Biology subjects

Perkins, M. L.

Publications and source records attributed to Perkins, M. L..

5 recordsLinked to original sources

bioRxiv2024

Chromatin enables precise and scalable gene regulation with factors of limited specificity

Biophysical constraints limit the specificity with which transcription factors (TFs) can target regulatory DNA. While individual nontarget binding events may be low affinity, the sheer number of such interactions could present a challenge for gene regulation by degrading its precision or possibly leading to an erroneous induction state. Chromatin can prevent nontarget binding by rendering DNA physically inaccessible to TFs, at the cost of energy-consuming remodeling orchestrated by pioneer factors (PFs). Under what conditions and by how much can chromatin reduce regulatory errors on a global scale? We use a theoretical approach to compare two scenarios for gene regulation: one that relies on TF binding to free DNA alone, and one that uses a combination of TFs and chromatin-regulating PFs to achieve desired gene expression patterns. We find, first, that chromatin effectively silences groups of genes that should be simultaneously OFF, thereby allowing more accurate graded control of expression for the remaining ON genes. Second, chromatin buffers the deleterious consequences of nontarget binding as the number of OFF genes grows, permitting a substantial expansion in regulatory complexity. Third, chromatin-based regulation productively co-opts nontarget TF binding for ON genes in order to establish a "leaky" baseline expression level, which targeted activator or repressor binding subsequently up- or down-modulates. Thus, on a global scale, using chromatin simultaneously alleviates pressure for high specificity of regulatory interactions and enables an increase in genome size with minimal impact on global expression error. Significance StatementReliably keeping a gene off is as important as controlling its expression level when the gene is on. Yet both tasks become challenging in the packed nuclear environment of a eukaryotic cell, where the numerous and diverse regulatory proteins that are present cannot bind enhancers for target genes with perfect specificity. While regulatory schemes based on prokaryotic models would be overwhelmed by errors in such conditions, we show that chromatin-based regulation, an evolutionary innovation of eukaryotic cells, successfully rescues precise gene expression control by reliably keeping desired genes off. Our systems-level computational analysis demonstrates that this result is nontrivial, because chromatin opening must itself be correctly regulated. We furthermore identify when and how chromatin-based regulation outperforms alternative schemes.

biophysics↗

bioRxiv2022

A bistable autoregulatory module in the developing embryo commits cells to binary fates

Positive autoregulation has been repeatedly proposed as a mechanism for cells to adopt binary fates during embryonic development through bistability. However, without quantitatively determining their parameters, it is unclear whether the plethora of positive autoregulatory modules found within developmental gene regulatory networks are actually bistable. Here, we combine in vivo live imaging with mathematical modeling to dissect the binary cell fate dynamics of the fruit fly pair-rule gene fushi tarazu (ftz), which is regulated by two known enhancers: the early (non-autoregulating) element and the autoregulatory element. Live imaging of transcription and protein concentration in the blastoderm revealed that binary Ftz cell states are achieved as ftz expression rapidly transitions from being dictated by the early element to the autoregulatory element. Moreover, we discovered that Ftz concentration alone is insufficient to activate the autoregulatory element, and that this element only becomes responsive to Ftz at a prescribed developmental time. Based on these observations, we developed a dynamical systems model, and quantitated its kinetic parameters directly from experimental measurements. Our model demonstrated that the ftz autoregulatory module is indeed bistable and that the early element transiently establishes the content of the binary cell fate decision to which the autoregulatory module then commits. Further analysis in silico revealed that the autoregulatory element locks the Ftz expression fate quickly, within 35 min of exposure to the transient signal of the early element. Overall, our work confirms the widely held hypothesis that autoregulation can establish developmental fates through bistability and, most importantly, provides a framework for the quantitative dissection of cellular decision-making based on systems dynamics models and real-time measurements of transcriptional and protein dynamics.

systems biology↗

bioRxiv2022

Enhancer architecture and chromatin accessibility constrain phenotypic space during development

Developmental enhancers are DNA sequences that when bound to transcription factors dictate specific patterns of gene expression during development. It has been proposed that the evolution of such cis-regulatory elements is a major source of adaptive evolution; however, the regulatory and evolutionary potential of such elements remains little understood, masked by selective constraints, drift and contingency. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most mutations in classical developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native cell- and tissue-types. In contrast, random sequences often acted as developmental enhancers, driving expression across a range of levels and cell-types, in patterns consistent with transcription factor motifs therein; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently and resulting in higher levels of expression. Together, our findings suggest that the adaptive phenotypic landscapes of developmental enhancers are constrained by both enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty. QUOTE"Chance and chance alone has a message for us." Milan Kundera, The Unbearable Lightness of Being

developmental biology↗

bioRxiv2020

Dynamic positioning and precision of bistable gene expression boundaries through diffusion and morphogen decay

1Traditional models for how morphogen gradients guide embryonic patterning fail to account for experimental observations of temporal refinement in gene expression domains. Dynamic positional information has recently emerged as a framework to address this shortcoming. Here, we explore two central aspects of dynamic positional information--the precision and placement of gene expression boundaries--in bistable genetic networks driven by morphogen gradients. First, we hypothesize that temporal morphogen decay may increase the precision of a boundary by compensating for variation in initial conditions that would otherwise lead neighboring cells with identical inputs to diverge to separate steady states. Second, we explore how diffusion of gene products may play a key role in placing gene expression boundaries. Using an existing model for Hb patterning in embryonic fruit flies, we show that diffusion permits boundaries to act as near-traveling wavefronts with local propagation speed determined by morphogen concentration. We then harness our understanding of near-traveling fronts to propose a method for achieving accurate steady-state boundary placement independent of initial conditions. Our work posits functional roles for temporally varying inputs and cell-to-cell coupling in the regulation and interpretation of dynamic positional information, illustrating that mathematical theory should serve to clarify not just our quantitative, but also our intuitive understanding of patterning processes. 2 Author SummaryIn many developmental systems, cells interpret spatial gradients of chemical morphogens to produce gene expression boundaries in exact positions. The simplest mathematical models for "positional information" rely on threshold detection, but such models are not robust to variations in the morphogen gradient or initial protein concentrations. Furthermore, these models fail to account for experimental results showing dynamic shifts in boundary placement and increased boundary precision over time. Here, we propose two theoretical mechanisms for enhancing boundary precision and placement using a bistable toggle switch. Distinct from existing research in "dynamic positional information", this work posits a functional role for temporal decay in morphogen concentration and for diffusion of gene expression products, the latter of which is often omitted in quantitative models. We suggest that future research into dynamic positional information would benefit from perspectives that link local (cellular) and global (patterning) behaviors, as well as from mathematical theory that builds our intuitive understanding alongside more data-driven approaches.

developmental biology↗

bioRxiv2019

Cell-in-the-loop pattern formation with optogenetically emulated cell-to-cell signaling

1Designing and implementing synthetic biological pattern formation remains a challenge due to underlying theoretical complexity as well as the difficulty of engineering multicellular networks bio-chemically. Here, we introduce a \"cell-in-the-loop\" approach where living cells interact through in silico signaling, establishing a new testbed to interrogate theoretical principles when internal cell dynamics are incorporated rather than modeled. We present a theory that offers an easy-to-use test to predict the emergence of contrasting patterns in gene expression among laterally inhibiting cells. Guided by the theory, we experimentally demonstrated spontaneous checkerboard patterning in an optogenetic setup where cell-to-cell signaling was emulated with light inputs calculated in silico from real-time gene expression measurements. The scheme successfully produced spontaneous, persistent checkerboard patterns for systems of sixteen patches, in quantitative agreement with theoretical predictions. Our research highlights how tools from dynamical systems theory may inform our understanding of patterning, and illustrates the potential of cell-in-the-loop for engineering synthetic multicellular systems.

synthetic biology↗