Jonathan R. Chubb

Considering Nuclear Compartmentalization in the Light of Nuclear Dynamics

Many proteins are concentrated in compartments within the nucleus. Chromatin is also compartmentalized at different nuclear sites. However, nuclear proteins have now been shown to be highly mobile. This review considers the formation and function of nuclear compartments in a situation in which proteins are rapidly moving through the nuclear volume.

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary “tunable” responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.

Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.

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 40 s 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. DOI: http://dx.doi.org/10.7554/eLife.13051.001

Digital nature of the immediate-early transcriptional response

Stimulation of transcription by extracellular signals is a major component of a cells decision making. Yet the quantitative relationship between signal and acute transcriptional response is unclear. One view is that transcription is directly graded with inducer concentration. In an alternative model, the response occurs only above a threshold inducer concentration. Standard methods for monitoring transcription lack continuous information from individual cells or mask immediate-early transcription by measuring downstream protein expression. We have therefore used a technique for directly monitoring nascent RNA in living cells, to quantify the direct transcriptional response to an extracellular signal in real time, in single cells. At increasing doses of inducer, increasing numbers of cells displayed a transcriptional response. However, over the same range of doses, the change in cell response strength, measured as the length, frequency and intensity of transcriptional pulses, was small, with considerable variation between cells. These data support a model in which cells have different sensitivities to developmental inducer and respond in a digital manner above individual stimulus thresholds. Biased digital responses may be necessary for certain forms of developmental specification. Limiting bias in responsiveness is required to reduce noise in positional signalling.

Regulation of Transcriptional Bursting by a Naturally Oscillating Signal

Summary 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.

A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development.

BACKGROUND Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins. RESULTS We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins. CONCLUSIONS RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.

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.

Chromosome Position: Now, Where Was I?

Is the nuclear organisation of chromosomes inherited through mitosis, when the nuclear membrane is broken down, and is it propagated to the nuclei of daughter cells? Two recent studies address this question using similar live cell imaging techniques, but reach different conclusions.