The Trithorax group protein ASH1 requires a combination of BAH domain and AT hooks, but not the SET domain, for mitotic chromatin binding and survival.

The Drosophila Trithorax group (TrxG) protein ASH1 remains associated with mitotic chromatin through mechanisms that are poorly understood. ASH1 dimethylates histone H3 at lysine 36 via its SET domain. Here, we identify domains of the TrxG protein ASH1 that are required for mitotic chromatin attachment in living Drosophila. Quantitative live imaging demonstrates that ASH1 requires AT hooks and the BAH domain but not the SET domain for full chromatin binding in metaphase, and that none of these domains are essential for interphase binding. Genetic experiments show that disruptions of the AT hooks and the BAH domain together, but not deletion of the SET domain alone, are lethal. Transcriptional profiling demonstrates that intact ASH1 AT hooks and the BAH domain are required to maintain expression levels of a specific set of genes, including several involved in cell identity and survival. This study identifies in vivo roles for specific ASH1 domains in mitotic binding, gene regulation, and survival that are distinct from its functions as a histone methyltransferase.

A theoretical model of Polycomb/Trithorax action unites stable epigenetic memory and dynamic regulation.

Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. Here we combine experiment and theory to examine the mechanistic basis of these different modes of regulation. We present a mathematical model comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. Our work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into  genome-wide Polycomb/Trithorax regulation.

Theoretical analysis of Polycomb-Trithorax systems predicts that poised chromatin is bistable and not bivalent.

Polycomb (PcG) and Trithorax (TrxG) group proteins give stable epigenetic memory of silent and active gene expression states, but also allow poised states in pluripotent cells. Here we systematically address the relationship between poised, active and silent chromatin, by integrating 73 publications on PcG/TrxG biochemistry into a mathematical model comprising 144 nucleosome modification states and 8 enzymatic reactions. Our model predicts that poised chromatin is bistable and not bivalent. Bivalent chromatin, containing opposing active and silent modifications, is present as an unstable background population in all system states, and different subtypes co-occur with active and silent chromatin. In contrast, bistability, in which the system switches frequently between stable active and silent states, occurs under a wide range of conditions at the transition between monostable active and silent system states. By proposing that bistability and not bivalency is associated with poised chromatin, this work has implications for understanding the molecular nature of pluripotency.

Noncoding RNAs in Polycomb and Trithorax Regulation: A Quantitative Perspective.

The question of how noncoding RNAs are involved in Polycomb group (PcG) and Trithorax group (TrxG) regulation has been on an extraordinary journey over the last three decades. Favored models have risen and fallen, and healthy debates have swept back and forth. The field has recently reached a critical mass of compelling data that throws light on several previously unresolved issues. The time is ripe for a fruitful combination of these findings with two other long-running avenues of research, namely the biochemical properties of the PcG/TrxG system and the application of theoretical mathematical models toward an understanding of the system's regulatory properties. I propose that integrating our current knowledge of noncoding RNA into a quantitative biochemical and theoretical framework for PcG and TrxG regulation has the potential to reconcile several apparently conflicting models and identifies fascinating questions for future research.

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response.

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.

The quest for mammalian Polycomb response elements: are we there yet?

A long-standing mystery in the field of Polycomb and Trithorax regulation is how these proteins, which are highly conserved between flies and mammals, can regulate several hundred equally highly conserved target genes, but recognise these targets via cis-regulatory elements that appear to show no conservation in their DNA sequence. These elements, termed Polycomb/Trithorax response elements (PRE/TREs or PREs), are relatively well characterised in flies, but their mammalian counterparts have proved to be extremely difficult to identify. Recent progress in this endeavour has generated a wealth of data and raised several intriguing questions. Here, we ask why and to what extent mammalian PREs are so different to those of the fly. We review recent advances, evaluate current models and identify open questions in the quest for mammalian PREs.

A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element.

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.

What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory.

In any biological system with memory, the state of the system depends on its history. Epigenetic memory maintains gene expression states through cell generations without a change in DNA sequence and in the absence of initiating signals. It is immensely powerful in biological systems - it adds long-term stability to gene expression states and increases the robustness of gene regulatory networks. The Polycomb group (PcG) and Trithorax group (TrxG) proteins can confer long-term, mitotically heritable memory by sustaining silent and active gene expression states, respectively. Several recent studies have advanced our understanding of the molecular mechanisms of this epigenetic memory during DNA replication and mitosis.

Quantitative in vivo analysis of chromatin binding of Polycomb and Trithorax group proteins reveals retention of ASH1 on mitotic chromatin.

The Polycomb (PcG) and Trithorax (TrxG) group proteins work antagonistically on several hundred developmentally important target genes, giving stable mitotic memory, but also allowing flexibility of gene expression states. How this is achieved in quantitative terms is poorly understood. Here, we present a quantitative kinetic analysis in living Drosophila of the PcG proteins Enhancer of Zeste, (E(Z)), Pleiohomeotic (PHO) and Polycomb (PC) and the TrxG protein absent, small or homeotic discs 1 (ASH1). Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy reveal highly dynamic chromatin binding behaviour for all proteins, with exchange occurring within seconds. We show that although the PcG proteins substantially dissociate from mitotic chromatin, ASH1 remains robustly associated with chromatin throughout mitosis. Finally, we show that chromatin binding by ASH1 and PC switches from an antagonistic relationship in interphase, to a cooperative one during mitosis. These results provide quantitative insights into PcG and TrxG chromatin-binding dynamics and have implications for our understanding of the molecular nature of epigenetic memory.

Epigenetics meets mathematics: towards a quantitative understanding of chromatin biology.

How fast? How strong? How many? So what? Why do numbers matter in biology? Chromatin binding proteins are forever in motion, exchanging rapidly between bound and free pools. How do regulatory systems whose components are in constant flux ensure stability and flexibility? This review explores the application of quantitative and mathematical approaches to mechanisms of epigenetic regulation. We discuss methods for measuring kinetic parameters and protein quantities in living cells, and explore the insights that have been gained by quantifying and modelling dynamics of chromatin binding proteins.

First annual meeting of the EpiGeneSys Network of Excellence: moving epigenetics towards systems biology.

This meeting united participants from the fields of experimental epigenetics, mathematics and computational biology, to discuss results and challenges in the endeavour to explore points of synergy between these fields. The design shown here expresses the idea of moving complex biological phenomena (left nucleosome) towards precise mathematical descriptions (right nucleosome).

In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells.

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. Here we quantify dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at Ser 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.

Intergenic Polycomb target sites are dynamically marked by non-coding transcription during lineage commitment.

Non-coding (nc) RNAs are involved both in recruitment of vertebrate Polycomb (PcG) proteins to chromatin, and in activation of PcG target genes. Here we investigate dynamic changes in the relationship between ncRNA transcription and recruitment of PcG proteins to chromatin during differentiation. Profiling of purified cell populations from different stages of a defined murine in vitro neural differentiation system shows that over 50% of regulated intergenic non-coding transcripts precisely correspond to PcG target sites. We designate these PcG recruiting elements as Transcribed Intergenic Polycomb (TIP) sites. The relationship between TIP transcription and PcG recruitment switches dynamically during differentiation between different states, in which transcription and PcG recruitment exclude each other, or in which both are present. Reporter assays show that transcribed TIP sites can repress a flanking gene. Knockdown experiments demonstrate that TIP ncRNAs 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.

Quantitative analysis of polycomb response elements (PREs) at identical genomic locations distinguishes contributions of PRE sequence and genomic environment.

BACKGROUND: Polycomb/Trithorax response elements (PREs) are cis-regulatory elements essential for the regulation of several hundred developmentally important genes. However, the precise sequence requirements for PRE function are not fully understood, and it is also unclear whether these elements all function in a similar manner. Drosophila PRE reporter assays typically rely on random integration by P-element insertion, but PREs are extremely sensitive to genomic position. RESULTS: We adapted the ΦC31 site-specific integration tool to enable systematic quantitative comparison of PREs and sequence variants at identical genomic locations. In this adaptation, a miniwhite (mw) reporter in combination with eye-pigment analysis gives a quantitative readout of PRE function. We compared the Hox PRE Frontabdominal-7 (Fab-7) with a PRE from the vestigial (vg) gene at four landing sites. The analysis revealed that the Fab-7 and vg PREs have fundamentally different properties, both in terms of their interaction with the genomic environment at each site and their inherent silencing abilities. Furthermore, we used the ΦC31 tool to examine the effect of deletions and mutations in the vg PRE, identifying a 106 bp region containing a previously predicted motif (GTGT) that is essential for silencing. CONCLUSIONS: This analysis showed that different PREs have quantifiably different properties, and that changes in as few as four base pairs have profound effects on PRE function, thus illustrating the power and sensitivity of ΦC31 site-specific integration as a tool for the rapid and quantitative dissection of elements of PRE design.

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners.

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function.

How do RNA sequence, DNA sequence, and chromatin properties regulate splicing?

Recent genome-wide studies have revealed a remarkable correspondence between nucleosome positions and exon-intron boundaries, and several studies have implicated specific histone modifications in regulating alternative splicing. In addition, recent progress in cracking the 'splicing code' shows that sequence motifs carried on the nascent RNA molecule itself are sufficient to accurately predict tissue-specific alternative splicing patterns. Together, these studies shed light on the complex interplay between RNA sequence, DNA sequence, and chromatin properties in regulating splicing.

Transgenesis in Drosophila melanogaster.

Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequent crossing of surviving adults. The necessity of crossing single flies to screen for transgenic events limits the range of useful transgenesis techniques to those that have a very high frequency of integration, so that about 1 in 10 to 1 in 100 surviving adult flies carry a transgene. Until recently, only random P-element transgenesis fulfilled these criteria. However, recent advances have brought homologous recombination and site-directed integration up to and beyond this level of efficiency. For all transgenesis techniques in Drosophila melanogaster, microinjection of embryos is the central procedure. This chapter gives a detailed protocol for microinjection, and aims to enable the reader to use it for both site-directed integration and for P-element transgenesis.

Non-coding RNAs in polycomb/trithorax regulation.

The Polycomb (PcG) and Trithorax (TrxG) proteins are epigenetic regulators that maintain correct expression patterns of several hundred developmentally important genes by binding to DNA regulatory elements called Polycomb/Trithorax Response Elements (PRE/TREs). Many PRE/TREs are transcribed into long non-coding RNAs. Furthermore, many PcG and TrxG proteins bind to RNA, and recent evidence suggests that these RNA interactions are essential for targeting both groups of proteins to specific sites, and modulating their effects on gene expression. We discuss current evidence for the molecular mechanisms by which non-coding RNAs may recruit PcG and TrxG proteins, switch gene expression states, and maintain epigenetic memory.