Epigenetic regulation by proteins of the Polycomb and Trithorax group
The aim of research in epigenetics is to understand how a single cell, with a single genomic DNA sequence, can give rise to and maintain the extraordinary diversity of cell identities and functions that comprise the adult organism. Modifications in chromatin and the binding of other chromatin proteins and non-coding RNAs provide a regulatory layer that modulates genome function, so that one genome gives rise to several ‘epigenomes’. The highly conserved Polycomb (PcG) and Trithorax (TrxG) groups of proteins are essential components of the epigenome in every cell type studied so far.
The PcG and TrxG proteins constitute an epigenetic “cellular memory” system that is essential for maintaining the correct identity of both stem cells and differentiated cells, and for orchestrating transitions between them. Aberrant expression of these proteins leads to developmental defects and cancer. PcG and TrxG proteins work antagonistically on several hundred developmentally important target genes to maintain repressed (PcG) or active (TrxG) transcription states. Since the PcG/TrxG proteins are able to maintain stable states of gene expression over several rounds of cell division, it was long believed that they must form stable static structures on their chromatin target sites. However, this system is extraordinarily dynamic: both PcG and TrxG proteins exchange rapidly on chromatin within seconds. This in turn offers opportunities for regulation, permitting switching or modulation of output in response to cell cycle, developmental, environmental or metabolic signaling. We focus on three key questions.
1) How does the system maintain memory? PcG and TrxG proteins bind mitotic chromatin.
Using quantitative live imaging, FRAP and FCS, we have established an “in vivo biochemistry” approach to perform absolute quantification and kinetic analysis of PcG and TrxG protein dynamics in living Drosophila in single defined cells that undergo mitosis and differentiation. This shows that both PcG and TrxG proteins bind mitotic chromatin, and enables us to address two important questions: What is the molecular mechanism by which these proteins attach to mitotic chromatin? Is mitotic chromatin attachment important for cell identity and viability in the living animal? These questions are closely interconnected. If we understand mechanisms, we can extrapolate specific interactions to address their function. Using quantitative live imaging in combination with genetic analysis in living Drosophila, we dissected and quantified molecular mechanisms by which the TrxG protein ASH1 binds to chromatin in vivo, identifying distinct protein domains that mediate chromatin binding in the interphase and during mitosis. Using genetic rescue experiments, we show that mitotic chromatin attachment of ASH1 is essential for survival to adulthood as well as for the maintenance of correct cell identity in living animals (Steffen et al., submitted).
2) How does the system switch between active and silent states? Non-coding RNA strand switching defines the PRE/TRE status.
PcG and TrxG proteins work through specialized DNA elements known as Polycomb/Trithorax Response Elements (PRE/TREs). We made the exciting discovery that, in both vertebrates and flies, specific developmentally regulated non-coding RNAs transcribed from these elements are involved in both silencing and activation by PcG/TrxG proteins. Remarkably, a Drosophila PRE/TRE switches its function by alternating between forward and reverse strands of its non-coding RNA. Our work identified a novel and potentially widespread class of PRE/TREs that switch their function by switching between forward and reverse strand non-coding RNA transcription (Herzog, Lempradl et al., in revision). Current work is focused on the mechanism by which these ncRNAs influence the properties of chromatin binding proteins.
3) What makes a PRE/TRE? Principles of DNA sequence in flies and mammals.
We use quantitative assays to identify novel motifs required for PRE/TRE function in flies (Okulski et al., in preparation). However, the analogous elements in mammals proved to be highly elusive. The race to understand the sequence principles of mammalian PRE/TREs is currently one of the most active and controversial areas in the field of PcG/TrxG. Based on previous work in flies, we established a computational tool that can accurately identify candidate PRE/TRE elements on the basis of the DNA sequence alone (collaboration with Marc Rehmsmeier, University of Bergen, Norway). We are currently testing these predictions in quantitative experimental assays. Future work will be focused on identifying mammalian PRE/TREs on a genome-wide basis and understanding the relationship between DNA sequence and function in quantitative assays (Trupke et al., in preparation).
In addition, we recently showed that PREs and CpG islands are distinct regulatory entities, in contrast to current models. CpG islands and CpG-rich sequences were found to be necessary. In some cases they are sufficient to recruit vertebrate PcG proteins. We now show that the CpG island potentiates while the PRE consolidates recruitment and confers regulation (Figure 3; Heinen et al., submitted). The interplay between enhancers, CpG islands and PREs will be a key issue in future investigations.