FIGURE 8.

Co-occupation of the H19 ICR by CTCF and Smads requires intact binding sites for CTCF on DNA. A, CTCF and Smads bind only to the maternally inherited allele of the H19 ICR. Primary hepatocytes were stimulated with TGFβ1 for 4 h (black bars) or left untreated (white bars) prior to ChIP with the indicated antibodies. Immunoprecipitated chromatin was amplified with primers specific for the H19 ICR, which can discriminate between the maternally inherited allele (♀) and the paternally inherited allele (♂). Input chromatin and nonspecific IgG controls are also shown after PCR amplification. B, MEFs were stimulated with TGFβ1 for 4 h (black bars) or left untreated (white bars) prior to ChIP. The genotype of the amplified allele is shown as wild type or mutant with respect to the H19 ICR point mutations engineered in the specific sequences where CTCF binds. C, nonspecific IgG controls and input chromatin controls for the same samples analyzed in B are shown. In all of the panels, the Q-PCR data show the average values and standard deviations calculated from triplicate determinations.