Similar to human Mash1 promoter, mouse Mash1 promoter also contains three putative HES1-binding sites and one E-box. Neuro2a cells were PF-04979064 transfected with constant amount of empty plasmid or with expression plasmid for Myc-HES1 PD-156707 together with or without increasing amounts of FLAG-HEN2 expression plasmid. Forty-eight hours after transfection, cross-linked chromatin was prepared and subjected to ChIP assay. As shown in Figure 4D, the anti-Myc tag immunoprecipitates contained genomic fragments including putative HES1-binding sites as well as E-box. The amounts of Myc-HES1 recruited onto HES1-binding sites and E-box significantly decreased in the presence of FLAG-HEN2 in a dose-dependent manner. Additionally, the anti-FLAG immunoprecipitates contained genomic fragments including putative HES1-binding sites and E-box in the absence of exogenous HES1. Co-expression of FLAG-HEN2 and Myc-HES1 inhibited recruitment of FLAG-HEN2 onto putative HES1-binding sites and E-box, however, its inhibition was efficiently abrogated by increasing amounts of FLAG-HEN2. These results suggest that HEN2 might compete with HES1 in binding to putative HES1- binding sites and E-box, and thereby inducing the expression of Mash1. To examine whether HEN2 could interact with HES1 in cells, we performed immunoprecipitation experiments. Cell lysates prepared from Neuro2a cells co-transfected with the indicated combinations of expression plasmids were subjected to immunoprecipitation. As clearly shown in Figure 5A, HES1 was coimmunoprecipitated with FLAG-HEN2. Consistent with these results, reciprocal experiments showed that the anti-Myc tag immunoprecipitates contain FLAG-HEN2. In vitro pull-down assay demonstrated that radio-labeled FLAG-HEN2 is coimmunoprecipitated with Myc-HES1. Additional immunoprecipitation experiments demonstrated that LMO3 also forms a stable complex with HES1. We have previously showed that LMO3 forms a stable complex with HEN2. To investigate the effect of LMO3 on binding of HEN2 and HES1 to putative HES1-binding sites and E-box, Neuro2a cells were transfected with Myc-HES1 or FLAG-HEN2 together with or without HA-LMO3 expression plasmid and subjected to ChIP assay.