Figure 3

Oct4 and ERG-associated protein with SET domain (ESET) synergistically repress Cdx2. (a) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of Oct4 on different positions along the Cdx2 promoter relative to the least enriched region (C3) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates. (b) Western blot shows upregulation of Cdx2 and downregulation of ESET upon depletion of Oct4 at day 2 of tetracycline (Tc) treatment of ZHBTc4 embryonic stem (ES) cells. (c) Q-PCR analysis of the levels of Oct4 and ESET enrichment at region C7 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for 1 day relative to untreated cells after normalising against their respective input. Error bars, SD of three independent experiments. (d) Carrier chromatin immunoprecipitation (ChIP) Q-PCR analysis of the levels of histone 3 lysine 9 trimethylation (H3K9me3) at region C1 and C2 of the Cdx2 promoter in ZHBTc4 ES cells treated with Tc for the indicated days relative to untreated cells after normalising against their respective input and IgG controls. Error bars, SD of three independent experiments. (e) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control.