Emergence of activation or repression in transcriptional control under a fixed molecular context.

For decades, studies have noted that transcription factors (TFs) can behave as either activators or repressors of different target genes. More recently, evidence suggests TFs can act on transcription simultaneously in positive and negative ways. Here we use biophysical models of gene regulation to define, conceptualize and explore these two aspects of TF action: "duality", where TFs can be overall both activators and repressors at the level of the transcriptional response, and "coherent and incoherent" modes of regulation, where TFs act mechanistically on a given target gene either as an activator or a repressor (coherent) or as both (incoherent). For incoherent TFs, the overall response depends on three kinds of features: the TF's mechanistic effects, the dynamics and effects of additional regulatory molecules or the transcriptional machinery, and the occupancy of the TF on DNA. Therefore, activation or repression can be tuned by just the TF-DNA binding affinity, or the number of TF binding sites, given an otherwise fixed molecular context. Moreover, incoherent TFs can cause non-monotonic transcriptional responses, increasing over a certain concentration range and decreasing outside the range, and we clarify the relationship between non-monotonicity and common assumptions of gene regulation models. Using the mammalian SP1 as a case study and well controlled, synthetically designed target sequences, we find experimental evidence for incoherent action and activation, repression or non-monotonicity tuned by affinity. Our work highlights the importance of moving from a TF-centric view to a systems view when reasoning about transcriptional control.

Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

Transcription factors (TFs) control gene expression, often acting synergistically. Classical thermodynamic models offer a biophysical explanation for synergy based on binding cooperativity and regulated recruitment of RNA polymerase. Because transcription requires polymerase to transition through multiple states, recent work suggests that "kinetic synergy" can arise through TFs acting on distinct steps of the transcription cycle. These types of synergy are not mutually exclusive and are difficult to disentangle conceptually and experimentally. Here, we model and build a synthetic circuit in which TFs bind to a single shared site on DNA, such that TFs cannot synergize by simultaneous binding. We model mRNA production as a function of both TF binding and regulation of the transcription cycle, revealing a complex landscape dependent on TF concentration, DNA binding affinity, and regulatory activity. We use synthetic TFs to confirm that the transcription cycle must be integrated with recruitment for a quantitative understanding of gene regulation.

Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency.

Discontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factors is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time-resolved measurements of gene expression at the single-cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time-varying acetylation state of p53's C-terminal lysine residues is critical for gene-specific regulation of stochastic bursting.

Resolving titin's lifecycle and the spatial organization of protein turnover in mouse cardiomyocytes.

Cardiac protein homeostasis, sarcomere assembly, and integration of titin as the sarcomeric backbone are tightly regulated to facilitate adaptation and repair. Very little is known on how the >3-MDa titin protein is synthesized, moved, inserted into sarcomeres, detached, and degraded. Here, we generated a bifluorescently labeled knockin mouse to simultaneously visualize both ends of the molecule and follow titin's life cycle in vivo. We find titin mRNA, protein synthesis and degradation compartmentalized toward the Z-disk in adult, but not embryonic cardiomyocytes. Originating at the Z-disk, titin contributes to a soluble protein pool (>15% of total titin) before it is integrated into the sarcomere lattice. Titin integration, disintegration, and reintegration are stochastic and do not proceed sequentially from Z-disk to M-band, as suggested previously. Exchange between soluble and integrated titin depends on titin protein composition and differs between individual cardiomyocytes. Thus, titin dynamics facilitate embryonic vs. adult sarcomere remodeling with implications for cardiac development and disease.

PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response and Cell Cycle Regulation in Individual Cells.

To enable reliable cell fate decisions, mammalian cells need to adjust their responses to dynamically changing internal states by rewiring the corresponding signaling networks. Here, we combine time-lapse microscopy of endogenous fluorescent reporters with computational analysis to understand at the single-cell level how the p53-mediated DNA damage response is adjusted during cell cycle progression. Shape-based clustering revealed that the dynamics of the CDK inhibitor p21 diverges from the dynamics of its transcription factor p53 during S phase. Using mathematical modeling, we predict and experimentally validate that S phase-specific degradation of p21 by PCNA-CRL4cdt2 is sufficient to explain these heterogeneous responses. This highlights how signaling pathways and cell regulatory networks intertwine to adjust the cellular response to the individual needs of a given cell.

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging.

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP-FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.

A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells.

Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.