PARP1 inhibits HES1 function and localizes to a HES1 binding site. (A) Cell type–specific function of HES1. T-ALL (SupT1) and B-ALL (JM1) cells were cotransfected with HES1-responsive luciferase reporter, Renilla luciferase control, and increasing amounts of pcDNA-FLAG-HES1. After 48 hours, Firefly/Renilla luciferase activity was measured. Significant dose-dependent repression was only seen in B-ALL cells. (P < .038, mean ± SD). (B) PARP1 inhibits HES1 function. B-ALL (JM1) cells were cotransfected with HES1-responsive luciferase reporter, Renilla luciferase control, and increasing amounts of PARP1 expression plasmid or pcDNA-FLAG-HES1 with or without PARP1. No significant effect was seen with PARP1 alone. As expected, significant dose-dependent repression was seen with HES1; however, the addition of PARP1 abrogated this effect, inhibiting HES1-mediated repression of this reporter (P < .05, mean ± SD). (C) PARP1 binds to a HES1 binding site via HES1. Schematic representation of the forward and reverse primers for ChIP-PCR. Forward “F” and reverse “R” primers produce a 325-base pair (bp) product for the known HES1 site (N-box) and a 216-bp product for a random site in intron 3 as a control. (D) ChIP-PCR analysis to determine the interaction of PARP1 and HES1. JM1 cells were transfected with empty vector, FLAG-HES1, PARP1, shRNA control, shRNA to HES1, or shRNA to PARP1 as indicated. After 48 hours, the cells were fixed, and IP with anti-PARP1 or anti-FLAG antibodies was performed with the use of the ChIP-IT Express kit. PCR products were resolved on an agarose gel and visualized with ethidium bromide/UV. These results show HES1 and endogenous PARP1 binding to a known HES1 binding site. ShRNA to HES1 decreased PARP1 binding to this site, suggesting that PARP1 binding depends on endogenous HES1.