Human Bach1 gene was generated and cloned into pcDNA3.1 as described previously (43). Briefly, the full-length cDNA of human Bach1 were generated by PCR and verified by DNA sequencing. Flag-epitope tags were introduced to the C termini of Bach1 gene. The EZH2-HA plasmid was provided by J. Xu (Fudan University, China). GST-Bach1 full or GST-Bach1–N terminal or GST-Bach1–C terminal fusion protein was constructed by inserting PCR-generated DNA fragments encoding regions of Bach1 full [1 to 736 amino acids (aa)] or Bach1 N-terminus (1 to 560 aa) or Bach1 C-terminus (127 to 736 aa) into PGEX-6P-1 vector. His-EZH2 fusion protein was constructed by inserting PCR-generated DNA fragments encoding regions of EZH2 into pET-28 (a) + plasmid. The TOPflash reporter vector was purchased from Millipore (Billerica, MA). T, Gata6, Wnt3, and Nodal promoter fragments were amplified from human genomic DNA with appropriate sets of primers. Mutations of the consensus Bach1 binding site that abolished Bach1 binding were introduced in the forward primer of these gene promoter construct. PCR products were cloned into the luciferase vector pGL3-basic. Primers for reporter and mutation are listed in table S2.2. All constructs were verified by sequencing.

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