of wt (lane 1-3) and H1536K mutant (lane 4-6). The corresponding autoradiograph is shown in the lower panel.

Mouse G9a has been shown to methylate H3K9 and K27 (Tachibana et al., 2001; Tachibana et al., 2002). To define substrate specificity of dG9a, H3 molecules carrying a lysine to alanine replacement at position 9 and 27 or both were used (Figure 4.21). Decreased methylation efficiency on H3K9A and H3 K27A compared to wild type H3 was observed. In a filter binding assay a 70% reduction for the K9 mutant and a 50% reduction for the K27 mutant was observed. When both H3 lysine residues were mutated (K9A and K27A) a lower activity was observed (efficiency of 27%) indicating that in absence of K9 and K27 dG9a was also able to methylate other lysines. When a highly active full-length mG9a (Patnaik et al., 2004) was used, it methylated wild type H3 and the H3 molecules carrying a single mutation on K9 or K27 with a similar efficiency. Mouse G9a also showed a decreased activity (27%) towards the double mutant (K9A/K27A). However, it can not be excluded that K9 was methylated faster than K27, as initial rate kinetics were not performed (Collins et al., 2005; Esteve et al., 2005).

Figure 4.21 dG9a methylates H3K9 and K27. (A)In vitro methylation of 2 µg of recombinant H3

(lane 1), H3 mutated at lysine 9 (lane 2), H3 mutated at lysine 27 (lane 3) or both (lane 4) using dG9a and a mock purification. Coomassie stained H3 is shown in the upper panel and a corresponding autoradiography in lower panel. A corresponding filter binding assay is shown to the right. The y-axis displays the percent radioactivity incorporated on 2 µg histone H3 and H3 mutants K9A, K27A and K9/K27A with radioactivity incorporated on H3 wt set to 100 % and the background is subtracted. (B)

HKMTase activity of mG9a on histone H3 molecules and H3K9A, H3 K27A and the double mutant K9A/K27A. A gel of Coomassie stained histones and the corresponding autoradiography is shown. On the right, a filter-binding assay showing percent radioactivity incorporated on two µg histone H3 and H3 mutants K9A, K27A and K9/K27A. The y-axis displays the percent radioactivity incorporated with activity on H3 wt set to 100 % and the background is subtracted.

Interestingly dG9a was also able to methylate histone H4 (Figure 4.19 and 4.20). This activity was not shown for mouse G9a (Figure 4.19.B) (Tachibana et al., 2001). The only lysine residue shown to be methylated in H4 is lysine 20 and the first HMTase identified with this activity was hPR-Set7/dSET8 (Fang et al., 2002; Rice et al., 2002). Other HMTases in Drosophila shown to methylate H4 lysine 20 are Ash1 and

Ash1 was in addition able to methylate lysine 4 and 9 in histone H3 (Beisel et al., 2002). Comparing the similarity within the SET domain, mouse NSD1 has closest homology to SET family member Ash1 (Huang et al., 1998) and NSD1 SET domain methylate H3K36 and H4K20 in vitro (Rayasam et al., 2003). The data indicated that

dG9a also was a multi-catalytic histone methyltransferase with specificity for lysine 9 and 27 on H3 and possibly lysine 20 on histone H4. Surprisingly when dG9a was incubated with H4 carrying a mutation of lysine 20 to alanine no reduction of activity was observed (Figure 4.22). To further investigate the specificity, dG9a activity was tested on different N-terminal H4 mutants (Clapier et al.,2002). dG9a could methylate the H4 N-terminus when the first five amino acids were deleted, excluding K5 as a possible substrate. However, the activity was lost when a H4 Δ10 mutant was used (Figure 4.22). This suggests that the substrate was K8, but knowing that the minimal substrate specificity for mG9a surrounding K9 contains seven amino acids (TARKSTG) (Chin et al., 2005) the substrate could also be K12 or K16.

Figure 4.22 dG9a methylates H4 K8, K12 or K16. Amino acid sequence of the H4 N-terminus is

shown at the top. Asterisk indicates possible substrates for dG9a in vitro. dG9a methylation assay

with 2 μg of recombinant H4 (lane 1), H4 K20A (lane 2), H4Δ5 (lane 3), H4Δ10 (lane 4), H4Δ15 (lane 5) and globular H4 (lane 6). Mock control (lane 7) is incubation of wt H4 without enzyme.

Using MALDI-TOF analysis to measure the activity of dG9a on H3 confirmed the finding that K9 was the major substrate (Figure 4.23). Four times more activity towards K9 compared to K27 was observed. As shown for mG9a (Patnaik et al., 2004, Collins et al., 2005) dG9a was able to add three methylgroups to H3K9.

Tri-methylation of H3 peptide 27-40 was very inefficient and could only be detected when 200 ng of enzyme was used (data not shown). This was consistent with MALDI-TOF data of mouse G9a where H3 K27 methylated peptides were slowly generated (Collins et al., 2005). All visible peptides by MALDI-TOF of methylated H3 and H4 were analyzed and no other modified lysines were detected (data not shown). Peptide 4-17 on H4 methylated by dG9a, was mono-, di-, and trimethylated providing a proof of a lysine methylation (Figure 4.23). This was a novel finding that a SET domain HMTase can methylate another lysine in the H4 N-terminus than K20. There is yet no strong evidence of any of these methyl marks being present on the H4 N-terminus in vivo, and it remains to be seen to what level these lysines are

methylated and what the function of this methylation is.

Figure 4.23 MALDI-TOF analysis of dG9a in vitro methylated H3 and H4. A MALDI-TOF

analysis of 500 ng H3 and H4 methylated by 100 ng dG9a. Peptides spanning amino acids 9-17 and 27-40 of H3 and 4-17 of H4 is represented by graphs. Mono-, di- and trimethylation is shown as percent of total H3 or H4. This figure is representative for at least three different methyltransferase assays.