Gene Regulation: Stable Noise

Transcriptional regulation is noisy, yet despite this variability, embryonic development reproducibly generates form and function. Recent work demonstrates that patterns of transcriptional activity in embryos are stably inherited through mitosis. These observations have implications for how accuracy arises in development

Generation of Single-Cell Transcript Variability by Repression

Gene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation

A continuum model of transcriptional bursting

Transcription occurs in stochastic bursts. Early models based upon RNA hybridisation studies suggest bursting dynamics arise from alternating inactive and permissive states. Here we investigate bursting mechanism in live cells by quantitative imaging of actin gene transcription, combined with molecular genetics, stochastic simulation and probabilistic modelling. In contrast to early models, our data indicate a continuum of transcriptional states, with a slowly fluctuating initiation rate converting the gene between different levels of activity, interspersed with extended periods of inactivity. We place an upper limit of 40s on the lifetime of fluctuations in elongation rate, with initiation rate variations persisting an order of magnitude longer. TATA mutations reduce the accessibility of high activity states, leaving the lifetime of on- and off-states unchanged. A continuum or spectrum of gene states potentially enables a wide dynamic range for cell responses to stimuli

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate

Regulation of Transcriptional Bursting by a Naturally Oscillating Signal.

Transcription is highly stochastic, occurring in irregular bursts [1-3]. For temporal and spatial precision of gene expression, cells must somehow deal with this noisy behavior. To address how this is achieved, we investigated how transcriptional bursting is entrained by a naturally oscillating signal, by direct measurement of transcription together with signal dynamics in living cells. We identify a Dictyostelium gene showing rapid transcriptional oscillations with the same period as extracellular cAMP signaling waves. Bursting approaches antiphase to cAMP waves, with accelerating transcription cycles during differentiation. Although coupling between signal and transcription oscillations was clear at the population level, single-cell transcriptional bursts retained considerable heterogeneity, indicating that transcription is not governed solely by signaling frequency. Previous studies implied that burst heterogeneity reflects distinct chromatin states [4-6]. Here we show that heterogeneity is determined by multiple intrinsic and extrinsic cues and is maintained by a transcriptional persistence. Unusually for a persistent transcriptional behavior, the lifetime was only 20 min, with rapid randomization of transcriptional state by the response to oscillatory signaling. Linking transcription to rapid signaling oscillations allows reduction of gene expression heterogeneity by temporal averaging, providing a mechanism to generate precision in cell choices during development

Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis.

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP6 or Phytic acid) and its derivative inositol pyrophosphates, IP7 and IP8. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP9 in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP5) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP8 was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP5K, doesn't' play a major role in the IP8 developmental increase

Promoter-mediated diversification of transcriptional bursting dynamics following gene duplication

During the evolution of gene families, functional diversification of proteins often follows gene duplication. However, many gene families expand while preserving protein sequence. Why do cells maintain multiple copies of the same gene? Here we have addressed this question for an actin family with 17 genes encoding an identical protein. The genes have divergent flanking regions and are scattered throughout the genome. Surprisingly, almost the entire family showed similar developmental expression profiles, with their expression also strongly coupled in single cells. Using live cell imaging, we show that differences in gene expression were apparent over shorter timescales, with family members displaying different transcriptional bursting dynamics. Strong “bursty” behaviors contrasted steady, more continuous activity, indicating different regulatory inputs to individual actin genes. To determine the sources of these different dynamic behaviors, we reciprocally exchanged the upstream regulatory regions of gene family members. This revealed that dynamic transcriptional behavior is directly instructed by upstream sequence, rather than features specific to genomic context. A residual minor contribution of genomic context modulates the gene OFF rate. Our data suggest promoter diversification following gene duplication could expand the range of stimuli that regulate the expression of essential genes. These observations contextualize the significance of transcriptional bursting

Moving the research forward: The best of british biology using the tractable model system dictyostelium discoideum

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system