Figure 6.

NeuroD1 binds to the intronic E-box (E600). A, Weri cells express modest amounts of endogenous NeuroD1, detected by Western blot. 293T cells lack detectable NeuroD1 but transfection of pSG5-NeuroD1 produces a NeuroD1 band similar to that in Weri cells. B, EMSA showing that NeuroD1 in nuclear extracts (NE) of Weri cells (w) binds to a wt probe containing E600. Competition with increasing amounts of unlabeled wt probe (com) abolishes the band (10-, 100-, 1000-fold excesses), whereas a mutant form of the probe (M600) competes weakly. Antibody (AB) against NeuroD1 but not irrelevant antisera (preimmune, pre) reduced the specific band. Control 293T cells transfected with pSG5-NeuroD1 (T+Nd) gave a similar band on EMSA as did endogenous NeuroD1 in Weri cells. C, Detection by ChIP of NeuroD1 bound to the intron control region. Location of ChIP probes (A, B, C, D) is shown above a diagram of the relevant part of the human THRB gene. The right panel shows an example of PCR products, visualized on an ethidium bromide-stained gel, generated for each probe in Weri cells on use of IgG control or anti-NeuroD1 serum. D, Quantitation of ChIP products obtained from Weri or 293T cells was performed by RT-PCR. NeuroD1 was enriched in the region of probe C, but not in more distal regions of the THRB gene in Weri cells. 293T cells, which do not express TRβ2, showed no enrichment of bound NeuroD1. Error bars indicate SEM.