Because MBH expression of both Cbx7 and Eed appears to be DNA methylation-dependent, and considering that EED is essential for PcG action 32, we selected Eed for further analysis. In silico analysis of the Kiss1 promoter (Supplementary Fig. 5a) demonstrated that it contains several motifs found to be present in PcG target genes, including the core motif for YY1 binding (CCAT), the GAF (GAGAG) and extended MPho (CNGCCATNDNND) motifs 36, the two BMI1 binding motifs (CCTTCC and GGNNNGNG) reported by Meng et al. 37, and the binding motif for HOTAIR, a long noncoding RNA, recently shown to serve as an anchor for PcG binding to gene promoters 38. To determine if these motifs have biological significance, we performed gene promoter assays and observed that EED indeed represses Kiss1 promoter activity and that this repressive effect is enhanced by YY1 (Supplementary 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
