Biology subjects

Lammers, N. C.

Publications and source records attributed to Lammers, N. C..

8 recordsLinked to original sources

bioRxiv2023

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

developmental biology↗

bioRxiv2022

Optogenetic dissection of transcriptional repression in a multicellular organism

Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive. Here we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible ([~]1 minute) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

biophysics↗

bioRxiv2022

Decoding of YAP levels and dynamics by pluripotency factors

YAP is a transcriptional regulator that controls pluripotency, germ layer specification, and proliferation. Different subsets of YAP target genes are engaged in each physiological setting, but how YAP selectively regulates different effectors in different contexts is not known. Here we use optogenetics to investigate how the levels and dynamics of YAP activation control its pluripotency effectors Oct4 and Nanog. We observe different thresholds for repression of Oct4 and Nanog, enabling differential control of both genes through YAP levels. Pluripotency factors also decode YAP dynamics. Oct4 preferentially responds to oscillatory YAP inputs that mimic endogenous pulsatile YAP dynamics. Using single-cell live imaging of Oct4 transcription and computational-theoretical analysis of transcriptional regulation, we demonstrate that YAP dynamics are decoded by an adaptive change sensor that modulates Oct4 transcription burst frequency. Our results reveal how the levels and timing of YAP activation enable multiplexing of information transmission for key regulators of cellular differentiation and pluripotency.

cell biology↗

bioRxiv2022

Competing constraints shape the non-equilibrium limits of cellular decision making

Gene regulation is central to cellular function. Yet, despite decades of work, we lack quantitative models that can predict how transcriptional control emerges from molecular interactions at the gene locus. Thermodynamic models of transcription, which assume that gene circuits operate at equilibrium, have previously been employed with considerable success in the context of bacterial systems. However, the presence of ATP-dependent processes within the eukaryotic transcriptional cycle suggests that equilibrium models may be insufficient to capture how eukaryotic gene circuits sense and respond to input transcription factor concentrations. Here, we employ simple kinetic models of transcription to investigate how energy dissipation within the transcriptional cycle impacts the rate at which genes transmit information and drive cellular decisions. We find that biologically plausible levels of energy input can lead to significant gains in how rapidly gene loci transmit information, but discover that the regulatory mechanisms underlying these gains change depending on the level of interference from non-cognate activator binding. When interference is low, information is maximized by harnessing energy to push the sensitivity of the transcriptional response to input transcription factors beyond its equilibrium limits. Conversely, when interference is high, conditions favor genes that harness energy to increase transcriptional specificity by proofreading activator identity. Our analysis further reveals that equilibrium gene regulatory mechanisms break down as transcriptional interference increases, suggesting that energy dissipation may be indispensable in systems where non-cognate factor interference is sufficiently large.

biophysics↗

bioRxiv2020

Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing in Drosophila

The spatial configuration of the eukaryotic genome is organized and dynamic, providing the structural basis for regulated gene expression in living cells. In Drosophila melanogaster, 3D genome organization is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, the process by which homologs identify one another and pair has remained mysterious. A recent model proposed that specifically interacting "buttons" encoded along the lengths of homologous chromosomes drive somatic homolog pairing. Here, we turned this hypothesis into a precise biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model and constrained its free parameters using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. Our analysis showed strong agreement between model predictions and experiments in the separation dynamics of tagged homologous loci as they transition from unpaired to paired states, and in the percentage of nuclei that become paired as embryonic development proceeds. In sum, as a result of this dialogue between theory and experiment, our data strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.

developmental biology↗

bioRxiv2018

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

developmental biology↗

bioRxiv2018

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shape complex developmental patterns.

developmental biology↗

bioRxiv2018

Binary transcriptional control of pattern formation in development

Predicting developmental outcomes from regulatory DNA sequence and transcription factor patterns remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, we found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe, and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies--bursting and the time window--obey different kinds of regulatory logic, suggesting that the stripe is shaped by the interplay of two distinct underlying molecular processes.

developmental biology↗