Specificity analysis of MLL3 in complex with WDR5, RBBP5 and

As it was already described for MLL1 in 4.3.1.2.1, a peptide array was used to investigate the influence of each amino acid in the H3(1-15) template for the substrate recognition by MLL3. In the case of MLL3, methylation of the substrate specificity array was performed in presence of complex partners WDR5, RBBP5 and ASH2L, as MLL3 was only active in presence of complex partners (4.3.2.1). After methylation of the peptide array with the MLL3-WRA

protein complex in the presence of radioactively labeled AdoMet, the methyl transfer was analyzed by autoradiography. The experiment was carried out twice and the average activities were calculated and plotted in Figure 27A. The calculation of the standard deviation for the methylation activity of each peptide from both experiments showed that the results are highly reproducible with a SD smaller than 20% for 92.5% of the peptides. 99% of the peptides have a SD < 30% and only 2 peptides showed a SD between 30 and 40% (Figure 27B).

The results show that MLL3-WRA recognizes the template H3 (1-15) residues from A1 to T6. At position -2, MLL3-WRA mainly tolerates the canonical arginine and only weakly accepts leucine. Alanine at position -3 can be replaced by all other hydrophobic residues, additionally serine and threonine were accepted. Beside glutamine at position +1, MLL3- WRA has also a good activity with asparagine, histidine and methionine. In summary, the substrate specificity recognition motif can be described as follows: A(ILFP)-R(L)-T(AILMFYV)-K-Q(NHM)-T(AQEILSV).

Figure 27: Substrate specificity analysis of MLL3 in complex with WRA. A) Two independent peptide array methylation

experiments were performed with MLL3+WRA and the data were averaged after normalizing the full activity to 1. The activity is displayed in the grey scale as indicated. The horizontal axis represents the template sequence (H3 1-15). Each residue was exchanged against all 20 natural amino acid residues as indicated by the vertical axis. B) Distribution of standard errors of the mean of MLL3+WRA on all peptides tested in the two independent assays.

N u m b e r o f s p o ts Error range A R T K Q T A R K S T G G K A A R N D C Q E G H I L K M F P S T W Y V Original Sequence E x c h a n g e t o Rel. activity

To screen the human proteome for possible non-histone targets of MLL3, a Scansite search with the substrate recognition motif was conducted and 42 potential non-histone substrates were identified (Obenauer et al. 2003). To check if the identified non-histone targets can be methylated at peptide level, 15 amino acid long peptides were synthesized on a cellulose membrane. The peptide array was incubated with MLL3 complex containing WDR5, RBBP5 and ASH2L in presence of radioactively labeled AdoMet and the methyl group transfer was detected by autoradiography. Out of the 42 putative non-histone targets (Table 5, see appendix), 24 were methylated (Figure 28A). Out of these, 10 target protein domains were selected (Table 2), overexpressed and purified by affinity chromatography (Figure 28B).

Figure 28: Screening of MLL3 non-histone targets. A) The peptide array was methylated with MLL3 together with WRA in

presence of radioactively labeled AdoMet. The transfer of the methyl group to the immobilized target lysine residues was detected by autoradiography. Target information is listed in Table 5. B) 16% SDS gel documents the purification of 10 non- histone targets. The corresponding bands of the expected size are labeled with asterisk.

Table 2: Selected non-histone targets from the peptide array (Figure 28A), which were further analyzed. Swiss

Prot. Nr. Original name Name Sequence

Protein- length MW (kDa) Target K position A6 Q8WYP5 ELYS_HUMAN Protein ELYS DKQLRIKHVRRVRGR 2266 253 1933

A13 P78412 IRX6_HUMAN

Iroquois-class homeodomain protein IRX-6

SGAGRRKNATRETTS 446 48 151

A16 Q7L590 MCM10_HUMAN Protein MCM10 homolog PALPRTKRVARTPKA 875 98 187

A19 Q8IYA7 MKX_HUMAN Homeobox protein

Mohawk NARRRLKNTVRQPDL 352 39 129

B9 Q8N488 RYBP_HUMAN RING1 and YY1-binding

protein TSRPRLKNVDRSTAQ 228 25 149

B11 P42229 STA5A_HUMAN

Signal transducer and activator of transcription 5A / 5B

MSLKRIKRADRRGAE 794 91 425

B17 Q8IY57 YAF2_HUMAN YY1-associated factor 2 KTRPRLKNVDRSSAQ 180 20 106

B19 Q86UD4 ZN329_HUMAN Zinc finger protein 329 MRLKMTTRNFPEREV 541 62 4

B20 O60290 ZN862_HUMAN Zinc finger protein 862 DGPRRIKRTYRPRSI 1169 132 457

C2 Q8IYH5 ZZZ3_HUMAN ZZ-type zinc finger-

containing protein 3 PVLKRIKRCLRSEAP 903 102 117/120

Equal amounts of RYBP, YAF2, MKX and ZN329 (Figure 29A) were incubated with the MLL3-WRA complex (Figure 29B) in a reaction mixture containing radioactively labeled AdoMet, to investigate methylation of the candidate substrate proteins. As positive control, H3 was included. To rule out any methylation signal from the MLL3 or its complex partners, MLL3-WRA was also incubated under the same conditions without any substrate. Strong methylation of the recombinant H3 was detected. In addition, methylation of ASH2L was observed. Further investigation is necessary to analyze if MLL1 catalyze the methylation of ASH2L or if ASH2L automethylation is observed. However, even after long exposure, none of the four selected non-histone targets was methylated by MLL3 in presence of complex partners (Figure 29C).

Figure 29: Methylation of non-histone proteins by MLL3. A) 16% SDS gel of all selected non-histone targets serving as

input control of the protein amounts of RYBP, YAF2, MKX and ZN329 that were used for methylation. B) 16% SDS gel of MLL3 with the complex proteins WDR5, RBBP5 and ASH2L showing that roughly equal protein amounts of all proteins were used for methylation C) Methylation of RYBP, YAF2, MKX and ZN329 by MLL3 in presence of WRA. Methyl transfer was detected by autoradiography. The corresponding bands of the expected size are labeled with asterisk. Exposure times are indicated. a= methylation of ASH2L; b= automethylation of MLL3; c= methylation of recombinant H3.

4.3.2.2.1. Conclusion and outlook of the MLL3 experiments

MLL3 substrate specificity peptide arrays were used to analyze the interaction of MLL3 complex with the amino acids of the H3 peptide (1-15). Using a Scansite search, 42 potential non-histone targets with the identified substrate recognition motif were found. Out of these, 24 peptides were methylated and 10 corresponding proteins were successfully overexpressed and purified. Methylation was performed for RYBP, YAF2, MKX and ZN329 with MLL3 in complex, but no methylation of any of the non-histone substrates could be identified. More research is necessary to test the potential methylation of the other non-histone protein domains (IRX, MCM, ZN862, ZZZ3, ELYS and STRA5A). In addition, more targets can be selected on the peptide array to test methylation at protein level. Furthermore, methylation of the targets should be performed with MLL3 in absence of complex partners, because it is possible that WRA influences the specificity of MLL3.