Panoply™ Human EED Over-expressing Stable Cell Line

Female puberty is subject to Polycomb Group (PcG)-dependent transcriptional repression. Kiss1, a puberty-activating gene, is a key target of this silencing mechanism. Using gain-of-function studies and systems biology approaches, researchers discovered that EED, a key component of PcG, acts in the hypothalamic arcuate nucleus, altering the functional organization of the gene network involved in regulating pubertal stimulation. The core node of this network is Kdm6b, which encodes an enzyme that removes the PcG-dependent histone modification H3K27me3. Kiss1 is a first-degree neighbor gene in this network; genes encoding glutamate receptors and potassium channels are second-degree neighbor genes. By inhibiting Kdm6b expression, EED increases the abundance of H3K27me3 in the promoter regions of these genes, thereby reducing the gene expression of the entire gene network controlling pubertal activation. These results indicate that inhibiting Kdm6b expression is a fundamental mechanism by which PcG regulates the biological output of the gene network involved in pubertal activation.

Here, researchers compared the gene expression profiles in the hypothalamus of in vivo control and EED-overexpressing animals with the gene expression profiles observed in in vitro EED-overexpressing hypothalamic R22 cells. Except for the Kcnn1 gene, whose mRNA levels were increased under in vivo EED overexpression but decreased in in vitro EED-overexpressing cells, the expression changes of all other analyzed genes were similar in both in vivo and in vitro conditions (Figure 1A). Surprisingly, two glutamatergic markers, Nell2 and Grm7, were highly expressed in the hypothalamus but not in R22 cells. Despite this difference, these results generally support the use of R22 cells as a valid in vitro system for studying hypothalamic gene interactions. Following EED overexpression, the recruitment of EED to the Kiss1 and Kdm6b promoters was significantly increased (Figure 1B), and similar phenomena were observed in glutamatergic genes and potassium channel genes. The levels of H3K27me3 also increased (Figure 1C), indicating that-consistent with its role in PRC2 function-EED promotes the deposition of H3K27me3 in the promoter regions of downstream target genes. In summary, these results support gene network predictions and the idea that the PRC2 complex downregulates not only the promoters of Kiss1 and Kdm6b but also the promoters of multiple glutamatergic-related genes and potassium channel genes, consistent with its previously demonstrated role as a negative regulator of pubertal timing.

Figure 1. Changes in Gene Expression and recruitment of EED and H3K27me3 to the promoter of network genes. (Wright, Hollis, et al.,2021)