A single stem cell, with a single genomic DNA sequence, can give rise to an extraordinary diversity of cell identities and functions. The highly conserved Polycomb (PcG) and Trithorax (TrxG) group proteins constitute an epigenetic “cellular memory” system that is essential for maintaining the correct identity of both stem cells and differentiated cells. We aim to understand how this dynamic system can ensure both flexibility and stability of cell identities.
The PcG and TrxG proteins regulate several hundred developmentally important genes in flies and mammals. These proteins act through Polycomb/Trithorax response elements (PRE/TREs) (Figure 1). PRE/TREs are switchable bi-stable regulatory DNA elements that can preserve a memory of the activated or silenced state of their associated genes over several cell generations. This year we have further investigated the role of individual motifs that were predicted computationally. Our findings point towards a structural role both at the DNA and RNA levels, of a previously uncharacterised PRE/TRE motif (Okulski et al., 2011). In addition, to examine the role of PRE/TREs in a dynamically developing tissue, we have studied the eyes absent (eya) gene in the developing Drosophila eye.
We show that the eya PRE/TRE is essential for converting the output of the eya enhancer into spatially different expression profiles of the two alternative promoters across the eye disc. Replacement of the eya PRE/TRE with other PRE/TREs reveals that different PRE/TREs have profoundly different properties in this assay. This work provides unexpected insights into the rich diversity of different PRE/TRE properties, and defines a novel function of PRE/TREs in fine-tuning enhancer output during differentiation. Elucidating the molecular mechanisms by which PRE/TRE sequence, DNA and RNA structure contribute to gene activation, silencing and switching will be important tasks for the future.
In contrast to fly PRE/TREs, the corresponding mammalian elements have so far proved highly elusive. During the past year we have use experimental and bioinformatic analysis to identify mammalian PRE elements, and to extract sequence principles of mammalian PRE/TREs (Figure 2). Our data reveal unexpected similarities between fly and vertebrate elements at the DNA sequence level. In future we will continue to combine experimental and computational analyses to tackle the question of what makes a mammalian PRE/TRE.
Our recent work in both fly and mouse has identified several novel long noncoding RNAs that are transcribed from Polycomb regulatory sites, and suggests an essential role for these RNAs in PRE/TRE regulation during development and differentiation. In mouse, we have shown by profiling of purified cell populations from different stages of a defined in vitro neural differentiation system that over 50% of regulated intergenic noncoding transcripts precisely correspond to PcG target sites.
We designate these PcG recruiting elements as Transcribed Intergenic Polycomb (TIP) sites. Reporter assays show that transcribed TIP sites can repress a flanking gene. Knockdown experiments demonstrate that TIP noncoding RNAs are themselves required for repression of target genes both in cis and in trans. We propose that TIP transcription may ensure coordinated regulation of gene networks via dynamic switching and recruitment of PcG proteins both in cis and in trans during lineage commitment. Our future work in fly and mouse will address the molecular mechanisms by which selected transcripts recruit PcG proteins in specific cell types and at specific developmental stages.
We have established an “in vivo biochemistry” approach to perform quantitative analysis of PcG and TrxG protein dynamics in living Drosophila in defined cell types that undergo mitosis and differentiation. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at serine 28 as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. We propose that regulation of the kinetic properties of PcG - chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities. This year we have extended our analysis to several other proteins including the TrxG protein Ash1, which shows a remarkably robust attachment to chromatin throughout mitosis (Figure 3). Future work on Ash1 aims to elucidate the molecular basis of this mitotic attachment and its role in propagating memory of cell identity through mitosis.