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Title: The Role of Multiple Marks in Epigenetic Silencing and the Emergence of a Stable Bivalent Chromatin State}
Article:
Abstract:
We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent( i. e., both active and silencing) marks under certain perturbations( knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants( e. g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.
Introduction:
To provide the relevant context of our approach to a wider audience, we briefly review the molecular biology of yeast silencing system and its central puzzles. In budding yeast, the Silent mating-type Information Regulator( SIR) proteins have been identified to play a pivotal role in heterochromatin formation[30],[37]–[39]. The Sir complex includes four proteins, of which Sir1 facilitates assembly of Sir2–4 at the Hidden Mating( HML/HMR) Cassettes, in concert with proteins factors like Orc, Rap1 an Abf1, all of which have specific binding sites at the silencer of HML/HMR. In telomeres, the players are slightly different; Sir1 is not needed for the nucleation of Sir complex[38],[39] and yKu DNA-end binding complexes is important. One of the proteins of the Sir complex, Sir2, is a NAD-dependent deacetylase[40]. The acetylation of Histone tails antagonizes silencing[21],[41]–[44]. The deacetylase activity of Sir2 is critical for the spread of silencing by presumably increasing Sir3-Sir4 affinity for nucleosomes[45]. Specifically, acetylation of H4K16 by Sas2( Something About Silencing) activity undermined the spread of silencing[6],[7],[46],[47]. Recent studies have established that besides the interaction of Sir proteins with nucleosomes[48], interactions between Sir proteins themselves are crucial in the spread of silencing[18],[45],[49]. This spreading in telomeres appears to be unhindered by silencer boundary element and is arguably stochastic[33],. In contrast, in the HML/HMR loci boundary elements prevent the spread of Sir complex[7],[53]. Sir spreading happens in only a few of the natural telomeres[15],[33],[54]. Moreover, mechanisms like the clustering of telomeres anchored to the nuclear envelope is perhaps important in subtelomeric silencing[52]. Sir2 does not exclusively deacetylate H4K16 residue. Other residues like H3K9 and H3K14 are also deacetylated moderately[34], and H4K56 extensively by Sir2[55]. However, the spreading of Sir2 compete primarily with H4K16Ac[46]. The compaction into silent heterochromatin requires further deacetylation of H4K56Ac[55]. The effect of spread of Sir proteins on silencing is subtle—spreading is necessary but not sufficient for heterochromatin formation and Sir binding is broader in scope[15]. Though the compaction of chromatin owing to stable Sir-complex-binding blocks association of RNA polymerase II thereby preventing transcriptional activity[7], Sir binding and spreading itself may be a very dynamic phenomenon. Not surprisingly, Sir protein binds at various loci which are not necessarily silenced, as deduced from Chromatin Immunoprecipitation( ChIP) studies[15],[56]. The association of Sir proteins does not immediately lead to gene silencing[57] arguing for added steps to the formation of heterochromatin[9],[10],[12],[58]. Nevertheless, Sir protein association is a prerequisite in establishing an inheritable pattern of silencing in the telomeric and HML/HMR loci. Active sites in budding yeast chromatin are not only hyper-acetylated at particular histone tail sites but also tend to be hyper-methylated at certain key residues[23],[32],[35],[51],[59]. One of the DOT( Disruptor Of Telomeric Silencing) proteins, Dot1, methylates H3K79 residue and competes in binding with Sir3 for the same basic patch on the histone core region( H3K79)[45],[59]–[61]. Dot1 can mono- di- or tri- methylate this residue distributively[62]. Studies have revealed that H4K16Ac displaces Sir3 binding, thereby aiding Dot1-mediated methylation of H3K79[60]. What other histone marks( or other factors) regulate transcriptional activity is a complex question[63]. The enzymatic activity of Dot1 itself is modulated by various other factors. For example, Paf1 complex( RNA Polymerase Associating Factor) which is known to be an important factor in transcription elongation plays a crucial role in Dot1 methylase activity[64]–[66]. In fact, such positive feedback from transcriptional elongation into establishing transcriptionally-active marks is not uncommon. For example, H3K4 methylation[67]–[69], which is an active mark in yeast, requires Paf1[66] for being methylated by Set1[70]. The phenomenon of yeast silencing has several other components many of which are poorly understood. Though the structure and interaction domains of Sir proteins are well-studied[45],[48],[49],[71]–[73], protein factors, histone modifications etc. which interact with the system continue to be uncovered. For example, Rad6 dependent ubiquitylation of H2B-K123 influences methylation by Dot1 and Set1[69],[74]. Ubiquitylation is a histone mark implicated in transcriptional initiation and elongation[75], and such a trans-histone regulatory pathway acts as a feedback into the establishment of transcriptional activity. Another such feedback in establishment of active marks is the interaction of Dot1 with a histone acetyltransferase[76]. Sumoylation is a silencing histone mark, and might play an important role in the silencing of subtelomeric regions[77],[78]. Variants of the histones[79],[80], particularly H2A. Z, plays a complex role in transcriptional activity, and has been shown to deter the ectopic spread of silencing[68]. The topic of histone variants invite a host of connections between chromatin assembly and transcriptional activity. These influences are perhaps peripheral to the central mechanism resolving epigenetic fates[81]–[83]. We argue that the epigenetic fates are reinforced both from transcriptional and silencing feedbacks—our focus is the core mechanism, summarized in Fig. 1. Recent experiments[15] have shown that binding of Sir3 protein is not indicative of heterochromatin and many genes in euchromatin, including highly transcribed genes, may show wide-spread Sir3 binding. Such a co-occurrence of silencing and active marks called bivalent chromatin states has been discussed in the context of Polycomb silencing[84] and that of HP1 binding in expressed exons[85]. We show how stable bivalent states emerge naturally on modelling the known interactions. In the next section we define the mathematical model based on this minimal picture.
Discussion:
We have introduced and analyzed a model which captures the distinct mechanisms of cooperativity at the molecular level in the budding yeast silencing system;( a) cooperativity in the histone modification states,( b) cooperative interactions of the Sir proteins in a chromatin-bound-complex. Both of these mechanisms have been proposed by different groups, sometimes as mutually-exclusive ones[1]–[3],[17],[18],[51]. We show that these two mechanisms complement each other in designing epigenetic stability. Our model is minimal—all the interactions included are essential for establishment and inheritance of stable epigenetic fates. The model is build entirely upon known biochemical interactions. Though we explore design principles of the system using inhibition/over-expression/knock-out of key proteins like Sir, Sas2 and Dot1, the model can be extended to include other silencing and active marks—and can inform, like it did for methylation, which of those marks are essential in resolving epigenetic fate, and which simply reinforces one fate over another. Our model makes the following predictions— Experiments focusing on direct measurement of Sir occupancy, preferably at single-cell resolution, as a function of tuning Dot1, Sas2 and Sir2's enzymatic activity will put to test several observations of the model—the emergence of the bivalent state, and the rich behavior of silencing domains under Sir titration. We have argued that inhibition/over-expression experiments can reveal key information about the engineering design of the system not observable in knock-out experiments. We hope that our model will sharpen experimental questions on the systems biology of budding yeast silencing. For the sake of brevity we have presented results for single perturbations; the model is especially informative for multiple perturbations, for example, double mutants. We are currently investigating such perturbations experimentally. After our paper was submitted, an experimental study by Grunstein lab[94] has further revealed the role of H3K79 methylation in the silencing state of telomeres. Specifically, the transcription-mediated positive feedback of H3K79 methylation was discussed, and indications of our bivalent state where Sir occupancy and H3K79 methylation coexist were provided, though the response of this state to perturbations is still to be studied.
Epigenetics is the study of heritable phenotypic variations that are not caused by changes in the genotype . Silent Information Regulator ( SIR ) silencing in budding yeast is an important model system for epigenetics . The standard model of silencing relies on feedback , mediated by chromatin modifications ( for example , deacetylation of histone residues ) which lead to enhanced recruitment of chromatin modifiers . However , the SIR mechanism is not completely understood and it is important to investigate whether as-yet-undiscovered components alter the systems design in a fundamental way . We address this question using minimal models constructed from experimentally known interactions . Rather than building a detailed network model with parameters to fit for quantitative predictions , we build an effective model and study its bifurcation diagram which leads to robust qualitative predictions on the nature of mutants . This minimal modeling delineates a phase space with qualitatively different epigenetic mechanisms and states; some of which arise from drug/genetic perturbations and exhibit large cell-to-cell variation in chromatin marks . Our methodology can be applied to the study of epigenetic chromatin silencing in other model systems , especially Polycomb silencing , and reveals engineering principles that may be of broad relevance .