Cloning and Mutational Analysis of BCL11A and NONO

The coding sequences of BCL11A-S (GenBank: NM_138559), BCL11A-L (GenBank: NM_018014), and NONO (GenBank: NM_001145408) were amplified from human fetal brain cDNA using the primers in Table S1 and cloned into pCR2.1-TOPO (Invitrogen). The missense mutations were introduced using the Quik-Change Lightning SDM kit (Agilent) and the primers in Table S2. For expression of fusion proteins with Renilla luciferase, YFP, and mCherry, cDNAs were subcloned into the pLuc, pYFP, and pmCherry expression vectors, respectively, which have been described previously, using the BamHI and XbaI sites.39 (link), 40 For the mammalian one-hybrid assay, a vector for expression of BCL11A fused in frame with the yeast GAL4 DNA-binding domain was created by cutting and re-ligating pBIND (Promega) at the ClaI sites to remove the Renilla luciferase expression cassette. Wild-type and mutant forms of BCL11A-L were subcloned into the BamHI and XbaI sites of this vector. A reporter plasmid was generated by inserting a KpnI-NcoI fragment of pG5luc (Promega) containing five GAL4 binding sites and a minimal adenovirus major late promoter into the vector pGL4.23 (Promega), which contains a codon-optimized firefly luciferase gene. A plasmid containing Renilla luciferase downstream of the herpes simplex virus thymidine kinase promoter (pGL4.74, Promega) was used for normalization.