Beisel et al.
Supplementary Information
Methods
Plasmids
Baculovirus expression plasmids: Baculovirus expression plasmids were generated by
inserting ash1 cDNA-fragments into pVLFLAG28. pVLFLAG-ASH1∆N and pVLFLAGASH1(SET) were generated by inserting PCR-amplified cDNA fragments encoding ASH1
amino acids 1032-2217 and 1032-1619, respectively. The ASH1N10, ASH1N21 and
ASH1N1142 single amino acid point mutations were introduced into the ash1 cDNA using
the “QuickChange Site-Directed Mutagenesis Kit” (Stratagene). Following mutant
oligonucleotides
were
used:
5’-cgcaattgggttcgcaagacttgttgacaaacctacaatcg
and
5’-
cgattgtaggtttgtcaacaagtcttgcgaacccaattgcg to generate the ash10-mutation in ASH1N10. 5’gcagcgattgtaggtttgtcatccattcttgcgaacccaattgc
cctacaatcgctgc
to
generate
the
and
5’-gcaattgggttcgcaagaatggatgacaaa
1142-mutation
in
ASH1N1142.
5’-
ccgcatggtttatacgaagtgctcgcccagc and 5’-gctgggcgagcacttcgtataaaccatgcgg to generate the
ash21 mutation in ASH1N21. Plasmids for the in vitro expression of ASH1N, ASH1N10,
ASH1N21 were generated by inserting the ash1 cDNA-fragments described above into
pTSTOP28. To express GST-TRX(SET) fusion-proteins in bacteria a trx cDNA fragment
encoding TRX amino acids 3389-3759 into pGEX2TKN (ref. 28). The expression plasmid for
TAFII110 has been described (ref. 28).
Schneider cell expression plasmids: A cDNA fragment encoding the DNA binding domain of
Gal4 [Gal4DBD] (amino acids 1-147), was inserted into pTSTOP (ref. 28) to create
pTSTOPGAL4DBD. cDNA fragments of ASH1∆N, ASHN110, ASHN121 were inserted
into pTSTOPGal4DBD to create pTSTOPGal4(DBD)ASH1∆N, -ASH1N10, -ASH1N21
respectively. SmaI-SmaI fragments from pTSTOPGal4DBDASH1∆N, -ASH1N10, ASH1N21 were inserted into pPactin (ref. 20) to generate the Schneider cell expression
plasmids pPactinGal4DBDASH1∆N, -ASH1∆N21, -ASH1∆N10.
Figure Legends
Figure 1
1
Beisel et al.
Coomassie-blue stained SDS-gel (top panel) or fluorogram (bottom panel) of HMTase-assays
programmed with ASH1(SET) and 2 g polynucleosomes, 10 g histone-mixture (H1, H2A,
H2B, H3, and H4), and 5 g of purified H4, H3, H2B, H2A or H1 (Roche).
Figure 2
Fluorogram of protein-protein interaction assays using the TRX SET-domain as a bait and
[35S]-methionine-labeled in vitro expressed ASH1N (a), ASH1N10 (b), ASH1N21, or
ASH1N1142. Input represents 5% of the input material. The arrow indicates the position of
[35S]-labeled ASH1-derivatives. The position and relative molecular weight (in kilodaltons) of
protein standards is indicated to the left (a, c-d).
2