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Dynamics of epigenetic regulation at the single-cell level.

ABSTRACT: Chromatin regulators play a major role in establishing and maintaining gene expression states. Yet how they control gene expression in single cells, quantitatively and over time, remains unclear. We used time-lapse microscopy to analyze the dynamic effects of four silencers associated with diverse modifications: DNA methylation, histone deacetylation, and histone methylation. For all regulators, silencing and reactivation occurred in all-or-none events, enabling the regulators to modulate the fraction of cells silenced rather than the amount of gene expression. These dynamics could be described by a three-state model involving stochastic transitions between active, reversibly silent, and irreversibly silent states. Through their individual transition rates, these regulators operate over different time scales and generate distinct types of epigenetic memory. Our results provide a framework for understanding and engineering mammalian chromatin regulation and epigenetic memory.

SUBMITTER: Bintu L

PROVIDER: S-EPMC5108652 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Dynamics of epigenetic regulation at the single-cell level.

Bintu Lacramioara L Yong John J Antebi Yaron E YE McCue Kayla K Kazuki Yasuhiro Y Uno Narumi N Oshimura Mitsuo M Elowitz Michael B MB

Science (New York, N.Y.) 20160201 6274

Chromatin regulators play a major role in establishing and maintaining gene expression states. Yet how they control gene expression in single cells, quantitatively and over time, remains unclear. We used time-lapse microscopy to analyze the dynamic effects of four silencers associated with diverse modifications: DNA methylation, histone deacetylation, and histone methylation. For all regulators, silencing and reactivation occurred in all-or-none events, enabling the regulators to modulate the fr ...[more]

PMID: 26912859

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Project description:We have analysed activation of nuclear factor-kappaB (NF-kappaB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-kappaB1 and inhibitor of kappaB (I-kappaB) alpha using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-kappaBalpha degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-kappaB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3-4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration. IL-1 stimulation caused translocation of only 20% of the relA, but resulted in degradation of up to 70% of the cytoplasmic I-kappaBalpha. RelA nuclear translocation in fibroblasts correlated with DNA-binding activity measured by electrophoretic mobility shift assay (EMSA), both with respect to kinetics and IL-1 concentration-dependence. Clonal populations of cells demonstrated a marked degree of heterogeneity in the response to IL-1. The single-cell assay revealed the presence of responder and non-responder subpopulations, with an enhanced proportion of responder cells, and prolonged responses at higher concentrations of IL-1. Comparing different cell types demonstrated that whereas HepG2 cells, as fibroblasts, showed good correlation between nuclear translocation of relA and activation of DNA binding by relA-containing dimers, EL4 thymoma cells showed no effect on relA localization, even during induction of significant levels NF-kappaB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-kappaB system, which cycles between the resting and active states with net redistribution of a minor proportion of its DNA-binding component. In addition, it demonstrates significant cell-to-cell variations, as well as cell-type-specific differences in net relA nuclear transport in response to stimuli. The data are consistent with NF-kappaB constituting a dynamic and versatile system, regulated to a significant degree by binary events involving bidirectional trafficking between the cytoplasmic and nuclear compartments during pathway activation.

Project description:Here we describe a method for whole genome bisulfite sequencing in very small cell populations (µWGBS) and in single cells (scWGBS). Our method is optimized for profiling DNA methylation in many samples at low coverage, and we developed a bioinformatic method that uses collections of single-cell methylomes to infer cell state dynamics. Using these technological advances, we studied epigenetic cell state dynamics in three in vitro models of cellular differentiation and pluripotency and identified patterns of epigenetic remodeling and cell-to-cell heterogeneity. 1-cell or 4-cell samples were collected from a variety of human and mouse cell lines (KBM7, CCE, K562, 32D, HL60). We also included negative controls (no cells), and cross controls (cells of one species aligned to the other). K562 cells were treated with 5-azacytidine in a final concentration of 1µM to induce demethylation. HL60 cells were treated with 1α,25-dihydroxyvitamin D3 in a final concentration of 10nM to induce differentiation. Naïve pluripotency of CCE ES cells was induced by culturing the cells in ESGRO-2i medium with the addition of 1% FCS. Differentiation of CCE ES cells was induced by treating the cells with all-trans retinoic acid (ATRA) in a final concentration of 0,3µM every third day along with complete LIF-free medium exchange. Embryoid body formation from CCE ES cells was induced by hanging-drop culture for 5 days, followed by 9 days on gelatinized dishes (see treatment annotation). Each experiment, with treated and untreated cells, was performed in multiple biological replicates. We then measured DNA methylation in the individual 1-cell or 4-cell experiments with bisulfite treatment and high-throughput sequencing. For sequencing, most experiments pooled a 4-cell experiment from one species with a 1-cell experiment from the other, but some replicates were sequenced as a single cell (indicated with SC in the replicate annotation). Two samples (marked as “deep”) were sequenced much deeper than the others. See Farlik et al. (2015) Cell Reports for complete study details.

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Dynamics of epigenetic regulation at the single-cell level.

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Dynamics of epigenetic regulation at the single-cell level.

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