Pentr1a no ccdb

BirA^R118G (BirA*) cDNA was amplified from pcDNA3.1-mycBioID (Addgene, catalog no. 35700). DOT1L wild type (WT) and mutant (G163R/S164C/G165R) cDNAs with HA-tag in pMIY plasmids were described previously [18 (link)]. In-frame BirA*-DOT1L fusion protein coding sequence was cloned into pENTR1A no ccDB (Addgene, catalog no. 17398) and transferred into expression plasmid pLEX-307 (Addgene, catalog no. 41392) via LR cloning (Invitrogen). pBabe-puro-AF10 wild-type (wt) and L107A mutant (mut) plasmids were gifts of Or Gozani (Stanford University). Wt- and mut-AF10 cDNAs were amplified with Phusion polymerase and inserted into pENTR1A no ccDB (Addgene, catalog no. 17398). OM-LZ domain (703–784) deleted plasmids were prepared with Q5-site directed mutagenesis kit (NEB) according to manufacturer’s instructions. All AF10 sequences were cloned into a lentiviral expression plasmid pLenti CMV/TO Hygro DEST (Addgene, catalog no. 17291) via LR cloning (Invitrogen). pcDNA5 GFP-AF10 wt and L107A mutant (mut) plasmids were gifts of Or Gozani (Stanford University). OM-LZ domain (703–784) deleted pcDNA5 GFP fusion AF10 OMLZ deleted mutant was prepared with Q5-site directed mutagenesis kit (NEB) according to manufacturer’s instructions.

Plasmids encoding full-length TRIOBP-1 and two N-terminally truncated versions of it, have been described previously [15 (link)]. Additional constructs encoding the coiled-coil regions of TRIOBP-1 were made by subcloning reading frames, and then transferring into either a Gateway entry vector, either pDONR/Zeo (using BP clonase recombination, enzyme and plasmids: Thermo Fisher Scientific, Waltham, NJ, USA) or pENTR1A no ccDB (Dr. Eric Campeau, supplied by AddGene, Watertown, MA, USA, clone 17398 [34 (link)], by ligation). Vectors encoding full-length TRIOBP-1 with internal deletions were produced by subcloning of 5′ and 3′ fragments of the gene, which were then sequentially ligated into pENTRA1A no ccDB. Reading frames were transferred from entry vectors into destination vectors using LR clonase II recombination (Thermo Fisher Scientific). Destination vectors used were pdcDNA-FlagMyc (B. Janssens, supplied by the BCCM/LMBP Plasmid Collection, Zwijnaarde, Belgium, clone LMBP 4705) and pdECFP (Dr. S. Wiemann, BCCM/LMBP Plasmid Collection, clone LMBP 4548 [35 (link)]). All vectors were confirmed by sequencing. Details of all plasmids used are in Supplementary Table S2 and the primers used to clone them are in Supplementary Table S3.