Fig. 5b). Next, we carried out ChIP assays to determine: a) If EED is recruited to the Kiss1 promoter in the MBH, and b) if this relationship changes during the onset of puberty. We observed that the EED protein was associated with the Kiss1 promoter in EJ and this association decreased at LJ, the time of initiation of puberty (Fig. 5d). Consistent with the notion that inhibition of DNA methylation leads to increased expression of PcG genes, which then repress downstream targets genes, the pubertal loss of EED association to the Kiss1 promoter failed to occur in Aza treated rats (Fig. 5d). The chromatin status of the Kiss1 promoter also changed at the time of puberty. Whereas the content of H3K27me3, a PcG-dependent repressive histone modification 39, 40 did not decrease significantly in LJ animals (Fig. 5e), the abundance of two activating histone modifications, H3K4me3 and H3K9,14ac 39, 41, 42 increased markedly at this time (Fig. 5f,g). Treatment with Aza, which prevented the eviction of EED from the Kiss1 promoter at LJ, also prevented the LJ increase in both H3K4me3 and H3K9,14ac abundance (Fig. 5f,g). To determine if the chromatin landscape of the Kiss1 promoter continues to change as the
