Regulation of cellular memory by non-coding transcription through Polycomb group response elements

Abstract

The formation of functionally distinct cell types from a single zygote is a fundamental aspect of metazoan development. The specification of cell identities usually occurs during early development, long before this information is used during differentiation. The molecular basis of cell fate specification is the activation of characteristic gene expression programs. A prerequisite for the appropriate differentiation of cells according to their fate is therefore the faithful transmission of once established gene expression states throughout phases of cell proliferation and growth. This task is accomplished by the so-called cellular memory. A paradigm to study the molecular basis of the cellular memory is the regulation of homeotic gene expression in Drosophila. A number of studies have shown that the cellular memory is an epigenetic mechanism based on the modification of the chromatin structure. This is accomplished through the antagonizing activities of repressive PcG (Polycomb group) and activating TrxG (Trithorax group) proteins. The recruitment of both PcG as well as TrxG proteins to their target loci depends on the presence of identical cis-regulatory elements termed Polycomb group response elements, or PREs. How is the decision between epigenetic silencing and activation at a PRE taken? Presumably, the default state of a PRE is to function as a PcG-dependent silencer, whereas the conversion into the active mode requires incoming signals. It has previously been shown that the epigenetically active state at a PRE correlates with non-coding transcription through these elements themselves. This raised the question whether the non-coding transcription is of functional significance for the epigenetic activation of a PRE, or if it reflects only a consequence of this process. The major aim of this thesis was to determine whether the transcription through the well-characterized Fab-7 PRE has any function in the regulation of the epigenetic state of this element. In the first part, a transgenic reporter system was used to answer this question. The results obtained show that the transcription through Fab-7 functions as a novel anti-silencing mechanism that counteracts the PcG-mediated repression by default. Depending on the tissue and/or on the locus, transcription through PREs may be required throughout development to prevent the re-establishment of PcG silencing. Importantly, PREs located outside the homeotic gene complexes are also transcribed, suggesting that the anti-silencing function of transcription through PREs may be a fundamental aspect of the cellular memory. In the second part, the transgenic reporters mentioned above were used to analyze the epigenetic consequences of transcription through the Fab-7 PRE. The results suggest that at least on the transgene, both repressive PcG as well as activating TrxG proteins associate with the Fab-7 PRE, irrespective of the epigenetic state of this element. The primary function of transcription through the Fab-7 PRE may therefore be to modulate the activities of PcG/TrxG complexes, rather than regulating their differential recruitment to the chromatin. To get insight into the precise function of non-coding transcription through PREs, the properties Fab-7 RNA were investigated in SF4 tissue culture cells in vitro, as well as in wildtype embryos in vivo. Although a sequence-specific function of Fab-7 RNA is unlikely, the results obtained do not exclude structural function of non-coding PRE transcripts. Futhermore, Fab-7 RNA can be detected on mitotic chromatin, suggesting that the transmission of epigenetically activated states through cell division might depend on the timing of non-coding transcription with respect to the cell cycle and/or the association of non-coding RNAs with the chromatin. Since the functions of PcG/TrxG proteins have been conserved during evolution, the results presented here might have implications on our understanding of epigenetic gene regulation in mammals.