Cooperative assembly confers regulatory specificity and long-term genetic circuit stability.

A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

Global decoupling of cell differentiation from cell division in early embryo development.

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. In the absence of cell division, differentiation slows down in some cell types, and cells exhibit global stress responses. While differentiation is robust to blocking cell division, the proportions of cells across cell states are not. This work simplifies our understanding of the role of cell division in development and showcases the utility of combining embryo-wide perturbations with single-cell RNA sequencing to uncover the role of common biological processes across multiple tissues.

The secreted neuronal signal Spock1 promotes blood-brain barrier development.

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 initiates BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.

Ureteral injury after posterior spinal fusion.

Entry into the retroperitoneal space during Minimally Invasive Transforaminal Lumbar Interbody Fusion (MI-TLIF) presents a rare possibility of iatrogenic ureteral injury. Rare complications of ureteral injury after previous open posterior spinal surgery have been reported however there is a paucity of reports after a minimally invasive technique. We now describe a case of ureteral injury after MI-TLIF.

Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies.

Eukaryotic genes are regulated by multivalent transcription factor complexes. Through cooperative self-assembly, these complexes perform nonlinear regulatory operations involved in cellular decision-making and signal processing. In this study, we apply this design principle to synthetic networks, testing whether engineered cooperative assemblies can program nonlinear gene circuit behavior in yeast. Using a model-guided approach, we show that specifying the strength and number of assembly subunits enables predictive tuning between linear and nonlinear regulatory responses for single- and multi-input circuits. We demonstrate that assemblies can be adjusted to control circuit dynamics. We harness this capability to engineer circuits that perform dynamic filtering, enabling frequency-dependent decoding in cell populations. Programmable cooperative assembly provides a versatile way to tune the nonlinearity of network connections, markedly expanding the engineerable behaviors available to synthetic circuits.

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules.

Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating "read-write" architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity.

Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this noise in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock factor 1 (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90, a known phenotypic capacitor and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation, rather than absolute Hsf1 activity, promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a range of phenotypic states.

Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response.

Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone. Previously we identified Hsp70 as the chaperone responsible for Hsf1 repression and constructed a mathematical model that recapitulated the yeast heat shock response (Zheng et al., 2016). The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validate these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next, we mapped a discrete Hsp70 binding site on Hsf1 to a C-terminal segment known as conserved element 2 (CE2). In vitro, CE2 binds to Hsp70 with low affinity (9 µM), in agreement with model requirements. In cells, removal of CE2 resulted in increased basal Hsf1 activity and delayed deactivation during heat shock, while tandem repeats of CE2 sped up Hsf1 deactivation. Finally, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the heat shock response.

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation.

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways.