Noncoding RNA-nucleated heterochromatin spreading is intrinsically labile and requires accessory elements for epigenetic stability.
ABSTRACT: The heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high positional precision, or fidelity, following replication. How the spreading reaction contributes to this fidelity is not clear. To resolve the origins of stable inheritance of repression, we probed the intrinsic character of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading triggered by noncoding RNA-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. This lack of stability correlates with high histone turnover. At the mating type locus, this unstable behavior is restrained by an accessory cis-acting element REIII, which represses histone turnover. Further, REIII safeguards epigenetic memory against environmental perturbations. Our results suggest that the most prevalent type of spreading, driven by noncoding RNA-nucleators, is epigenetically unstable and requires collaboration with accessory elements to achieve high fidelity.
Project description:Heterochromatin formation requires several steps involving: nucleation followed by spreading of large, silent domains and its faithful re-establishment and maintenance after each replication cycle. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, little is known about the specificity of FACT in this process. Here, by applying single cell assays, ChIP and genetics, we show that FACT mutants have defects in the spreading of heterochromatin at subtelomeres and mating type locus in fission yeast. Further, we demonstrate that the transition of H3K9me2 to me3 is impaired in the FACT mutant and we link the spreading function of FACT to its role in histone turnover suppression. We also identify Epe1 as a specific factor that counteracts heterochromatin spreading defects in the FACT mutants. Together, our studies identify FACT as a first histone chaperone that promotes heterochromatin spreading and add to the current models on histone turnover in propagation of epigenetic marks. Overall design: ChIP-seq experiments in wild type and pob3∆ mutant fission yeast.
Project description:BACKGROUND: The activity of a single gene is influenced by the composition of the chromatin in which it is embedded. Nucleosome turnover, conformational dynamics, and covalent histone modifications each induce changes in the structure of chromatin and its affinity for regulatory proteins. The dynamics of histone modifications and the persistence of modification patterns for long periods are still largely unknown. RESULTS: In this study, we present a stochastic mathematical model that describes the molecular mechanisms of histone modification pattern formation along a single gene, with non-phenomenological, physical parameters. We find that diffusion and recruitment properties of histone modifying enzymes together with chromatin connectivity allow for a rich repertoire of stochastic histone modification dynamics and pattern formation. We demonstrate that histone modification patterns at a single gene can be established or removed within a few minutes through diffusion and weak recruitment mechanisms of histone modification spreading. Moreover, we show that strong synergism between diffusion and weak recruitment mechanisms leads to nearly irreversible transitions in histone modification patterns providing stable patterns. In the absence of chromatin connectivity spontaneous and dynamic histone modification boundaries can be formed that are highly unstable, and spontaneous fluctuations cause them to diffuse randomly. Chromatin connectivity destabilizes this synergistic system and introduces bistability, illustrating state switching between opposing modification states of the model gene. The observed bistable long-range and localized pattern formation are critical effectors of gene expression regulation. CONCLUSION: This study illustrates how the cooperative interactions between regulatory proteins and the chromatin state generate complex stochastic dynamics of gene expression regulation.
Project description:Heterochromatin, a highly compact chromatin state characterized by histone H3K9 methylation and HP1 protein binding, silences the underlying DNA and influences the expression of neighboring genes. However, the mechanisms that regulate heterochromatin spreading are not well understood. In this study, we show that the conserved Mst2 histone acetyltransferase complex in fission yeast regulates histone turnover at heterochromatin regions to control heterochromatin spreading and prevents ectopic heterochromatin assembly. The combined loss of Mst2 and the JmjC domain protein Epe1 results in uncontrolled heterochromatin spreading and massive ectopic heterochromatin, leading to severe growth defects due to the inactivation of essential genes. Interestingly, these cells quickly recover by accumulating heterochromatin at genes essential for heterochromatin assembly, leading to their reduced expression to restrain heterochromatin spreading. Our studies discover redundant pathways that control heterochromatin spreading and prevent ectopic heterochromatin assembly and reveal a fast epigenetic adaptation response to changes in heterochromatin landscape.
Project description:To investigate the possible range of additional RNase P substrates in vivo a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turnover pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively non-specific capacity to bind and cleave mixed sequence RNAs, these data suggest nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.
Project description:The combinatorial pattern of DNA and histone modifications constitutes an epigenetic 'code' that shapes gene-expression patterns by enabling or restricting the transcriptional potential of genomic domains. DNA methylation is associated with histone modifications, particularly the absence of histone H3 lysine 4 methylation (H3K4me0) and the presence of H3K9 methylation. This article focuses on three protein domains (ATRX-Dnmt3-Dnmt3L [ADD], Cys-X-X-Cys [CXXC] and the methyl-CpG-binding domain [MBD]) and the functional implications of domain architecture in the mechanisms linking histone methylation and DNA methylation in mammalian cells. The DNA methyltransferase DNMT3a and its accessory protein Dnmt 3L contain a H3K4me0-interacting ADD domain that links the DNA methylation reaction with unmodified H3K4. The H3K4 methyltransferase MLL1 contains a CpG-interacting CXXC domain that may couple the H3K4 methylation reaction to unmethylated DNA. Another H3K4 methyltransferase, SET1, although lacking an intrinsic CXXC domain, interacts directly with an accessory protein CFP1 that contains the same domain. The H3K9 methyltransferase SETDB1 contains a putative MBD that potentially links the H3K4 methylation reaction to methylated DNA or may do so through the interaction with the MBD containing protein MBD1. Finally, we consider the domain structure of the DNA methyltransferase DNMT1, its accessory protein UHRF1 and their associated proteins, and propose a mechanism by which DNA methylation and histone methylation may be coordinately maintained through mitotic cell division, allowing for the transmission of parental DNA and for the histone methylation patterns to be copied to newly replicated chromatin.
Project description:The dosage compensation complex in Drosophila is composed of at least five MSL proteins and two noncoding roX RNAs that bind hundreds of sites along the single male X chromosome. The roX RNAs are transcribed from X-linked genes and their RNA products "paint" the male X. The roX RNAs and bound MSL proteins can spread in cis from sites of roX transcription, but the mechanism controlling spreading is unknown. Here we find that cis spreading from autosomal roX1 transgenes is coupled to the level of roX transcription. Low to moderate transcription favors, and vigorous transcription abolishes local spreading. We constructed a roX1 minigene one third the size of wild type as a starting point for mutagenesis. This allowed us to test which evolutionarily conserved motifs were required for activity. One short repeat element shared between roX1 and roX2 was found to be particularly important. When all copies were deleted, the RNA was inactive and unstable, while extra copies seem to promote local spreading of the MSL complex from sites of roX1 synthesis. We propose that assembly of the MSL proteins onto the extreme 3' region of elongating roX1 transcripts determines whether the MSL complex spreads in cis.
Project description:A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.
Project description:Eukaryotic genomes are pervasively transcribed, resulting in the production of many unstable nuclear long noncoding RNAs such as nuclear Short-Lived noncoding Transcripts (nSLiTs). However, the biological significance and turnover mechanism of nSLiTs are largely unknown. We used microarrays to detail the global program of gene expression that is regulated by enSLiT upon Salmonella infection Overall design: To assess the role of enSLiT against Salmonella infection, we knockout enSLiT07573 by using CRISPR/Cas9. KO was performed replicate for each transcripts. We comparied Salmonella infected/not infected HeLa cells and KO cells. All data was obtained biological triplicate.
Project description:Eukaryotic genomes are pervasively transcribed, resulting in the production of many unstable nuclear long noncoding RNAs such as nuclear Short-Lived noncoding Transcripts (nSLiTs). However, the biological significance and turnover mechanism of nSLiTs are largely unknown. We used microarrays to detail the global program of gene expression that is regulated by LinSLiT08211 upon Salmonella infection Overall design: To assess the role of LinSLiT against Salmonella infection, we knockout LinSLiT08211 by using CRISPR/Cas9. KO was performed replicate for each transcripts. We comparied Salmonella infected/not infected HeLa cells and KO cells. All data was obtained biological triplicate.
Project description:The combinatorial pattern of DNA and histone modifications and their associated histone variants constitute an epigenetic code that shapes gene expression patterns by increasing or decreasing the transcriptional potential of genomic domains. The epigenetic coding status, at any given chromosomal location, is subject to modulation by noncoding RNAs and remodeling complexes. DNA methylation is associated with histone modifications, particularly the absence of histone H3 lysine 4 methylation (H3K4me0) and the presence of histone H3 lysine 9 methylation (H3K9m). We briefly discuss four protein domains (ADD, CXXC, MBD, and SRA), and the functional implications of their architecture in linking histone methylation to that of DNA in mammalian cells. We also consider the domain structure of the DNA methyltransferase DNMT1, its accessory protein UHRF1, and their associated proteins. Finally, we discuss a mechanism by which methylation of DNA and of histones may be coordinately maintained during mitotic cell division, allowing for the transmission of parental methylation patterns to newly replicated chromatin.