Lateral confined growth of cells activates Lef1 dependent pathways to regulate cell-state transitions.

Long-term sustained mechano-chemical signals in tissue microenvironment regulate cell-state transitions. In recent work, we showed that laterally confined growth of fibroblasts induce dedifferentiation programs. However, the molecular mechanisms underlying such mechanically induced cell-state transitions are poorly understood. In this paper, we identify Lef1 as a critical somatic transcription factor for the mechanical regulation of de-differentiation pathways. Network optimization methods applied to time-lapse RNA-seq data identify Lef1 dependent signaling as potential regulators of such cell-state transitions. We show that Lef1 knockdown results in the down-regulation of fibroblast de-differentiation and that Lef1 directly interacts with the promoter regions of downstream reprogramming factors. We also evaluate the potential upstream activation pathways of Lef1, including the Smad4, Atf2, NFkB and Beta-catenin pathways, thereby identifying that Smad4 and Atf2 may be critical for Lef1 activation. Collectively, we describe an important mechanotransduction pathway, including Lef1, which upon activation, through progressive lateral cell confinement, results in fibroblast de-differentiation.

Actomyosin contractility as a mechanical checkpoint for cell state transitions.

Cell state transitions induced by mechano-chemical cues result in a heterogeneous population of cell states. While much of the work towards understanding the origins of such heterogeneity has focused on the gene regulatory mechanisms, the contribution of intrinsic mechanical properties of cells remains unknown. In this paper, using a well-defined single cell platform to induce cell-state transitions, we reveal the importance of actomyosin contractile forces in regulating the heterogeneous cell-fate decisions. Temporal analysis of laterally confined growth of fibroblasts revealed sequential changes in the colony morphology which was tightly coupled to the progressive erasure of lineage-specific transcription programs. Pseudo-trajectory constructed using unsupervised diffusion analysis of the colony morphology features revealed a bifurcation event in which some cells undergo successful cell state transitions towards partial reprogramming. Importantly, inhibiting actomyosin contractility before the bifurcation event leads to more efficient dedifferentiation. Taken together, this study highlights the presence of mechanical checkpoints that contribute to the heterogeneity in cell state transitions.

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models.

Cell geometry dictates TNFα-induced genome response.

Cells in physiology integrate local soluble and mechanical signals to regulate genomic programs. Whereas the individual roles of these signals are well studied, the cellular responses to the combined chemical and physical signals are less explored. Here, we investigated the cross-talk between cellular geometry and TNFα signaling. We stabilized NIH 3T3 fibroblasts into rectangular anisotropic or circular isotropic geometries and stimulated them with TNFα and analyzed nuclear translocation of transcription regulators -NFκB (p65) and MKL and downstream gene-expression patterns. We found that TNFα induces geometry-dependent actin depolymerization, which enhances IκB degradation, p65 nuclear translocation, nuclear exit of MKL, and sequestration of p65 at the RNA-polymerase-II foci. Further, global transcription profile of cells under matrix-TNFα interplay reveals a geometry-dependent gene-expression pattern. At a functional level, we find cell geometry affects TNFα-induced cell proliferation. Our results provide compelling evidence that fibroblasts, depending on their geometries, elicit distinct cellular responses for the same cytokine.

Superresolution imaging of nanoscale chromosome contacts.

Co-expression of a specific group of genes requires physical associations among these genes, which form functional chromosomal contacts. While DNA fluorescence in situ hybridization (FISH) pinpoints the localization of genes within the 3D nuclear architecture, direct evidence of physical chromosomal contacts is still lacking. Here, we report a method for the direct visualization of transcription-dependent chromosomal contacts formed in two distinct mechanical states of cells. We prepared open chromatin spreads from isolated nuclei, ensuring 2D rendering of chromosome organization. Superresolution imaging of these chromatin spreads resolved the nanoscale organization of genome contacts. We optimized our imaging method using chromatin spreads from serum+/- cells. We then showed direct visualization of functional gene clusters targeted by YAP (Yes-associated protein) and SRF (Serum response factor) transcription factors. In addition, we showed the association of NF-κB bound gene clusters induced by TNF-α addition. Furthermore, EpiTect ChIP qPCR results showed that these nanoscale clusters were enriched with corresponding transcription factors. Taken together, our method provides a robust platform to directly visualize and study specific genome-wide chromosomal contacts.

Stochastic signalling rewires the interaction map of a multiple feedback network during yeast evolution.

During evolution, genetic networks are rewired through strengthening or weakening their interactions to develop new regulatory schemes. In the galactose network, the GAL1/GAL3 paralogues and the GAL2 gene enhance their own expression mediated by the Gal4p transcriptional activator. The wiring strength in these feedback loops is set by the number of Gal4p binding sites. Here we show using synthetic circuits that multiplying the binding sites increases the expression of a gene under the direct control of an activator, but this enhancement is not fed back in the circuit. The feedback loops are rather activated by genes that have frequent stochastic bursts and fast RNA decay rates. In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes. Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

Construction of cis-regulatory input functions of yeast promoters.

Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic constructs and measure gene expression to deliver data suitable for quantitative analysis. Synthetic genetic constructs are well suited to precisely control and measure gene expression to construct cis-regulatory input functions. These functions can be used to predict gene expression based on signal intensities transmitted to activators and repressors in the gene regulatory region. Simple models of gene expression are presented for competitive and noncompetitive repressions. Complex phenomena, exemplified by synergistic silencing, are modeled by reaction-diffusion equations.

Spatial epigenetic control of mono- and bistable gene expression.

Bistability in signaling networks is frequently employed to promote stochastic switch-like transitions between cellular differentiation states. Differentiation can also be triggered by antagonism of activators and repressors mediated by epigenetic processes that constitute regulatory circuits anchored to the chromosome. Their regulatory logic has remained unclear. A reaction-diffusion model reveals that the same reaction mechanism can support both graded monostable and switch-like bistable gene expression, depending on whether recruited repressor proteins generate a single silencing gradient or two interacting gradients that flank a gene. Our experiments confirm that chromosomal recruitment of activator and repressor proteins permits a plastic form of control; the stability of gene expression is determined by the spatial distribution of silencing nucleation sites along the chromosome. The unveiled regulatory principles will help to understand the mechanisms of variegated gene expression, to design synthetic genetic networks that combine transcriptional regulatory motifs with chromatin-based epigenetic effects, and to control cellular differentiation.

Control and signal processing by transcriptional interference.

A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context.

Synergy of repression and silencing gradients along the chromosome.

The expression of a gene is determined by the transcriptional activators and repressors bound to its regulatory regions. It is not clear how these opposing activities are summed to define the degree of silencing of genes within a segment of the eukaryotic chromosome. We show that the general repressor Ssn6 and the silencing protein Sir3 generate inhibitory gradients with similar slopes over a transcribed gene, even though Ssn6 is considered a promoter-specific repressor of single genes, while Sir3 is a regional silencer. When two repression or silencing gradients flank a gene, they have a multiplicative effect on gene expression. A significant amplification of the interacting gradients distinguishes silencing from repression. When a silencing gradient is enhanced, the distance-dependence of the amplification changes and long-range effects are established preferentially. These observations reveal that repression and silencing proteins can attain different tiers in a hierarchy of conserved regulatory modes. The quantitative rules associated with these modes will help to explain the co-expression pattern of adjacent genes in the genome.

Structure of biosynthetic N-acetylornithine aminotransferase from Salmonella typhimurium: studies on substrate specificity and inhibitor binding.

Acetylornithine aminotransferase (AcOAT) is one of the key enzymes involved in arginine metabolism and catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate. It belongs to the Type I subgroup II family of pyridoxal 5'-phosphate (PLP) dependent enzymes. E. coli biosynthetic AcOAT (eAcOAT) also catalyzes the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate, one of the steps in lysine biosynthesis. In view of the critical role of AcOAT in lysine and arginine biosynthesis, structural studies were initiated on the enzyme from S. typhimurium (sAcOAT). The K(m) and k(cat)/K(m) values determined with the purified sAcOAT suggested that the enzyme had much higher affinity for AcOrn than for ornithine (Orn) and was more efficient than eAcOAT. sAcOAT was inhibited by gabaculine (Gcn) with an inhibition constant (K(i)) of 7 microM and a second-order rate constant (k(2)) of 0.16 mM(-1) s(-1). sAcOAT, crystallized in the unliganded form and in the presence of Gcn or L-glutamate, diffracted to a maximum resolution of 1.90 A and contained a dimer in the asymmetric unit. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop L(alphak-) (betam) in only one subunit (subunit B; the loop from subunit B comes close to the phosphate group of PLP in subunit A). Structural and spectral studies of sAcOAT with Gcn suggested that the enzyme might have a low affinity for PLP-Gcn complex. Comparison of sAcOAT with T. thermophilus AcOAT and human ornithine aminotransferase suggested that the higher specificity of sAcOAT towards AcOrn may not be due to specific changes in the active site residues but could result from minor conformational changes in some of them. This is the first structural report of AcOAT from a mesophilic organism and could serve as a basis for drug design as the enzyme is important for bacterial cell wall biosynthesis.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the biosynthetic N-acetylornithine aminotransferases from Salmonella typhimurium and Escherichia coli.

Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5'-phosphate-dependent enzyme catalyzing the conversion of N-acetylglutamic semialdehyde to N-acetylornithine in the presence of alpha-ketoglutarate, a step involved in arginine metabolism. In Escherichia coli, the biosynthetic AcOAT also catalyzes the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli, overexpressed in E. coli and purified using Ni-NTA affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X-rays to 3.5 A and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 A and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular-replacement method.