HES1 domains essential for repressor function and PARP1 interaction. (A) Schematic of human HES1 domains and mutants. The conserved domains are the bHLH region, orange domain, proline-rich region, and C-terminal WRPW region. (B) HES1 mutant reporter assay. B-ALL (JM1) cells were transfected with the HES1-responsive luciferase reporter as in Figure 2A-B, with the increasing amounts of the indicated wild-type or truncated versions of FLAG-HES1 plasmids. Only the full-length HES1 and proline mutant showed repressor activity, indicating the critical role for the bHLH, orange, and WRPW domains in HES1 repressor function (P < .001, mean ± SD). (C) HES1 mutant DNA binding. B-ALL (JM1) cells were transfected with the HES1 mutants, and ChIP assays were performed as in Figure 2C-D. All mutants, except the bHLH mutant (which contains the DNA binding domain), are able to bind the native HES1 binding site. (D) Co-IP with HES1 mutants. B-ALL (JM1) cells were transfected with full-length and HES1 mutants. Co-IP was performed as in Figure 1C. The bHLH and orange domain mutants were less efficient at binding PARP1. Similar results were observed whether IP was performed with anti-FLAG or anti-PARP1.