Pluripotent stem cells often adopt a unique developmental program while retaining certain flexibility. The molecular basis of such properties remains unclear. Using differentiation of pluripotent Drosophila imaginal tissues as assays, we examined the contribution of epigenetic factors in ectopic activation of Hox genes. We found that over-expression of Trithorax H3K4 methyltransferase can induce ectopic adult appendages by selectively activating the Hox genes Ultrabithorax and Sex comb reduced in wing and leg discs, respectively. This tissue-specific inducibility correlates with the presence of paused RNA polymerase II in the promoter-proximal region of these genes. Although the Antennapedia promoter is paused in eye-antenna discs, it cannot be induced by Trx without a reduction in histone variants or their chaperones, suggesting additional control by the nucleosomal architecture. Lineage tracing and pulse-chase experiments revealed that the active state of Hox genes is maintained substantially longer in mutants deficient for HIRA, a chaperone for the H3.3 variant. In addition, both HIRA and H3.3 appeared to act cooperatively with the Polycomb group of epigenetic repressors. These results support the involvement of H3.3-mediated nucleosome turnover in restoring the repressed state. We propose a regulatory framework integrating transcriptional pausing, histone modification, nucleosome architecture and turnover for cell lineage maintenance.

Polycomb group (PcG) repressors confer epigenetically heritable silencing on key regulatory genes through histone H3 trimethylation on lysine 27 (H3K27me3). How the silencing state withstands antagonistic activities from co-expressed trithorax group (trxG) activators is unclear. Upon overexpression of Trx H3K4 methylase, to perturb the silenced state, we find a dynamic process triggered in a stepwise fashion to neutralize the inductive impacts from excess Trx. Shortly after Trx overexpression, there are global increases in H3K4 trimethylation and RNA polymerase II phosphorylation, marking active transcription. Subsequently, these patterns diminish at the same time as the levels of Set1, an abundant H3K4 methylase involved in productive transcription, reduce. Concomitantly, the global H3K27me3 level is markedly reduced, corresponding to an increase in the amount of Utx demethylase. Finally, excess Pc repressive complex 1 (PRC1) is induced and located to numerous ectopic chromosomal sites independently of H3K27me3 and several key recruitment factors. The observation that PRC1 becomes almost completely colocalized with Trx suggests new aspects of recruitment and antagonistic interaction. We propose that these events represent a feedback circuitry ensuring the stability of the silenced state.