Anders S. Hansen

Promoter decoding of transcription factor dynamics involves a trade‐off between noise and control of gene expression

Numerous transcription factors (TFs) encode information about upstream signals in the dynamics of their activation, but how downstream genes decode these dynamics remains poorly understood. Using microfluidics to control the nucleocytoplasmic translocation dynamics of the budding yeast TF Msn2, we elucidate the principles that govern how different promoters convert dynamical Msn2 input into gene expression output in single cells. Combining modeling and experiments, we classify promoters according to their signal‐processing behavior and reveal that multiple, distinct gene expression programs can be encoded in the dynamics of Msn2. We show that both oscillatory TF dynamics and slow promoter kinetics lead to higher noise in gene expression. Furthermore, we show that the promoter activation timescale is related to nucleosome remodeling. Our findings imply a fundamental trade‐off: although the cell can exploit different promoter classes to differentially control gene expression using TF dynamics, gene expression noise fundamentally limits how much information can be encoded in the dynamics of a single TF and reliably decoded by promoters.

Control of Olefin Geometry in Macrocyclic Ring-Closing Metathesis Using a Removable Silyl Group

Introducing a silyl group at one of the internal olefin positions in diolefinic substrates results in E-selective olefin formation in macrocyclic ring-forming metathesis. The application of this method to a range of macrocyclic (E)-alkenylsiloxanes is described. Protodesilylation of alkenylsiloxane products yields novel Z-configured macrocycles.

A dynamic mode of mitotic bookmarking by transcription factors

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking. DOI: http://dx.doi.org/10.7554/eLife.22280.001

High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. By using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, while collecting data for thousands of single cells. Compared with other protocols, the present protocol is relatively easy to adopt and of higher throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.

Limits on information transduction through amplitude and frequency regulation of transcription factor activity

Signaling pathways often transmit multiple signals through a single shared transcription factor (TF) and encode signal information by differentially regulating TF dynamics. However, signal information will be lost unless it can be reliably decoded by downstream genes. To understand the limits on dynamic information transduction, we apply information theory to quantify how much gene expression information the yeast TF Msn2 can transduce to target genes in the amplitude or frequency of its activation dynamics. We find that although the amount of information transmitted by Msn2 to single target genes is limited, information transduction can be increased by modulating promoter cis-elements or by integrating information from multiple genes. By correcting for extrinsic noise, we estimate an upper bound on information transduction. Overall, we find that information transduction through amplitude and frequency regulation of Msn2 is limited to error-free transduction of signal identity, but not signal intensity information. DOI: http://dx.doi.org/10.7554/eLife.06559.001

Encoding four gene expression programs in the activation dynamics of a single transcription factor

Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3-5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs.

Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing1–3. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)—which consists of CDK9 and cyclin T1—is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation1,4,5. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase—DYRK1A6—also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD—which in isolation does not phase separate, despite being a low-complexity domain—is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH1–3. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.The histidine-rich domain of cyclin T1 promotes phase separation into liquid droplets, which facilitates the hyperphosphorylation of the C-terminal domain repeats of RNA polymerase II.

Recent evidence that TADs and chromatin loops are dynamic structures

ABSTRACT Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle.

Robust model-based analysis of single-particle tracking experiments with Spot-On

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce ‘Spot-On’, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

RNA polymerase II clustering through CTD phase separation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters at active genes through interactions between CTDs and with activators, and that CTD phosphorylation removes Pol II enzymes from clusters for transcription elongation.