Expert Review, Nature Chemical Biology | Exploring a New Mechanism of Cell Fate Determination by Quantitative Synthetic Biology

Cell differentiation allows cells with the same genotype to produce cells that differ in morphology, structure, and physiological function. For the occurrence of cell differentiation, the classical formulation suggests that the gene function of cells and the complex regulatory network they form control the expression of genes in time and space, thereby programming the process of cell fate determination. Although we can analyze the function of most genes, measure the spatiotemporal dynamics of gene expression, and plot a sketch of the gene regulatory network; in the process of cell fate determination, we still do not understand the source of gene differential expression, nor can we accurately predict the direction of the fate determination process.

On March 24, Beijing time, Fu Xiongfei's team of the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, published a research achievement in Nature Chemical Biology under the title of “Unbalanced response to growth variations reshapes the cell fate decision Landscape”. In this study, the research paradigm of organism creation for knowledge was used to explore the impact of cell growth rate on the bistability of the classical artificial synthetic genetic circuit - mutual inhibition circuit1 - through quantitative experiments and a mathematical model. It was found that the expression of different genes exhibited unbalanced and asynchronous responses to growth rate, thereby reshaping the Landscape of cell fate determination. The study suggested that growth rate can globally up-regulate the expression of each gene in the gene expression network and thus change cell fate, without necessarily relying on specific regulatory factors. This study provides a new perspective for the study of the regulatory mechanism of fate determination, and also provides a new idea for quantitatively controlling cell fate by synthetic biological methods for medical and industrial purposes.

 

 

Screenshot of the published paper

Link of the paper: https://www.nature.com/articles/s41589-023-01302-9

By biochemical and molecular biological methods, scientists have studied the functions and regulatory relationships of genes, as well as the signaling mechanism, plotted a sketch of the cell fate determination network; and synthesized and reconstructed functional gene networks, such as: bistable switch1, biological oscillation2, and pattern formation3, so that humans have taken a leap forward on the road of creation. However, leaving aside the details of genes in the network, we still do not understand how information is transmitted in the cell fate determination network; nor is it possible to perfectly predict the state of operation of the network and then infer the direction of cell fate. If we go beyond the details of the network and look at the whole network from a global perspective, are there any special factors that can reshape the cell fate determination Landscape and even emerge with new functions?

 

Phenotypic bistability dependent on cell growth rate

To investigate whether the growth rate of cells may have an impact on gene networks, the research team investigated this possibility by using the classical synthetic genetic circuit - the toggle switch1. The toggle switch is composed of two mutually repressing genes, so that only one gene is highly expressed in the stable states of the circuit. Such network topology also exists widely in nature, for example: bacteriolysis - lysogen determination of λ phage, and differentiation of left and right gustatory nerves in nematodes. In addition, the elements that make up this circuit have been characterized extensively and quantitatively and are orthogonal to the host's authigenic gene regulatory network, so direct regulation of the genetic circuit by the host's own state can be excluded, and the homeostatic behavior of the network can be analyzed quantitatively.

The research team accidentally found that in SOB medium, no matter the initial state of cells was red or green, in the one-step growth process, the cells in the plateau phase would be in a red state (Figure 1). Therefore, it is speculated that there may be some global physiological changes that affect cell fate determination decisons .

 

Figure [1] No matter the initial state was red or green, the cells were in the red state in the plateau phase, and green cells would spontaneously switch to the red state before entering the plateau phase.

 

Inspired by research4-6 on bacterial physiology, the research team observed the homeostatic properties and bistability of toggle switches under different physiology steady states of cells. The experimental results showed that the bistability of the genetic circuit was correlated with the cell growth rate (Figure 2). When the cell growth rate was greater than 0.5 h-1, the toggle switch exhibited bistability. When the cell growth rate was below this critical growth rate, the bistability of the toggle switch exhibited a bifurcation, that is, cells could only maintain a red steady state in the slow growth state, and the bistability of the circuit disappeared. Furthermore, it seems possible that changes in growth rate can cause the homeostatic properties of fate determination the network and influence the direction of cell fate.

 

Figure [2] Under different culture conditions, the homeostatic state and quantity of cells would change. When the cell growth rate was below 0.5h-1, the cells only had one stable state, namely red.

 

Unbalance in response of gene expression to changes in growth rate

To further reveal how changes in the physiological state of cells dominate the cell fate determination process, the research team quantitatively characterized the expression of two repressor proteins in toggle switches under different growth states. First, the research team found that the expression of the two genes showed an upward trend as the cell growth rate slowed down, and the rate of increase was greater than the impact of the decreasing dilution rate caused by the slowing down of cell growth; accordingly, the research team inferred that the translation rate of the two genes also changed with the cell growth rate. By using quantitative characterization of fluorescent protein and transcriptomics data, the research team found that although the expression of both showed a negative correlation with cell growth rate in the general trend, the peak and relative variation of expression rate were not the same (Figure 3a). By using a mathematical model, the research team evaluated the impact of this unbalanced growth rate dependent gene expression pattern on the stability of genetic circuits (Figure 3 b, c), and demonstrated that this growth rate dependence created the possibility of bifurcation to the bistability of the toggle switch (Figure 3 d).

 

Figure [3] Experimental and model analysis showed that growth rate dependent gene expression could reshape the fate determination landscape of cells.

 

Determining mechanism of critical growth rate

By further analyzing the mathematical model, the research team found that the critical growth rate at which the toggle switch bifurcated could also be regulated by the repression threshold (dissociation constant) of the repressor protein. By altering the LacI binding site sequence, the repression threshold of LacI protein on TetR protein expression could be altered; thus, the team obtained two mutants, namely LO2 and LO3, with stronger repression intensity than the original sequence (LO1). Through quantitative experiments, they determined the repression thresholds of the three, as well as their homeostatic states and quantities under different physiological conditions (Figure 4). The research team found that compared with the bistability shown by LO1 and LO2 within the range of experimentally measurable growth rate, LO3 showed monostability with a green state when the growth rate was greater than the critical growth rate, while showed bistability when the growth rate was lower than the critical growth rate. This result was consistent with the prediction of the mathematical model.

 

Figure [4]. Altering the repression threshold of the repressor protein could translate the bifurcation diagram of the response of the genetic circuit to the growth rate.

 

Dynamic behavior near bifurcation points

Under homeostatic conditions, the growth rate can reshape the fate determining landscape of cells. Another important question is: how do decisions about cell fate occur in a changing environment, as the life activity of cells is an unbalanced system?

The research team explored this issue by perturbing the cell growth rate. They switched the cell growth rate by dynamically changing the composition of the culture medium, and tracked the state of cells in real time. They first cultured the cells to a physiologically stable state under rapid growth conditions, then switched to a nutrient-poor medium (growth downshift), and then switched back to a nutrient-rich medium (growth upshift) when the cells returned to a physiologically stable state. It was found that in the initial stage of growth downshift, both red and green states of the LO1 strain with an initial green state increased simultaneously; as the red state increased, the green state began to decrease and eventually stabilized in the red state. By contrast, the LO3 strain maintained a green state throughout the whole process due to its bistability under slow growth conditions. The research team performed quasi-homeostatic approximation between the expression rates of LacI and TetR to their instantaneous growth rate, and constructed a deterministic dynamic model of the genetic circuit, which could well capture the dynamic characteristics of cells during upshift and downshift.

Interestingly, during the growth downshift of the LO2 strain, its cells differentiated with some cells still able to maintain a green state and some cells differentiating into red, although the strain exhibited bistability within any range of growth rate. This phenomenon could not be predicted by deterministic dynamic models. The research team quantitatively studied the impact of the noise of gene expression on cell fate determination under different growth rates by using the potential landscape. They then found that when the cell growth rate was slow or fast, the energy barrier between the two stable states in the potential Landscape was low, which meant that the cells were more likely to switch states due to noise; therefore, in the downshift process of the LO2 strain, some cells switched states under random drive.

Therefore, the fate determination process of cells could be determined in two ways: 1. Deterministic mechanism. The changes in the homeostatic properties of the network caused all of the cell population to switch their states; 2. By noise driving, the state jump near the critical point controlled part of the cell population to switch their states.

 

Figure [5] Different genetic circuits had irreversible or reversible fate determination trajectories for growth fluctuations.

 

Summary

To determine the relationship between growth rate and fate determination, the research team used quantitative synthetic biology to analyze the growth rate relationship of gene expression in bistable genetic circuits and the subsequent changes in the state of the genetic circuits. Under different nutritional conditions, they obtained a large number of experimental results by quantifying the number of stable states of circuits and the dynamic process of cell fate determination, and stated how to reshape the fate determination Landscape with growth changes:

1. The global interdependence between gene expression and cell growth rate was not fully reciprocal in the regulatory network. The asynchronous response to growth changes led to a bifurcation of the fate determination Landscape. This reshaping process did not rely on specific molecular signals or regulatory circuits, but was an intrinsic characteristic of the regulation of fate determination network by the global regulatory mechanism of cell physiology.

2. The phenotypic state transition driven by growth changes was controlled by changes in the fate determination Landscape. Based on quantitative observations of the experiments, a phenomenological mathematical model was constructed, the mechanism behind this phenomenon was revealed, and the dynamic process of cell fate determination mediated by growth rate.

3. The research team used classical synthetic circuits to simulate key nodes in the fate determination regulatory network, eliminated the potential impact of other gene regulation in the network, and enabled precise control of key parameters in the network. In addition, they also provided a method to improve the robustness of genetic circuit to growth perturbations, promoting the application of genetic circuit in biological computing and dynamic control of biological processes.

Fu Xiongfei, a researcher of the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, is the corresponding author of the paper, and assistant researcher Zhu Jingwen and doctoral candidate Chu Pan are the co-first authors of the paper. The research was supported by several programs, including the National Key Research and Development Program, the National Natural Science Foundation of China, the Priority Special Program B of the Chinese Academy of Sciences, the Natural Science Foundation of the Guangdong Basic and Applied Basic Research Foundation, Shenzhen Institute of Synthetic Biology, and the Major Technological Infrastructure for Synthetic Biology.

 

Expert comments: Zhao Guoping (academician of CAS)

The fate determining mechanism of cells is a fundamental problem in life science. Fu Xiongfei's team of the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, successfully decoupled the growth rate and cell fate determination network state using the quantitative synthetic biology research paradigm by synthetic biology technology, and found that the response of individual genetic circuit nodes to the unbalance of growth rate determines cell fate, thus enabling scientists to explore the universal mechanism of cell fate. This study provides systematic and quantitative insights for evaluating the robustness of genetic circuits.

This interesting study delved into the impact of cell growth rate on classical synthetic genetic circuits by combining quantitative experiments and mathematical models. This study explored the regulatory mechanism of cell fate from an innovative perspective, providing a new idea for quantitatively controlling cell fate by synthetic biological methods for medical and industrial purposes, and has significant impact on its related research fields.

Expert comments: Tang Leihan (Professor of Hong Kong Baptist University)

The fact that offspring after cell division can exhibit different phenotypes is a fundamental and ancient problem in developmental biology. C. H. Waddington, the founder of epigenetics, proposed the Waddington Landscape in the 1940s, in which a ball rolling down a gully slope was used to describe the gradual process of cell differentiation during embryonic development. With the development of modern biology, more and more details of the continuation and alteration of maternal traits by substances other than DNA in cells have been discovered, and the analogy of the Waddington Landscape has also been constantly enriched and improved. The progress of experiments has stimulated the establishment of quantitative models and theories, especially the study of gene expression dynamics, which has provided important interpretations and supplements to the Waddington Landscape.

The epigenetics of microorganisms also have several similar properties, and their research can learn from each other with the development of multicellular organisms, which has important biological theory and application value. Fu Xiongfei's team of the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, used quantitative biological technology to observe the genetic characteristics of classical synthetic genetic circuits - toggle switches at different growth rates, and found that too fast or too slow growth rates can cause asymmetric gene expression, and thus change the fate of daughter cells. Based on quantitative experimental data, researchers characterized the gene network dynamics under growth rate regulation through a concise mathematical model, and explained and experimentally verified various homeostatic and dynamic behaviors through bifurcation analysis, energy well analysis, and other means. This study connected the growth rate with the fate of daughter cells for the first time, providing a quantitative theoretical basis and implementation plan for the future design that the growth rate directly or indirectly controls the phenotype of daughter cells.

 

References

1. Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature403, 339–342 (2000).

2. Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature403, 335 (2000).

3. Liu, C. et al. Sequential Establishment of Stripe Patterns in an Expanding Cell Population. Science334, 238–241 (2011).

4. Klumpp, S., Zhang, Z. & Hwa, T. Growth Rate-Dependent Global Effects on Gene Expression in Bacteria. Cell139, 1366–1375 (2009).

5. Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z. & Hwa, T. Interdependence of Cell Growth and Gene Expression: Origins and Consequences. Science330, 1099–1102 (2010).

6. Zheng, H. et al. General quantitative relations linking cell growth and the cell cycle in Escherichia coli. Nat Microbiol5, 995–1001 (2020).

 

 

PI introduction:

Fu Xiongfei, researcher, doctoral supervisor, deputy director of the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, deputy director of the Key Laboratory of Quantitative Engineering Biology, Chinese Academy of Sciences, and vice president of Shenzhen Institute of Synthetic Biology. The main research direction of the research group: By combining quantitative theory and synthetic reconstruction, conduct research on issues such as the interaction between intracellular genetic circuits and chassis, and the formation principle of unicellular and multicellular ordered spatial structure.

The research group is now recruiting postdoctoral fellows, research assistants, technicians, visiting students, etc. with relevant research backgrounds in mathematics, physics, chemistry, biology, fluid mechanics, complex systems, etc. If you are interested, please contact yang.bai@siat.ac.cn. The subject line of the resume and email should be “Position applied - School Name - Major - Name”.

Laboratory homepage:

http://isynbio.siat.ac.cn/fulab