Molecular mechanisms involving PcG proteins have attracted significant attention in the stem cell field due to their critical role in controlling cultured ESCs (Boyer et al., 2006
; Lee et al., 2006
; Pasini et al., 2007
). By studying the role of Ezh2 in epidermal development and differentiation, we have uncovered new insights into the in vivo relevance of PRCs in the context of lineage determination and differentiation within a tissue. We have shown that Ezh2-dependent histone modifications act in embryonic epidermal progenitors to control both proliferative potential and differentiation. Our results are consistent with a role for PRCs in mediating repression of Ink4A/Ink4B
in epidermal stem cells and further unveil a role for PRCs in regulating terminal differentiation of these cells by repressing genes governing epidermal barrier formation.
Our findings are intriguing in light of a recent study reporting enhanced differentiation of organotypic human epidermal cultures by overexpressing or RNAi knockdown of the histone demethylase Jmjd3 (Agger et al., 2007
; Sen et al., 2008
). In contrast to Ezh2, Jmjd3
levels do not appear to change in basal versus suprabasal cells of mouse epidermis. It is tempting to speculate that suprabasal Jmjd3 might ensure proper differentiation through efficient removal of the Ezh-mediated triMeK27-H3 mark, and in the future, it will be interesting to explore the possible interplay between histone methylases and demethylases in this process. However, from our physiological and molecular evidence, we favor the view that the primary mechanism for controlling epidermal differentiation in mouse development is by temporal expression of histone methylase Ezh2, which in basal cells, interferes with the recruitment of transcriptional activators to their differentiation-specific targets.
Our study also uncovered several notable differences in the mechanisms by which PcGs control differentiation in ESCs versus embryonic epidermal progenitors. In ESCs, triMeK27-H3 is present at and globally represses key transcriptional regulators that promote diverse cell lineages (Boyer et al., 2006
; Lee et al., 2006
). Such widespread repression is critical since ESCs are pluripotent. That said, ESCs must also retain the ability to respond to external cues and activate any one of the differentiation lineages repressed by PcG complexes. Once they make a commitment to differentiate, they must repress pluripotent regulators, e.g., Oct4, Nanog, and Sox2, and this process also appears to be governed either indirectly or directly by PcG-mediated events (Pasini et al., 2007
). By contrast, embryonic epidermal stem cells maintain permanent repression of pluripotent regulators and selectively activate only the terminal differentiation program of the skin barrier.
Our results suggest that, like ESCs, embryonic basal progenitors use PcG-mediated repression to maintain repression of both nonepidermal and epidermal differentiation programs. However, in ESCs, bivalent active and repressive marks exist over many of the lineage-regulatory genes that are governed by PRCs, such that once repressive marks are relieved, the genes are poised to become activated (Pasini et al., 2007
). By contrast, although Ezh2 loss in embryonic basal cells reduced triMeK27-H3 on many of the same genes as in ESCs, this was not sufficient for either maintaining or relieving their transcriptional repression. Rather than use a bivalent chromatin mark, embryonic basal epidermal progenitors appear to employ PRCs to prevent recruitment of AP1 and perhaps other key transcriptional activators required for terminal differentiation (). This additional layer of regulation ensures that, as development proceeds, most lineages remain permanently silenced while permitting the epidermal lineage to be selectively activated.
By invoking additional mechanisms to restrict activation of differentiation programs, developing tissues are poised to take advantage of relieving PRC modifications as a means to fine-tune the transcriptional program of the desired lineage. In this regard, it was notable that Ezh2 was highest in proliferative, undifferentiated basal cells. Upon induction of the basal-to-spinous switch, Ezh2 was reduced, and by the time the spinous-to-granular transcriptional switch was executed, little or no Ezh2 was detected. By employing gene-specific transcriptional activators that can operate when Ezh2 and triMeK27-H3 wane, the correct differentiation stage genes can be induced, and the undesirable nonepidermal lineages can remain silenced.
Controlling the balance between proliferation and differentiation requires different challenges in embryonic versus adult skin. In the rapidly growing embryo, basal proliferation must be high, and the program of epidermal differentiation and barrier acquisition must be orchestrated from scratch. By contrast, during homeostasis of the postnatal epidermis, proliferation rates are considerably reduced, and the program of terminal differentiation merely requires maintenance and not establishment. In this regard, it may be relevant that postnatal human basal epidermal cells exhibit low immunoreactivity to the active chromatin mark acetylated-H4 and appreciable reactivity to the repressive chromatin mark triMeK9-H3 (Frye et al., 2007
). Additionally, our studies showing that Ezh2 expression wanes after birth suggest that either the Ezh2 paralog Ezh1 or alternative mechanisms come into play to control homeostasis in the mature epidermis. Although distinguishing between these possible models in vivo is predicated upon Ezh1
double cKO mice, our in vitro and engraftment studies suggest that Ezh2 may provide the bulk of triMeK27-H3 modification in basal epidermal cells, and Ezh1 may function in fine-tuning histone modifications at later stages of terminal differentiation during embryogenesis and in homeostasis of postnatal epidermis.
In closing, it is worth considering the potential clinical impact of our findings. The epidermal barrier does not form until shortly before birth. Prematurely born infants lack this essential shield, and hence, accelerating barrier acquisition becomes a critical necessity in reducing the medical risks of these infants. Our discovery that conditional loss of Ezh2 accelerates epidermal barrier formation in the embryo but does not impair postnatal development offers a hitherto unanticipated target for the development of therapies that might be useful to infant survival. More-over, Ezh2’s exquisite ability to function in rapidly proliferating, tissue-restricted progenitor cells provides an intriguing explanation for why Ezh2 is overexpressed in many tumors wherein hyperproliferation and reduced differentiation are often likened to an embryonic state.